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Characterization of target cell entry by murine leukemia viruses

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Characterization of target cell entry by murine leukemia viruses

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CHARACTERIZATION OF TARGET CELL ENTRY BY MURINE
LEUKEMIA VIRUSES
by
Louis John Katen QI
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biochemistry and Molecular Biology)
December 1998
© 1998 Louis John Katen IH
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V
/
CHARACTERIZATION OF TARGET CELL ENTRY BY MURINE
LEUKEMIA VIRUSES
Louis John Katen III
W. French Anderson, M.D. (Adviser)
It is important to understand the pathways being utilized for target cell
entry by naturally occurring viruses and retroviral vectors both for the
development of anti-viral therapies, as well as for the development of retroviral
vectors for gene therapy. The two possible entry pathways are believed to be
either by endocytosis or by direct entry through the plasma membrane. The
former is thought to provide a necessary acidic environment that induces a
conformational change in the viral envelope protein which facilitates fusion
between the viral and cell membranes. This pathway is referred to as pH
dependent. The latter pathway is thought to reflect the ability of viral particles to
fuse with the plasma membrane in the absence of any acidic environment and is
referred to as pH independent. Lysosomotropic bases, as well as proton
ionophores, have been used to determine the requirement for an acidic
compartment during viral entry based on the ability of these agents to raise the pH
in intracellular acidic compartments. A reduction of viral infectivity resulting
from treatment of target cells lysosomotropic drugs has been interpreted to
indicate the use of an endosomal entry pathway and the requirement for an acidic
environment for viral entry. Based on such assays, ecotropic murine leukemia
viruses (MLV-E) have been classified as pH dependent and amphotropic murine
leukemia viruses as pH independent However, these classifications are based on
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partial sensitivity of both viruses to lysosomotropic drugs. Additionally, there has
been significant variability in the reports from such assays. I have reexamined
viral entry assays utilizing lysosomotropic drugs and have determined that these
assays are measuring predominantly the inherent instability o f the viral particles
and not the requirement for an acidic compartment during entry. Furthermore, the
data presented here suggests that MLV-A and MLV-E are using an endocytic
entry pathway.
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90007
This dissertation written by
under the direction of fcia. Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School, in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
Dean of Graduate Studies
Date l / j l f a l
DISSERTATION COMMITTEE
Chairperson f
A ,
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Table of Contents
List of Figures and Tables iv
Chapter 1: Introduction I
Chapter 2: Materials and Methods 21
2.1 Lysosomotropic Drugs, Antibodies, and Virus Stocks 21
2.2 Cell Lines and Viral Vector Production 22
2.3 Mixed Vector Titer Assays with NH4 CI or BFLA1 30
2.4 Viral Vector Stability Assay 33
2.5 Viral Vector Inactivation Assay 35
2.6 Viral Envelope Protein Immunofluorescence Assay 37
2.7 Viral Vector Internalization in the Presence of NH4 CI
and BFLA1 38
Chapter 3: Effect of Lysosomotropic Drugs on the Transduction
ofNIH 3T3 Cells by Ecotropic and Amphotropic
Murine Leukemia Virus-Based Vectors 40
3.1 Relative viral vector titer is reduced by NH4 CI and
BFL A1 in the mixed vector titer assay 40
3.2 Results in the NH4 CI and BFL Al mixed vector titer
assay are the same for vectors generated from PA317
and PE501 packaging cells and vectors generated
with MoMLV and 4070A RCR 44
Chapter 4 : in vitro Instability of Viral Vectors Correlates with
the Loss in Relative Vector Titer in the NH4 CI and
BFLA1 Titer Assays 48
4.1 Increasing the duration of MLV-A, MLV-E, and
MoMLV(VSV-G) infection ofNIH 3T3 cells in the
presence of either NH4 CI or BFLA1 results in
a reduction of relative viral vector titer 48
4.2 The in vitro instability o f MLV-A, MLV-E, and
MoMLV(VSV-G) viral vectors correlates with
the reduction in relative viral vector titer in the
NH4 CI and BFLA1 titer assays 59
ii
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Chapter S: Internalization of Ecotropic and Amphotropic
Murine Leukemia Virus-Based Vectors into Target
Cells 66
S. I Viral vectors are inactivated by pH 3 citric acid buffer 66
S.2 MLV-A and MLV-E are internalized in the NH4CI
and BFLAl titer assays 72
Chapter 6: Discussion 79
References 96
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List of Figures and Tables
Table 1 Previously published results from NH4 CI viral
entry assays
Figure 1 Possible pathways for viral entry into target cells
are either by endocytosis or by direct fusion with the
plasma membrane
Figure 2 Mechanism of action on endosomal and lysosomal
acidification by NH4C1 and BFLAl
Figure 3 Both NH4C1 and BFLAl have been shown to inhibit
transport through the endosomal pathway
Figure 4 Model for MLV entry into target cells
Figure 5 Schematic of MLV-E, MLV-A, and MoMLV(VSV-G)
viral vector production
Figure 6 Schematic for production of MLV-E and MLV-A viral
vectors using the replication competent retroviruses
MoMLV and 4070A, respectively
Figure 7 Schematic of mixed viral vector titer assay with
NH4CI or BFLAl
Figure 8 Schematic of viral vector stability assay
Figure 9 Schematic of the citric acid viral vector inactivation assay
Figure 10 Effect of S O mM NH4CI on transduction ofNIH 3T3 cells
by MLV-A and MLV-E
Figure 11 Effect of S O nM BFLAl on transduction ofNIH 3T3 ceils
by MLV-A and MLV-E
Figure 12 Effect of S O mM NH4 CI on transduction ofNIH 3T3 cells
by 4070A(G1 nBgS VNa) and MoMLV(LAPSN)
Figure 13 Effect of S O nM BFLAl on transduction ofNIH 3T3 cells
by 4070A(GlnBgSVNa) and MoMLV(LAPSN)
Figure 14 Schematic ofNHtCl and BFLAl titer assay variations
7
2
5
14
16
23
27
31
34
36
42
43
46
47
50
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Figure IS Effect of S O mMNHtCl on MLV-A, MLV-E, and
MoMLV(VSV-G) transduction ofNIH 3T3 cells for
a S minute infection and no drug chase
Figure 16 Effect of S O mM NH4 CI on MLV-A, MLV-E, and
MoMLV(VSV-G) transduction ofNIH 3T3 cells for
a 2 hour infection and no drug chase
Figure 17 Effect of 50 mM NH4 CI on MLV-A, MLV-E, and
MoMLV(VSV-G) transduction ofNIH 3T3 cells for
a 2 hour infection and a 2 hour drug chase
Figure 18 Effect of S O nM BFLAl on MLV-A, MLV-E, and
MoMLV(VSV-G) transduction ofNIH 3T3 cells for
a S minute infection and no drug chase
Figure 19 Effect of 50 nM BFLAl on MLV-A, MLV-E, and
MoMLV(VSV-G) transduction ofNIH 3T3 cells for
a 2 hour infection and no drug chase
Figure 20 Effect of 50 nM BFLAl on MLV-A, MLV-E, and
MoMLV(VSV-G) transduction ofNIH 3T3 cells for
a 2 hour infection and a 2 hour drug chase
Figure 21 Stability of MLV-A at 37°C in medium containing H2 O,
S O mMNHtCI, 0.1 % DMSO, and S O nM BFLAl
Figure 22 Stability of MLV-E at 37°C in medium containing HjO,
50 mM NH4CI, 0.1% DMSO, and 50 nM BFLAl
Figure 23 Stability of MoMLV(VSV-G) at 37°C in medium
containing H20, 50 mMNHtCI, 0.1% DMSO, and
50 nM BFLAl
Figure 24 Correlation between the in vitro instability of MLV-A,
MLV-E, and MoMLV(VSV-G) viral vectors in 50 mM
NH4CI and the reduction in relative titer in the
NH4CI titer assay
Figure 25 Correlation between the in vitro instability of MLV-A,
MLV-E, and MoMLV(VSV-G) viral vectors in 50 nM
BFLAl and the reduction in relative titer in the
BFLAl titer assay
53
54
55
56
57
58
61
62
63
64
65
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Figure 26
Figure 27
Figure 28
Figure 29
Figure 30
Figure 3 1
Inactivation of cell-bound MLV-A and MLV-E by
citric acid treatment from pH 3 to 7 68
Effect of citric acid treatment on the transducability
ofNIH 3T3 cells 70
Effect of pH 3 citric acid treatment on the
immunofluorescent detection of the MoMLV envelope
protein on MoMLV infected NIH 3T3 cells 73
Effect of pH 3 citric acid treatment on the
immunofluorescent detection of the 4070A envelope
protein on 4070A infected NIH 3T3 cells 74
Viral vector internalization into N IH 3T3 cells in
the presence of H2 O and S O mM NH4CI based on
resistance to pH 3 citric acid treatment 76
Viral vector internalization into NIH 3T3 cells in
the presence of DMSO and S O nM BFLAl based on
resistance to pH 3 citric acid treatment 77
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Chapter 1: Introduction
In order to engineer efficient retroviral vectors for gene therapy, it is
important to understand the target cell entry mechanisms used by these vectors.
Furthermore, a general knowledge of host cell entry processes employed by
naturally occurring retroviruses is important for the understanding of the
infectious properties of the viruses and for developing strategies for anti-viral
therapy. The present study focuses on the elucidation of target cell entry
pathways being utilized by retroviral vectors based on the ecotropic and
amphotropic murine leukemia virus (MLV).
Enveloped viruses enter their host cells by fusion of the viral and cell lipid
bilayer following binding of the virus to its cellular receptor. Following fusion of
the viral and cell membranes, the viral core is released into the cytosol ultimately
resulting in nuclear import of the viral genome. The receptor specificity and
membrane fusion is a function of the viral envelope protein. Viral entry is
believed to proceed by either of two pathways (Fig. 1). The first is by receptor-
mediated endocytosis following receptor binding, where acidification of
endocytic vesicles is believed to trigger a conformational change in the viral
envelope protein that renders it fosogenic. However, the absolute necessity for
acidic activation of viral fusion in endocytosed virions is controversial(44).
1
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Endocytoiii
(pH-dependent)
Direct Membrane Fusion
(pH-in dependent)
Plasma Membrane
Early
Endosome
pH <6.2
Endosomal
Carrier
Vesicle
Late
Endosome
pH <5.5
Lysosome
Figure I. Possible pathways for viral entry into target cells are either by
endocytosis or by direct fusion with the plasma membrane. The utilization of an
endocytic entry pathway is generally assumed to indicate the requirement for an
acidic compartment during entry. Accordingly, those viral particles believed to
use this pathway are referred to as pH-dependent. Likewise, those particles that
are believed to enter target cells by direct fusion with the plasma membrane are
referred to as pH-independent.
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The second pathway is the direct entry through the plasma membrane, where
fusion would occur at extracellular pH. Entry via endocytosis is thus referred to
as “pH-dependent” entry and direct plasma membrane entry as “pH-independent”
entry. Examples of pH-dependent viruses are Semliki Forest virus (SFV)(2l),
vesicular stomatitis virus (VSV)(35,57), ecotropic MLV (MLV-E)(3,35,4l), and
influenza virus(S7). In the case of influenza virus, a prototypical pH-dependent
virus, a spring-like conformational change in the hemagglutinin (HA) envelope
protein at low pH has been observed with X-ray crystallography(8). Examples of
pH-independent viruses are human immunodefiency virus type 1 (HIV-1X34,35),
human T-cell leukemia virus (HTLV)(35), the amphotropic MLV (MLV-A),
4070A(35,41), and the feline endogenous retrovirus, RD114(35).
Methods typically used to distinguish between pH-dependent and pH-
independent viral entry are (1 ) inhibition of viral infection by lysosomotropic
weak bases and carboxylic ionophores that raise the pH of acidic vesicles within
target cells(l,3,18,20,21,25,35,41), (2) the ability of a low pH pulse to induce
fusion of virions bound to cells or vesicles(6,20,25,32,40), (3) the pH-sensitivity
of viral envelope protein mediated cell-cell fiision(48,58), and (4) the ability of
acid treatment to inactivate cell-free viral particles(35). The sensitivity of viral
infection to lysosomotropic weak bases (e.g. NH4 CI, chloroquine, amantidine)
and carboxylic ionophores (e.g. monensin) is, by far, the most commonly used
criterion for pH-dependent entry. Lysosomotropic weak bases, in their
unprotonated form, are able to cross the membranes of cells and vesicles (Fig.
3
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2A). Upon entering an acidic compartment, they are protonated and cannot
readily diffuse back out of the vesicles. Thus, the bases raise the pH within these
vesicles by functioning as a proton sink(12,36,42). The carboxylic ionophores
function by intercalating into membranes and facilitating the exchange of protons
in acidic vesicles for potassium ions in the cytoplasm, which also results in an
elevation of the pH in acidic vesicles(12,36,42). Since it is the viral envelope
protein that determines the target cell receptor and subsequent entry pathway, it
also determines the sensitivity of viral vectors and pseudotyped viral particles to
lysosomotropic agents such as NH4CI.
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Plasma
Membrane
N H 4 * H+ 1 EndoMMt/LyMMMe
NH3
• Raises pH in Acidic Vesicles
B
Plasma
Membrane
vacuolar H*-
ATPase
Endwaate/Lymame
BFLAl
• Prevents Acidification of Endosomes
and Lysosomes
Figure 2. Mechanism of action on endosomal and lysosomal acidification by
NH4 CI and BFLAl. Ammonia, from NH4 CI, diffuses into acidic vesicles where it
becomes protonated to ammonium ion (A). Ammonium ion cannot diffuse out of
the vesicles. BFLAl blocks the pumping of protons into endosomes and
lysosomes by inhibiting the vacuolar IT-ATPase (B).
5
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Recently, bafilomycin A1 (BFLAl) and concanamycin A, specific
inhibitors of the vacuolar H * ” -ATPase have been used to determine the
requirement of acidic endosomal compartments for viral entry(19,45). These are
both macrolide antibiotics that are specific and potent inhibitors of the vacuolar
IT- ATPase at concentrations below 1 nM, resulting in the inhibition of endosome
and lysosome acidification (Fig. 2BX7,13).
To date, MLV-E is the only mammalian retrovirus classified as pH-
dependent, based on assays utilizing lysosomotropic bases (Table 1). These
previously reported results are based on studies with the replication competent
ecotropic MLV C57MC(3), replication competent VSV particles that were
pseudotyped with the ecotropic Moloney murine leukemia virus (MoMLV)
envelope protein(35), and a retroviral vector where the ecotropic envelope protein
was expressed on MoMLV-based particles in a transient vector production
system(41). This classification, however, is based on a partial inhibition of
infection, where approximately 20% of the infectious viral particles are still
functional following treatment of target cells with NEtCl (Table 1).
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Virus/Vector [NH«CI]
(mM)
Target
Cells
* / • Reductioa ia
Relative Titer
Refereace
C57MC
(Ecotropic MLV)
56 BALB
3T3
80
Andersen, K„ and B. Nexo.
1983. Virology. 123:83-98.
VSV(MLV-E) 30 NIH3T3 95
McClure, M.O., M .A
Sommerfelt, M. M irsh, and
R. A W eiss. 1990. J.G en.
Virol. 71:767-773.
VSV(MLV-A) 30 NIH3T3 15
McClure, M.O., M .A
Sommerfelt, M. Marsh, and
R.A Weiss. 1990. J.G en.
Virol. 71:767-773.
VSV 30 NIH3T3 97
McClure. M.O., M .A
Sommerfelt, M. Marsh, and
R.A. Weiss. 1990. J.G en.
Virol. 71:767-773.
HIV-1 30 C8166 0
McClure, M.O., M. Marsh,
and R. A. Weiss. 1988.
EMBOJ. 7:313-318
HIV-1 30 CEM 14
McClure, M.O., M.A.
Sommerfelt, M. Marsh, and
R.A. Weiss. 1990. J.G en.
Virol. 71:767-773.
HIV-1 20 JM
(CD4+)
95
Maddon, P.J., A G . Dalgleish,
J.S. McOougal, P.R.
Clapham, R. A Weiss, and R.
Axel. 1986. Cell. 47:333-
348.
Ecotropic MLV Vector
(GPL Producers)
30 NIH3T3 80-90
Nussbaum, O ., A Roop, and
W.F. Anderson. 1993. J.
Virol. 67:7402-7405.
Amphotropic MLV
Vector (GPL Producers)
50 NIH3T3 0-20
Nussbaum, O ., A Roop, and
W.F. Anderson. 1993. J.
Virol. 67:7402-7405.
Table 1. Previously published results from NH4CI viral entry assays.
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This is in striking contrast to other pH-dependent viruses such as VSV, where
there is almost a complete inhibition of wild-type VSV infection upon treatment
withNH4 CI(34,35).
Additionally, infection by MLV-A has been observed to be sensitive to
NH4 CI, although to a lesser extent than MLV-E. These results are based on
studies with VSV particles pseudotyped with the 4070A MLV-A envelope
protein(3S) and a retroviral vector in which the amphotropic envelope protein was
expressed on MoMLV-based particles in a transient vector production system
(Table 1X41). Although in both reports, infection ofNIH 3T3 cells by MLV-A
virions was inhibited from 15-20%, MLV-A is considered to be a pH-independent
virus. There have been conflicting reports of the effect of NH4 CI treatment on
infection by HIV-1. The values have varied from no inhibition at all to 95%
inhibition (Table 1X30) (34,35). Despite the extreme variability for HIV-1, these
data have led to the classification of MLV-A and HTV-1 as pH-independent
viruses.
A confusing issue regarding the pH-dependent entry by MLV’s, is the
observation that the MLV-E envelope protein, when expressed on a variety of cell
types or when added in the form of viral particles to cells in culture, is able to
induce cell-cell fusion at neutral pH(22,48). This anomaly has been compared to
cell-cell fusion studies with VSV, SFV, and influenza, which display an increase
in fusion following a low pH pulse of virus added to cells(58). This data suggests
8
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that the MLV-E envelope protein may function at neutral pH during viral entry.
However, it is unclear if the MLV envelope protein mediated cell-cell fusion
reflects virus-cell fusion associated with infectious target cell entry. In this
regard, it has been observed that mutations in the cytoplasmic tail of the MLV-E
envelope protein that display wild-type levels of cell-cell fusion have resulted in
decreased transduction ofNIH 3T3 cells when expressed on MoMLV-based
retroviral vectors(22). These mutant envelope proteins were incorporated into the
viral particles at wild-type levels and were generated by a transient production
system in cells lacking the virus receptor. Thus, it is unlikely that the reduced
vector titer is the result of a low yield due to fusion of the vector producer cells.
The validity of cell-cell fusion data to be used as an indicator of virus-cell fusion,
therefore, remains in question.
MLV-E is also unique among pH-dependent viruses in that host cell entry
of bound particles cannot be facilitated by a low pH pulse and cell-free virions
have not been observed to be inactivated by moderately low pH treatment (pH
SX35,41). In comparison, VSV has been observed to be inactivated by incubation
in medium of pH 6 or lower(35). Additionally, reducing the pH of the
extracellular medium allows receptor-bound VSV and influenza virus to enter
target cells by directly fusing with the cell membrane(32,40,58).
Electron microscopy and immunofluorescence microscopy have also been
used to distinguish whether viral particles enter cells through the plasma
membrane or by endocytosis. However, this data has provided conflicting
9
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information. For example, HIV-1 has been shown to enter cells by both direct
fusion with the target cell plasma membrane(47,Sl), and by receptor mediated
endocytosis(44,47). A disadvantage of assessing mechanisms of virus entry by
these methods is that they typically require in excess of 5,000 to 10,000 physical
viral particles per cell. However, the ratio of infectious to non-infectious
retroviral particles has been reported to be very low, ranging from approximately
1:200 for C57MC in tissue culture medium(3) and 1:400 for MLV-based vectors
in tissue culture medium, to 1:500,000 for HIV in the plasmas of infected
patients(27). Because of this, the studies of large numbers of physical viral
particles may not necessarily reflect the infectious process. The present study
focuses on the characterization o f productive target cell entry pathways because it
is believed that this is more relevant to our understanding of viral, and viral
vector, infection.
In light of the existing data, I have chosen to re-examine the pH-
dependence of MLV-E entry of target cells and have focused on the use of NH4 CI
and BFLAl as inhibitors of endosome and lysosome acidification. Prior analyses
of the effect of NH4 CI on viral entry have typically consisted of a 30 minute pre
incubation of target cells with or without NH4 CI. The cells were then exposed to
the virus and an infection allowed to proceed in the presence or absence of NH4CI
for 1 to 2 hours at 37°C. Medium was then replaced with fresh medium with or
without NH4 CI and further incubated at 37°C for 2 to 5 hours. As previously
described, a reduction in the relative infectivity of viral particles following drug
1 0
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treatment has been interpreted to indicate a requirement for an acidic
compartment during target cell entry. This is assumed to indicate an endocytic
route of entry.
However, upon reconsideration of these assays, it would appear that the
current interpretations are based on an assumption that may not be valid in all
cases. This assumption being that treatment with a lysosomotropic drug would
cause a pH-dependent virus to enter some abortive entry pathway that would
remove that virus from the viable virus pool, resulting in a drop in the relative
infectivity. Otherwise, drug treatment would inhibit viral entry until the drug was
removed at the end of the assay, at which time the virus would be expected to
resume a productive infectious entry pathway. If this assumption is not valid, the
question remains as to what would cause a reduction in the relative viral titer in
these pH-dependent viral entry assays. A loss of relative titer can be explained by
the possibility of viral degradation while particles are arrested during target cell
entry. Thus, by eliminating an essential component of viral entry due to treatment
with a lysosomotropic drug, the viral particles would not be capable of proceeding
toward a productive infection. While arrested at such a step during entry, virions
would be subject to degradation, possibly as a result of their inherent instability
over the course of the assay. In fact, such a scenario is supported by previous
reports of the cellular effects of lysosomotropic drugs as well as the reported half-
lives of viral particles.
11
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The first requirement for such a model to be true, is that treatment with
lysosomotropic agents must inhibit progression of viral particles through their
entry pathway. In support of this, it has been reported that in baby hamster
kidney (BHK) cells, both BFLAl and NH4 CI inhibit the transport of endocytosed
horseradish peroxidase (HRP), a fluid phase marker, from early to late
endosomes(10). The drugs did not significantly affect the initial internalization or
recycling back to the plasma membrane. It has also been reported in HEP G2
cells that BFLAl inhibits trafficking of endocytosed markers beyond late
endosomes(56). In this study, it was also observed that sorting and recycling of
transferrin (Tf) was not inhibited. Additionally, treatment of Hep-2 cells with
BFLAl has been shown to reduce the transfer of cationized gold to lysosomes
without affecting uptake and recycling(55). BFLAl has been shown in another
study utilizing Chinese hamster ovary (CHO) cells to have no effect on transferrin
receptor (TfR) internalization kinetics while slowing the receptor extemaiization
rate(23). These data indicate that the lysosomotropic drugs, NH4 CI and BFLAl,
are able to inhibit the progression of markers through the endosomal pathway in a
variety of cells (Fig. 3).
The inhibition of marker transport through the endosomal pathway by
NH4CI and BFLAl suggests that the transport of viral particles through the
endocytic pathway would be affected similarly. There have been two reports
where the direct effect of lysosomotropic drugs on viral entry was studied.
Importantly, these agents inhibited viral infection while not inhibiting
12
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internalization into target cells. The first is a report of chloroquine inhibiting
infection of BHK-21 cells by SFV(20). Infectivity was reduced while the binding
and rate of internalization were not affected. Secondly, it has been observed that
the lysosomotropic drugs chloroquine, NH4 CI, amantadine, tributylamine, and
methylamine all inhibit the infection of BALB 3T3 cells by the ecotropic MLV
CS7MC without significantly inhibiting virus binding or intemalization(3).
13
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Plasma Membrane
n h 4 c i
BFLA1
Figure 3. Both NH4 CI and BFLA1 have been shown to inhibit transport through
the endosomal pathway
Early
Endosome
Endosomal
Carrier
Vesicle
Late
Endosome
X
Lysosome
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Taken together, there is significant support in the literature for the notion
of lysosomotropic drugs arresting the transport of fluid phase markers, ligands,
receptors, and viruses through the endocytic pathway. In all these cases, it is
important to note that, while trafficking through the endocytic pathway is
inhibited, internalization is not. This is key to the interpretation of viral entry
assays employing lysosomotropic drugs. Any observed inhibition of viral entry
by a lysosomotropic drug suggests that an endocytic pathway is being used by
that virion and does not necessarily indicate the direct involvement of endosomal
pH on the viral entry mechanism.
I have provided evidence supporting the first requirement of a model for
how lysosomotropic drugs may be inhibiting viral entry (Fig. 4). That is, by
blocking endosomal acidification, transport of endocytosed virions is arrested at a
stage in the endosomal pathway preceding the stage normally required for viral
entry. My model then predicts that, while the virus is arrested in the endosomal
pathway, it is subject to degradation due to the inherent instability of the viral
particle over the course of the assay.
15
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Plasma Membrane
• Drugs Block Endocytosis
• Viral Entry is Arrested
• Virus Degrades while
Stuck in Endosome
NH.C1
and
BFLA1
Figure 4. Model for MLV entry into target cells.
The second part of this model predicts that the in vitro instability of the
viral particles should correlate with the decrease in relative viral titer observed in
entry assays using lysosomotropic drugs. There is evidence in the literature to
support this hypothesis. As mentioned previously, in the typical viral entry assay
utilizing lysosomotropic agents, virus is on target cells in the presence of the drug
for anywhere from 3 to 7 hours. Thus, if viral particles spontaneously denature
with a half-time significantly greater than the duration of incubation with drug,
one would predict no significant reduction in relative viral titer. In other words,
lysosomotropic drugs would arrest the transport of the viral particle through the
16
Endosome
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
endosomal pathway. While arrested in some endosomal compartment, the viral
particle would be subject to spontaneous denaturation that was dependent on the
instability of that particle in the environment of such a compartment. So, particles
that are very stable for the period of time that they are stuck in this compartment
would not denature and would not lose the ability to resume a productive
infection once the lysosomotropic agent was removed. However, virions that are
relatively unstable would be expected to degrade while arrested in this endosomal
compartment. Then, when the lysosomotropic agent was removed at the end of
the assay, those viral particles that had degraded during the assay would not be
able to resume a productive infection. Based on this interpretation, one would
predict that viral particles which display a reduction in relative titer in titer assays
with lysosomotropic agents would be less stable under the conditions of the assay
than those viral particles that are not affected by those agents.
In fact, such a prediction is supported by previously published in vitro
viral vector stability data. The half-life for MLV-A vectors has been reported to
be from 4.1 to 6.5 hours at 37°C in tissue culture medium(4,5,17,43). The vectors
used in these studies were generated from the amphotropic PA317(38) or
v|/CRIP(l 1) packaging cell lines. Both of these cell lines express the MLV-E
(Mo-MLV) structural proteins, reverse transcriptase, and integrase (i.e. gag and
pot) and the MLV-A (4070A) envelope protein (i.e. env). Likewise, the half-life
of MLV-E vectors has been reported to be from 3.5 to 9.5 hours at 37°C in tissue
culture medium(29,43). The vectors used in these studies were generated from
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the ecotropic v|/-2 packaging cell line(31). This cell line expresses all the Mo-
MLV proteins. Thus, the reported stabilities of ecotropic and amphotropic MLV-
based vectors would be expected to be directly responsible for partial reductions
in relative viral titer over the course of a 4 hour titer assay with a lysosomotropic
drug. This is precisely the result that has been observed.
In addition, the in vitro stability of HIV-1 is also consistent with this
interpretation. The half-life of HIV-l has been reported to be 24 to 30 hours at
37°C in tissue culture medium(27,52). This greater virus stability, relative to the
MLV-based vectors, would be expected to yield insignificant reductions in
relative viral titer over the course of a 4 hour titer assay with a lysosomotropic
drug. As previously mentioned, this is the observed effect. Further supporting
such a hypothesis is the observation that the relative titer of HIV-1 has been
reported to be reduced if the duration of infection in the presence of NH4CI is
increased to 18 and 60 hours(34). These experiments, however, were only semi-
quantitative.
The data presented here calls into question the current interpretation of
viral entry assays relying on the action of lysosomotropic drugs. While the
primary effect of these drugs on endosomal and lysosomal pH has been
acknowledged in these studies, their ability to interfere with trafficking through
the endosomal pathway has not been addressed. However, alterations in
trafficking through the endosomal pathway is of critical importance in an assay
that is intended to determine the role of this pathway in viral entry. It seems that a
18
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reduction in relative viral titer by lysosomotropic agents has been assumed to
indicate the requirement for an acidic compartment, and therefore endocytic
uptake, without any explanation for why virions would be rendered non-
infectious. I have presented evidence in the literature to support a model that
takes into account this loss of viral infectivity and the implications of assays using
lysosomotropic drugs for viral entry pathways (Fig. 4). This model predicts that,
by treating cells with lysosomotropic agents, transport through the endosomal
pathway is inhibited at an early or late endosome. However, internalization into
this pathway is not significantly inhibited. In the presence of such drugs, entry of
viral particles that normally infect target ceils via endocytosis is also blocked at
an early or late endosome. While arrested in an endosomal compartment, virions
undergo spontaneous denaturation as a function of their inherent instability in this
environment. Thus, this inherent instability of the viral particles would determine
how many of those particles would still be infectious when the lysosomotropic
agent was washed away. At that time, only those particles that had not been
denatured would be able to resume a productive infection.
Based on these studies, MLV-E has been determined to be sensitive to
lysosomotropic drugs. Because of this, it has been classified as a “pH-dependent”
virus and is the only mammalian retrovirus classified as such. The model that I
propose, however, predicts that assays with lysosomotropic drugs are measuring
predominantly viral instability rather than pH-dependent entry. Therefore, viruses
that have previously been determined to be insensitive to treatment with
19
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lysosomotropic drugs, with respect to target cell infectivity, are likely to be
relatively stable. It would then be incorrect to assume that these virions did not
use an endocytic entry pathway, based on such experiments.
The present study is based on the hypothesis that viral entry assays using
lysosomotropic drugs are measuring the instability of viral particles while those
particles are arrested in their entry pathway. Additionally, that MLV-based
vectors are using an endocytic entry pathway into target cells. Data is presented
here to support such a hypothesis and suggests that previous interpretations of
viral entry assays with lysosomotropic drugs may have been incorrect. The
implications of the results presented here are that target cell entry via an endocytic
pathway may extend to all enveloped viruses and that viral entry assays using
lysosomotropic agents cannot necessarily be used to distinguish between plasma
membrane and endosomal viral entry if there is no effect of such agents.
20
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Chapter 2: Materials and Methods
2.1 Lysosomotropic Drugs, Antibodies, and Virus Stocks. A stock solution
of 2 M ammonium chloride (NH4 CI) (Sigma) was prepared in H2 O that had been
purified through a Millipore Milli-Q UV Plus water purification system
(Millipore, Inc., Bedford, MA). It was sterile filtered through 0.22 pm cellulose
acetate syringe filters (Millipore) and aliquots were frozen at -20°C. A fresh
aliquot was thawed for each experiment. The stock solution was diluted in
medium to a working concentration of S O mM. A stock solution of S O pM
BFLA1 was prepared in DMSO and stored at -20°C in a sealed container with
desiccant. The stock solution was diluted in medium to a working concentration
of 0.05 pM. The mouse monoclonal antibodies (mAbs) S73,273, and S14 were
obtained from Bruce Chesebro (National Institute of Allergy and Infectious
Diseases, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories,
Hamilton, Montana). The mAb S73 reacts with the envelope protein SU subunit
(gp70) of all MLVs. The mAb S38 reacts specifically with the MoMLV SU and
not with that of amphotropic, polytropic, or xenotropic MLVs and the mAb S14
reacts with the SU of all known polytropic MLV’s. The plasmid pMoMuLV-K,
which contains a complete genome of MoMLV, was obtained from A. Dusty
Miller (Fred Hutchinson Cancer Research Center, Seattle, WA). The plasmid was
21
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transfected into NIH 3T3 cells, and MoMLV was harvested 2 days later. 4070A
1 IRC replication competent virus was obtained from Genetic Therapy Inc.,
Gaithersburg, MD. NIH 3T3 cells that were confluently infected with either
replication competent retrovirus were screened with the mAbs 573, 538, and 514
and the viral envelope protein immunofluorescence assay (see description in
materials and methods) was used to confirm the identity of each virus.
2.2 Cell Lines and Viral Vector Production. All cell lines were grown in
Dulbecco’s modified Eagle’s medium supplemented with 5% heat inactivated calf
serum (Gibco/BRL, Grand Island, N.Y.) and 2 mM L-glutamine (Gibco/BRL).
The cell line 293T/17 was obtained from the American Type Culture Collection
(CRL 11268).
MLV-A and MLV-E vectors were generated from PA317(38) and
PE501(39) pre-packaging cells, respectively (Fig. 5). PA317 cells are based on
NIH 3T3 TK' cells. They contain the expression plasmid, pPAM3, which
expresses the ecotropic MoMLV gag and part of pol with the remainder of pol
and env being from the amphotropic 4070A. This proviral genome has
modifications in the 5’ and 3’ long terminal repeat (LTR) in order to prevent the
possibility of homologous recombination with endogenous retroviral sequences
which may generate replication competent retrovirus (RCR). In addition, the
packaging signal sequence (q/) has been mutated so that the resulting genomic
RNA cannot be packaged into the retroviral particles generated from these cells.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Ecotropic MLV
(MLV-E)
Amphotropic MLV
(MLV-A)
MoMLV(VSV-G)
f VSV- C
293T Cells PA317 Cells PESO 1 Cells
Figure 5. Schematic of MLV-E, MLV-A, and MoMLV(VSV-G) viral vector
production.
23
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The resulting retroviral particles from these cells contain the amphotropic
envelope protein, ecotropic core proteins, and are devoid of any genome. I have
transduced these cells with an ecotropic MLV>based viral particle containing the
GlnBgSVNa retroviral vector (Genetic Therapy, Inc.) to generate the producer
cell line PA317/GlnBgSVNa. This vector contains the £ coli (3-galactosidase
gene with the S V40 large T antigen nuclear localization signal (n(3-gal) driven by
the cytomegalovirus (CMV) promoter. It also encodes the neomycin
phosphotranferase (NeoR ) gene driven by the SV40 promoter. Following
transduction of these cells, they were selected with 0.6 mg/ml G418 (Gibco/BRL)
in order to obtain a pool of cells that were all producing viral vectors. The
resulting virions have amphotropic host specificity due to the 4070A envelope
protein and carry the GlnBgSVNa retroviral vector which contains the n(3-gal and
NeoR marker genes. PES01 cells are also generated from NIH 3T3 TK* cells.
These cells contain a defective retroviral genome that is identical to that used to
make PA317 cells, except that the amphotropic pol-env region of the provirus has
been replaced with the ecotropic region from MoMLV. I have transduced these
cells with an amphotropic MLV-based viral particle that contains the LAPSN
retroviral vector (Clontech Laboratories, Inc., Palo Alto, CA) to generate the
producer cell line PE501/LAPSN. This vector contains the human placental
alkaline phosphatase (AP) gene driven by the MoMLV LTR as well as the NeoR
gene driven by the SV40 promoter. Following transduction of these cells, they
24
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were selected with 0.6 mg/ml G418 (Gibco/BRL) in order to obtain a pool of cells
that were all producing viral vectors. The resulting virions have ecotropic host
specificity due to the MoMLV envelope protein and carry the LAPSN retroviral
vector which contains the AP and NeoR marker genes.
Published half-lives for MLV-E and MLV-A vectors were taken into
consideration in the collection of supernatants from PA317 and PES01 cells so
that supernatants with as little debris from denatured viral particles as possible
would be obtained. It was determined that a collection for approximately 12
hours from producer cells that had just reached confluency yielded sufficiently
high viral titers over a relatively short duration of collection. Cells were grown in
800 cm2 roller bottles (Coming, Inc., Coming, NY) and supernatants were
collected in 30 mis of medium. Supernatants were then filtered through 0.4S urn
cellulose acetate syringe filters (Millipore) and frozen immediately at -80°C.
A transient three-plasmid expression system was used to generate MLV-
based vectors pseudotyped with the envelope protein of VSV (G-protein) (Fig.
5X50). 293T/17 cells were transfected with the plasmids pHIT60, pHITl 12
(obtained from A. Kingsman, University of Oxford), and pVSV-G. The pHIT60
plasmid expresses the MoMLV gag and pol proteins from the CMV promoter and
possesses an SV40 origin of replication. The pHITl 12 plasmid contains a
retroviral vector carrying the gene for np-gal driven by the CMV promoter and
also possesses the SV40 origin of replication. The pVSV-G plasmid contains the
gene for the VS V-G protein driven by the CMV promoter. Plasmid DNA was
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isolated from bacterial cultures by using alkaline lysis and a Qiagen tip-500
(Qiagen, Chatsworth, CA) according to the manufacturer’s protocol.
The day before transfection, 293T/17 cells were seeded on 10 cm2 dishes
to give a maximum of 70% confluence per dish on the day of transfection. Ten
micrograms each of the three plasmids was then transfected into 293T/17 cells by
overnight CaP( > 4 precipitation. After incubation for 18 hours at 37°C/5% CO2 , the
medium on each plate was replaced with medium containing 10 mM sodium
butyrate (Sigma, St. Louis, MO) and incubated for another 12 hours. Medium on
each dish was then replaced with 6 mis of fresh medium. After 12 hours,
supernatants were collected and filtered through a 0.45 pm cellulose acetate
syringe filter prior to immediate freezing in liquid nitrogen.
MLV-based vectors were also generated by utilizing MoMLV and 4070A
RCR as has been previously reported (Fig. 6)(37). NIH 3T3 cells were
transduced with the MLV-A viral vector carrying either GlnBgSVNa or LAPSN.
Cells were then incubated with 0.6 mg/ml G418 in order to select for those stably
expressing each retroviral vector. The resulting cell lines were NIH
3T3/GlnBgSVNa and NIH 3T3/LAPSN, respectively. Each cell line was then
infected with RCR MoMLV or 4070A and passed for approximately 2 weeks in
order to obtain confluently infected cultures. Confluency was determined by the
viral envelope protein immunofluorescence assay (see materials and methods)
utilizing the mAb 573.
26
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W TktaM LV
Ecotropic MLV
(Eco)
NIH 313 Cells
*
AP
Amphotropic MLV
(Ampho)
*
wruraA
NIH 3 T3 Cells
*
■ M
Figure 6. Schematic for production of MLV-E and MLV-A viral vectors using
the replication competent retroviruses MoMLV and 4070A, respectively.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The RCR is able to package the retroviral vector in each cell line due to the
presence of the \|/ sequence in the vectors. The supernatants from these cells
contain both wild-type RCR corresponding to the virus used to infect those cells,
as well as viral vector that consists of completely 4070A or MoMLV particles
except for the genomic RNA. The genome of these particles consists of the
retroviral vector RNA from the infected cell line. The primary difference
between these viral vectors and those produced from the PA317 and PES01
packaging cells is that the particles from this system are completely ecotropic or
amphotropic, with respect to their structural components, whereas those from the
PA317 and PES01 cells have structural components that are both based on
MoMLV and have had manipulations in the pol-env portion of the provirus.
Titers of all supernatants were determined on NIH 3T3 TK+ cells. Cells were
seeded onto 60 mm2 dishes (Coming) at 1.2 x 105 cells per dish. At 18-20 hours
following seeding, the medium on each dish was replaced with 1 ml of viral
supernatant, either undiluted or diluted in fresh medium, with 8 pg/ml polybrene
(Sigma). Supernatants were left on cells for 2 hours at 37°C/5% CO2 , after which
time 2 mis of fresh medium was added to each dish. Approximately 24 hours
later, the medium on each dish was replaced with fresh medium and the cells were
grown to confluency (approximately S days). Cells were then stained to detect
foci expressing either nf3-gal or AP in order to determine the number of cells that
28
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had been transduced by either a GlnBgSVNa or LAPSN vial vectors,
respectively.
The substrates X-gal (5-bromo-4-chloro-3-indolyl P-D-galactopyranoside)
(Denville Scientific) and NBT/BCIP (nitro blue tetrazolium chloride/5-bromo-4-
chloro-3-indolyl phosphate, toluidine salt)(Boehringer Mannheim) were used to
stain for nP-gal and AP foci, respectively. For X-gal staining, cells were washed
with phosphate-buffered saline containing calcium and magnesium salts (PBS +
Ca2 7Mg2 + )(Irvine Scientific, Irvine, Calif.) and then fixed in dishes for
approximately 10 minutes with 0.5% glutaraldehyde (Sigma). Cells were then
washed with PBS without calcium and magnesium salts (PBS w/o Ca2 + /Mg2 + ) and
2 mis of X-gal solution was aliquotted onto each dish. This solution contains 5
mM potassium ferrocyanide (Sigma), 5 mM potassium ferricyanide (Sigma), 2
mM MgCh (Sigma), and 1 mg/ml X-gal in PBS w/o Ca2 + /Mg2 + . Cells were
incubated at 37°C for approximately 24 hours for development of blue foci. Foci
were counted on a Nikon Eclipse E800 microscope using a Nikon 4X objective
under bright-field lighting. Each 60 mm dish was counted in its entirety.
The method of Fields-Berry, S.C., et al., (I6)was used for AP staining and
was essentially the same as for nj3-gal up to the glutaraldehyde fixation.
Following fixing, cells were washed with PBS w/o Ca2 + /Mg2 + and incubated at
60°C for 10 minutes in order to reduce background staining from NIH 3T3 cells.
Cells were then incubated for 10 minutes at room temperature with AP buffer
29
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(100 mM Tris-HCL, pH 9.5 /100 mMNaCl / 5 mM MgCfe). Buffer was then
replaced with 0.38 mg/ml NBT and 0.2 mg/ml BCIP in AP buffer and incubated
at room temperature in the dark for 2-24 hours. Each 60 mm dish was counted in
its entirety, as for the nP-gal foci, and scored for purple AP-positive foci.
2.3 Mixed Vector Titer Assays with NH4 Q or BFLA1. An assay was
developed to simultaneously measure the transduction of two different viral
vectors, each containing either the n(3-gal or AP marker gene, in the presence or
absence of the lysosomotropic drugs NH«C1 or BFLA1 (Fig. 7). This assay has
been based on a previous assay that utilized (3-gal and AP-containing ecotropic
MLV vectors for lineage studies of murine retinal cells(l6). Supernatant
containing the MLV-E (LAPSN) vector was combined with either the MLV-A
(GlnBgSVNa) or VSV-G (GlnBgSVNa) for all titer assays, such that
transduction by each different vector in a single assay could be distinguished by
either n(3-gal or AP staining. NIH 3T3 cells were seeded onto 60 mm dishes
(Coming) at 1.2 x 105 cells per dish 18-20 hours prior to the addition of viral
supernatants. In the case ofNHtCl titer assays, cells were incubated for 30
minutes with either 50 mMNHtCI or MQ H2 O at 37°C/5% CO2 before the
addition of viral vector supernatant.
30
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NIH3T3 Cells
1.2 x10s
Pre-incubate with drug
for30m inat37*C
Replace medium with viral
vector mixture
+
drug
Replace medium
with fresh medium
+ drug for chase
Wash with
fresh medium
Aliquot fresh
medium and
grow to con-
flucncy
Stain with
X-gal
and
NBT/BCIP
Figure 7. Schematic of mixed viral vector titer assay with NH4CI or BFL A1.
Viral vector mixtures were placed onto NIH 3T3 cells that had been pre-incubated
with drug or solvent for 30 minutes. Following the infection period, virus was
either washed off and replaced with fresh medium or simply replaced with
medium containing drug or solvent for a 2 hour chase prior to washing with fresh
medium.
31
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The medium on each dish was then replaced with a 1 ml aliquot of the supernatant
mixture containing 8 pg/ml polybrene with either NH4 CI or H2 O and incubated at
37°C/5% COj for the specified duration. Following the infection period,
supernatant on each dish was either replaced with 1 ml of medium containing
NH4 CI or H2 O for a drug chase or washed once with 3 mis of fresh medium.
After the wash, S mis of fresh medium was aliquotted onto each dish and the cells
were subsequently incubated at 37°C/S% CO2 until confluent (approximately S
days). Those cells subjected to a drug chase were washed in the same manner
following the chase.
In the case of titer assays with BFLA1, the treatments were the same as
those with NH4 CI, except that 0.0S pM BFLA1 was used in place of NH4 CI and
DMSO was used in place of H2 O. The concentration of DMSO was 0.1% in all
assays. All assay dishes were scored for (3-gal and AP positive foci as described
except that, for double-staining, X-gal staining was performed first. After
counting n(3 -gal positive foci, dishes were washed with PBS w/o Ca2 + /Mg2 + ,
incubated at 60°C for 10 minutes, and then stained with NBT/BCIP substrate
solution for development of AP positive foci. As with the standard titer assays,
each dish was counted in its entirety under bright-field lighting. Optimal dilutions
to be used for each viral vector were determined from a pilot experiment and this
dilution was used for all subsequent experiments in replicates of from 3 to 6
dishes per data point.
32
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2.4 Viral Vector Stability Assay. NIH 3T3 cells were seeded out on 60 mm
dishes (Coming) at 1.2 X 103 cells per dish approximately 18-20 hours before
addition of viral supernatants. Pre-determined optimal dilutions of vims mixtures
were made in medium containing 8 |ig/ml polybrene and either drug or solvent at
concentrations equal to those used in the titer assays. Stock vials of vector
mixtures were incubated in a 37°C water bath (Fig. 8). At appropriate time
points, medium on replicate dishes of cells was replaced with 1 ml aliquots of
viral vector mixtures. Infection of target cells was allowed to take place for S
minutes at 37°C/S% CO2. Cells were then washed with 3 mis of fresh medium
before placing 5 mis of fresh medium onto each dish. Cells were incubated at
37°C/S% CO2 until confluent (approximately S days), at which time dishes were
stained for n(3-gal and AP positive foci and scored as in the titer assays.
33
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in vitro Incubation with Drug
Virus Mixture+ Drug or Solvent
Time (H n)
NIH3T3
t * 0 t* 2 H r s t = 4 Hrs
5 Minute Infection
Wash
Figure 8. Schematic of viral vector stability assay. Aliquots of each viral vector
mixture + drug or solvent were taken from the 3TC incubation and placed onto
NIH 3T3 cells. Particles were allowed to infect cells for S minutes prior to
washing with fresh medium.
34
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2.5 Viral Vector Inactivation Assay. NIH 3T3 cells were seeded out on 60
mm dishes (Coming) at 1.2 X 105 cells per dish approximately 18-20 hours before
addition of viral supernatants (Fig. 9). Prior to the addition of vims mixtures to
cells, cells were pre-chilled at 4°C for 1 hour. Medium on each dish was then
replaced with 1 ml of the viral vector mixture in medium containing 8 pg/ml
polybrene. Dishes were then incubated at 4°C for 2 hours to allow binding of viral
particles to cells. Following binding, dishes were transferred onto ice and washed
with 3 mis ice cold PBS + Ca2 + /Mg2 + . Dishes were then incubated with 3 mis of
ice cold citric acid buffer (40 mM citric acid, 10 mM KC1,135 mM NaCIX54) at
the appropriate pH for 30 seconds on ice. After the aspiration of the citric acid
buffer, all dishes were washed with 3 mis fresh medium and then 5 mis fresh
medium was aliquotted onto each. Cells were incubated at 37°C/5% CO2 until
confluent (approximately 5 days), at which time dishes were stained for nP-gal
and AP positive foci and scored as in the titer assays.
35
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Virus
Mixture
NIH 313 Cels
Wash with medium
37*C until confluent
Bind @ 4*C for 2 hrs
Citric Acid on ice for 30 sec
Figure 9. Schematic of the citric acid viral vector inactivation assay.
36
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In order to determine any cellular effects of citric acid buffer treatment,
dishes of NIH 3T3 cells were treated with citric acid buffer as described for vector
inactivation. Following a wash with 3 mis of fresh medium and placement of 5
mis fresh medium onto cells, medium was replaced with 1 ml per dish of viral
vector mixture in medium containing 8 pg/ml polybrene. The supernatant was
incubated with cells for 2 hours at 37°C/S% CO2 , after which each dish was
washed with 3 mis of fresh medium. Each dish then received S mis of fresh
medium and cells were incubated at 37°C/S% C02 until confluent (approximately
S days), at which time dishes were stained for n(3-gal and AP positive foci and
scored as in the titer assays.
2.6 Viral Envelope Protein Immunofluorescence Assay. NIH 3T3 TK+
cells were infected with MoMLV clone K or 4070A 1 IRC. Cells were passaged
for approximately 2 weeks in order to achieve confluently infected cultures.
Confluently infected cells were then washed with PBS + Ca2 + /Mg2 + and
approximately 0.5 ml of undiluted mAb 573, 538, or 514 was aliquotted onto cells
and incubated at 37°C/5% CO2 for 30 minutes. Antibody was then aspirated and
cells were washed with PBS + Ca2 */Mg2 + . FITC-conjugated goat anti-mouse IgG
was diluted 1:200 in PBS + Ca2 + /Mg2 + and approximately 0.5 ml was aliquotted
onto cells and incubated at 37°C/5% CO2 for 30 minutes. Cells were then washed
2X with PBS + Ca2 + /Mg2 + and fixed in 0.7% formaldehyde (Sigma) in PBS +
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Ca2 + /Mg2 + . The extent of RCR infection was determined based on mAb 573 -
dependent F1TC epifluorescence on a Nikon Eclipse E800 fluorescence
microscope (4X objective magnification; excitation wavelength of465-495 nm
and barrier wavelength o f515-555 nm).
In the case of immunofluorescent detection of viral envelope proteins
following citric acid treatment, confluent cells on 60 mm dishes were washed
with PBS + Ca2 + /Mg2 + and then treated with citric acid buffer on ice for 30
seconds. Dishes were immediately washed with 3 mis fresh medium and 5 mis
was then aliquotted into each. The procedure for immunofluorescent detection of
viral envelope proteins was then used as described.
2.7 Viral Vector Internalization in the Presence of NH4CI and BFLA1.
N IH 3T3 cells were seeded onto 60 mm dishes (Coming) 18-20 hours prior to
addition of viral supernatants. Cells were pre-incubated with 50 mM NH4CI,
H20, 0.05 pM BFLA1, or 0.1% DMSO in medium for 30 minutes at 37°C/5%
CO2. Medium was then replaced with a viral vector supernatant mixture in
medium containing either drug of solvent and 8 pg/ml polybrene. An infection
was carried out at 37°C/5% CCfefor 2 hours, after which time dishes were either
washed with 3 mis fresh medium prior to aliquotting 5 mis fresh medium to each
dish or were treated with citric acid buffer, pH 3.0, as described for the viral
vector inactivation. Cells were then incubated at 37°C/5% CO2 until confluent
38
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(approximately S days), at which time dishes were stained for nP-gal and AP
positive foci and scored as in the titer assay.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Chapter 3: Effect of Lysosomotropic Drugs on the Transduction of NIH
3T3 Cells by Ecotropic and Amphotropic Murine Leukemia Virus-Based
Vectors
3.1 Relative viral vector titer is reduced by NH4CI and BFLAl in the
mixed vector titer assay. Since the mixed vector titer assay with NH4CI and
BFLAl developed here is a modified version of previously described assays, it
was important to confirm that results with these assays were similar to previously
reported results. The NH4CI and BFLAl titer assay conditions used here were
similar to previously reported NH4CI titer assays. The duration of the infection in
the presence of NH4CI and BFLAl was a total of 4 hours, replacing the viral
supernatant mixture after the first 2 hours with medium containing drug.
As expected, similar results to those reported previously were observed.
Both MLV-A and MLV-E displayed a reduction in relative titer with NH4CI and
BFLAl compared to the titers with the corresponding solvents (i.e. H2 O or
DMSO) (Figs. 10 and 11). A notable difference from previous reports, however,
was that the MLV-A relative titer was approximately equal to that of MLV-E with
both drugs. Transduction by both virions was inhibited by 60-70%, in
comparison to controls. This may be due to the fact that variables between the
MLV-A and MLV-E supernatants have been controlled for in the mixed vector
assay. Being that the model proposed here predicts that the NH4CI and BFLAl
40
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assays are measuring the instability of viral particles, the most likely variable
would be some component of the MLV-E supernatant that makes the viral
particles less stable under assay conditions. This component would then be
decreasing the stability of the MLV-A vector in the mixed assay infection. In
fact, it has been reported that proteoglycans secreted by ecotropic and
amphotropic MLV packaging cell lines inhibit the infectivity of NIH 3T3 cells by
amphotropic MLV vector(28). It is also possible that, for some unknown reason,
the inherent instability of the viral vectors produced in this system differs from
that of vectors produced in previous reports such that the MLV-A in my system is
less stable. Vectors have been assayed individually for sensitivity to NH4CI and
BFLAl and the results obtained were similar to those published by other groups.
However, variability of the data was such that the statistical significance of the
relative titer values for MLV-A and MLV-E was in question. It is precisely for
this reason that the mixed viral vector titer assay described here was developed.
This assay has eliminated the variability and greatly increased the significance of
the data due to the controlling for the potential differences between supernatants
and the ability to assay and process multiple replicates of each data point.
41
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Si’ 0 0
1=3 MLV-A
H MLV-E
4 Hour Infection
Avg. % # Independent
____________________Transduction_______ SEM_________ n________ Experiments
MLV-A 41.70 4.25 12 2
MLV-E 24.70 3.64 20 4
Figure 10. Effect of S O 1 1 1 MNH4 CI on transduction of NIH 3T3 cells by MLV-A
and MLV-E. Infection in the presence of NH4 CI was as described in materials
and methods for a 2 hour infection with a 2 hour chase in the presence of NH4 CI.
The 100% titer values were 1.4 x 10 4 -1.6 x 105 for MLV-A and 1.4 x 104 -1.8 x
10s for MLV-E.
42
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
EtS so
□ MLV-A
■■MLV-E
4 Hour Infection
Avg. % # Independent
___________________ Transduction_______ SEM_________n________ Experiments
MLV-A 46.75 9.55 II 2
MLV-E 40.73 0.73 21 4
Figure 11. Effect of S O nM BFLAl on transduction of NIH 3T3 cells by MLV-A
and MLV-E. Infection in the presence of BFLAl was as described in materials
and methods for a 2 hour infection with a 2 hour chase in the presence of BFLAl.
The 100% titer values were 6.8 x 10 4 -9.1 x 10 4 for MLV-A and 8.3 x 104 - 9.8 x
104 for MLV-E.
43
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3.2 Results in the NH4CI and BFLAl mixed vector titer assay are the
same for vectors generated from PA317 and PES01 packaging cells and
vectors generated with MoMLV and 4070A RCR. Because the proviral
sequences used to make PA317 and PES01 pre-packaging cells have been
modified from their original form, and the PA317 provirus contains a chimeric
pol-env derived from MoMLV and 4070A sequences, it was necessary to confirm
the amphotropic and ecotropic functionality of vectors generated from PA317 and
PES01 cells, respectively. In order to address this question, viral vectors were
generated by infecting retroviral vector-containing NIH 3T3 cells with either
MoMLV or 4070A1 IRC, as described in materials and methods (Fig. 6). The
primary difference between vectors generated in this way and those from
packaging cell lines is that these vectors are composed of either complete
MoMLV or complete 4070A viral particles that contain the retroviral vector RNA
as their genome. Thus, there is no question of any chimeric viral components
altering the functionality of these particles as ecotropic and amphotropic MLVs
with respect to target cell entry.
As was anticipated, these viral vectors displayed the same sensitivity as
those from PA317 and PES01 cells to NH4 CI and BFLAl in the mixed vector titer
assay following a total infection time of 4 hours in the presence of each drug
(Figs. 12 and 13). Again, both MLV-A and MLV-E vectors were affected equally
by both drugs. The reduction in relative titer of each was approximately 60%
with NH4 CI and S0% with BFLAl. These results indicate that the vectors
44
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generated from PA317 and PESOl cells are not altered in their ability to function
as amphotropic and ecotropic MLVs, respectively. Therefore, the results
presented in this study are believed to be valid and relevant to the characteristics
of wild-type MLV-A and MLV-E target cell entry.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
120
5 1
100
EtS
80
60
* 5
40
20
0
C=l407QA(G1nBgSVNa)
MMoMLV(LAPSN)
4 Hour Infection
Avg. % # Independent
_______________________Transduction SEM_______ n________Experiments
4070A(GlnBgSVNa) 38.SS 2.91 12 4
MoMLV(LAPSN) 34.09 3.15 8 ____________ 2
Figure 12. Effect of S O mMNH^Cl on transduction of NIH 3T3 cells by
4070A(GlnBgSVNa) and MoMLV(LAPSN). Infection in the presence of NHtCl
was as described in materials and methods for a 2 hour infection with a 2 hour
chase in the presence of NH4CI. The 100% titer values were 6.S x 105 - 8.S x 105
CFU/ml for 4070A(GlnBgSVNa) and 8.4 x 106 - 8.5 x 106 CFU/ml for
MoMLV(LAPSN).
46
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C Z I 4070A(G1 nBgSVNa)
HMoMLV(LAPSN)
4 Hour Infection
Avg.%
Transduction SEM
# Independent
Experiments
4070A(GlnBgSVNa) 60.09 7.69 8
MoMLV(LAPSN) 49.51 7.83 8
2
2
Figure 13. Effect of 50 nM BFLAl on transduction of NIH 3T3 cells by
4070A(G1 nBgSVNa) and MoMLV(LAPSN). Infection in the presence of
BFLAl was as described in materials and methods for a 2 hour infection with a 2
hour chase in the presence of BFLAl. The 100% titer values were 4.3 x 10s - 5.1
x 10s CFU/ml for 4070A(GlnBgSVNa) and 5.0 x 10 6 - 5.8 x 106 CFU/ml for
MoMLV(LAPSN).
47
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Chapter 4
4.1 Increasing the duration of MLV-A, MLV-E, and MoMLV(VSV-G)
infection of N IH 3T3 cells in the presence of either NH4CI or BFLAl results
in a reduction of relative viral vector titer. The model that I have presented
here predicts that the reduction in relative viral vector titer in the NH4CI and
BFLAl titer assays is a function primarily of the inherent instability of the viral
particles. This is because the lysosomotropic drugs are believed to be arresting
entry of viral particles at an early or late endosome and the virions are then
believed to be stuck in one of these compartments, where they undergo
spontaneous denaturation. If this is the case, then it would stand to reason that
increasing the duration of infection in the presence of lysosomotropic agents
would result in a greater reduction of the relative vector titer.
In order to test this hypothesis, I have developed variations of the NH4 CI
and BFLAl titer assays, as described in materials and methods (Fig. 14). All titer
assays consisted of a 30 minute pre-incubation period with either drug or solvent
prior to addition of the viral vector mixture. Assays then consisted of either a 5
minute or 2 hour infection period in the presence of drug or solvent. In each case
the length of time that target cells were exposed to drug or solvent was controlled.
In the case of the 5 minute infection, the viral vector mixture was added during
the last S minutes of a 2 hour incubation with drug. Thus, the cells were exposed
to either drug or solvent for 2 hours following the 30 minute pre-incubation,
48
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regardless of the specified infection time. Additionally, in order to more closely
replicate assay conditions reported in the literature, the duration of infection in the
presence of drug or solvent was increased to 4 hours. Following a 2 hour
infection period containing drug or solvent, the virus was replaced with medium
containing drug or solvent and incubated for an additional 2 hours before washing
with fresh medium.
49
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5 min infection/no drug chue
1 v k u . i
Drag
Thne (H n)
-A5 •
2 hour infection/no drug chare
2 hour htfectkm/2 hour drug chare ^ vfeui ]
Drag
lim e (H n)
I I
-•.5 • 2 4
Figure 14. Schematic of NH4CI and BFLAl titer assay variations. NIH 3T3 cells were pre
incubated with either drug of solvent for 30 minutes in all assays. In the case of 5 minute or 2
hour infections without a drug chase, viral vector supernatant was washed off and replaced with
fresh medium at t = 2 hours. In the case of a 2 hour infection followed by a 2 hour drug chase, the
supernatant was replaced with fresh medium containing drug and incubated for another 2 hours
before washing and replacement with fresh medium.
Results from these assays indicated a correlation between the duration of
infection in the presence of either drug and a reduction in the relative viral titer.
When the infection time of viral vector supernatant mixtures was only S minutes
in the presence of NH4CI, there was no significant reduction of the relative MLV-
A and MLV-E titer (Fig. IS). However, there was an approximately 40%
reduction in the relative MoMLV(VSV-G) titer. When the infection time was
increased to 2 hours in the presence of NH4CI, there was a noticeable reduction in
the relative titers of all three viral vectors (Fig. 16). A titer reduction o f30-40%
was observed for MLV-A and MLV-E, whereas the relative titer of
MoMLV(VS V-G) was reduced by 80%. When a 2 hour NH4CI chase was added
to the 2 hour infection, a corresponding drop in the relative titer of all virions was
observed (Fig. 17). The relative titers were reduced by 58% for MLV-A, 75% for
MLV-E, and 94% for MoMLV(VSV-G). This fits nicely with the proposed
model, where each drug arrests viral entry and results in the spontaneous loss of
infectious viral particles due to viral vector denaturation during this arrest.
The results from the BFLAl titer assay were similar to those obtained with
NH4 CI. Following a 5 minute infection in the presence of BFLAl, all three viral
vectors exhibited a partial reduction in relative titer (Fig. 18). Reductions of 23%
for MLV-A 33% for MLV-E, and 48% for MoMLV(VSV-G) were observed.
The more pronounced reduction in relative titer under the conditions of this short
infection time in the presence of BFLAl, in comparison to the 5 minute infection
in the presence of NH4CI, may be partially due to a slight cytotoxic effect on the
51
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NIH 3T3 cells used in the assay (data not shown). Increasing the infection time to
2 hours in the presence of BFLAl reduced relative titers by 37% for MLV-A,
48% for MLV-E, and 78% for MoMLV(VSV-G) (Fig. 19). As expected, the
addition of a 2 hour BFLAl chase to the 2 hour infection resulted in a more
significant reduction of relative vector titers (Fig. 20). Under these conditions the
MLV-A relative titer was reduced by 53%, MLV-E by 59%, and MoMLV(VSV-
G) by 92%.
Interestingly, despite the fact that the MLV-A and MLV-E relative titers
are approximately equal under all assay conditions, in all cases except the 5
minute infection with no drug chase in the presence of NH4 CI, the MLV-E is
slightly lower than MLV-A Additionally, the relative MoMLV(VSV-G) titer in
all assays is significantly lower than that of MLV-A and MLV-E. According to
the proposed model, this would be suggestive of a substantially shorter half-life
for the MoMLV(VS V-G) particles under these assay conditions.
52
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!= □ MLV-A
M MLV-E
M MoMLV(VSV-G)
Avg.%
Transduction SEM n
# Independent
Experiments
MLV-A 105.36 8.76 15 3
MLV-E 115.10 5.17 2 0 5
MoMLV(VSV-G) 62.87 9.09 9 2
Figure 15. Effect of 50 mM NH4CI on MLV-A, MLV-E, and MoMLV(VSV-G)
transduction of NIH 3T3 cells for a 5 minute infection and no drug chase.
Infection in the presence of NH«C1 was as described in materials and methods.
The 100% titer values were 6.9 x 103 -1.8 x 104 CFU/ml for MLV-A, 6.2 x 103 -
1.4 x 104 CFU/ml for MLV-E, and 5.4 x 10 3 -1.5 x 104 CFU/ml for
MoMLV(VSV-G).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
C = l MLV-A
M MLV-E
m MoMLV(VSV-G)
2 Hour Infection/No Chase
Avg. % # Independent
Transduction SEM n_________Experiments
MLV-A 69.35 3.76 15 3
MLV-E 59.55 3.56 2 0 4
MoMLV(VSV-G) 19.31 4.27 15 3
Figure 16. Effect of 50 mM NH4CI on MLV-A, MLV-E, and MoMtV(VSV-G)
transduction of NIH 3T3 cells for a 2 hour infection and no drug chase. Infection
in the presence of NH4CI was as described in materials and methods. The 100%
titer values were 1.6 x 104 -1.5 x 105 CFU/ml for MLV-A, 1.3 x 104 -1.5 x 105
CFU/ml for MLV-E, and 3.8 x 104- 6.0 x 10 4 CFU/ml forMoMLV(VSV-G).
54
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S I
11
o g
1 2 0 - 1
100 -
80-
60-
c n MLV-A
■ I MLV-E
2 Hour lnfection/2 Hour Chase
Avg. % # Independent
Transduction SEM n_________Experiments
MLV-A 41.70 4.25 1 2 2
MLV-E 24.70 3.64 2 0 4
MoMLV(VSV-G) 6.08 0.93 1 2 2
Figure 17. Effect of 50 mMNKjCI on MLV-A, MLV-E, and MoMLV(VSV-G)
transduction of NIH 3T3 cells for a 2 hour infection and a 2 hour drug chase.
Infection in the presence of NH4CI was as described in materials and methods.
The 100% titer values were 1.4 x 104 -1 .6 x 105 CFU/ml for MLV-A, 1.4 x 104 -
1.8 x 10s CFU/ml for MLV-E, and 3.8 x 10 4 - 4.7 x 10 4 CFU/ml for
MoMLV(VSV-G).
55
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* 1 100
l= 3 MLV-A
■■M LV -E
M B MoMLV(VSV-G)
5 Minute Infection/No Chase
Avg. % # Independent
Transduction SEM n_________Experiments
MLV-A 77.04 4.97 1 1 2
MLV-E 6 6 . 8 6 8.26 19 4
MoMLV(VSV-G) 51.66 11.14 1 0 2
Figure 18. Effect of 50 nM BFLAl on MLV-A, MLV-E, and MoMLV(VSV-G)
transduction of NIH 3T3 cells for a 5 minute infection with no drug chase.
Infection in the presence of BFLAl was as described in materials and methods.
The 100% titer values were 8.8 x 10 3 -1.2 x 10 4 CFU/ml for MLV-A, 9.5 x 103 -
1.3 x 104 CFU/ml for MLV-E, and 4.6 x 103 - 5.4 x 10 3 CFU/ml for
MoMLV(VSV-G).
56
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* g 100
C = l MLV-A
■ 1 MLV-E
WM MoMLV(VSV-G)
2 Hour Infection/No Chase
Avg. % # Independent
Transduction SEM n_________Experiments
MLV-A 63.05 6 . 6 8 1 0 2
MLV-E 52.03 4.91 1 2 2
MoMLV(VSV-G) 2 2 . 1 0 1 . 8 1 0 2
Figure 19. Effect of 50 nM BFLAl on MLV-A, MLV-E, and MoMLV(VS V-G)
transduction of NIH 3T3 cells for a 2 hour infection with no drug chase. Infection
in the presence of BFLAl was as described in materials and methods. The 100%
titer values were 8.4 x 104 -1.7 x 10 5 CFU/ml for MLV-A, 8.3 x 104 - 7.4 x 10 4
CFU/ml for MLV-E, and 3.6 x 10 4 - 9.8 x 10 4 CFU/ml for MoMLV(VSV-G).
57
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C ts 80
* £ 4 0
[= 1 MLV-A
■ 1 MLV-E
B S I MoMLV(VSV-G)
2 Hour lnfection/2 Hour Chase
Avg. % # Independent
Transduction SEM n_________Experiments
MLV-A 46.75 9.55 1 1 2
MLV-E 40.73 0.73 2 1 4
MoMLV(VSV-G) 7.49 2 . 8 6 1 1 2
Figure 20. Eflfect of 50 nM BFLAl on MLV-A, MLV-E, and MoMLV(VSV-G)
transduction of M H 3T3 cells in a 2 hour infection with a 2 hour drug chase.
Infection in the presence of BFLAl was as described in materials and methods.
The 100% titer values were 6.8 x 104 - 9.1 x 104 CFU/ml for MLV-A, 8.3 x 104 -
1.0 x 103 CFU/ml for MLV-E, and 4.4 x 104 - 6.5 x 104 CFU/ml for
MoMLV(VSV-G).
58
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4.2 The in vitro instability of MLV-A, MLV-E, and MoMLV(VSV-G)
viral vectors correlates with the reduction in relative viral vector titer in the
NH4CI and BFLAl titer assays. The proposed model suggests that virion
instability accounts for the reduction in relative titer observed in the NH4CI and
BFLAl titer assays. Data presented here from NH4CI and BFLAl titer assays
indicates that the relative viral vector titer decreases as the duration of infection in
the presence of either drug is increased. Thus, supporting the notion of viral
particles being spontaneously degraded while stuck in an endosomal compartment
due to the action of the drugs. Based on such a notion, I have predicted that in
vitro incubations of the viral vectors under similar conditions as those of the titer
assays should result in a reduction in the relative titer that corresponds with that
seen in the NH4CI and BFLAl titer assays. In order to address this possibility, the
in vitro viral vector stability was determined for MLV-A, MLV-E, and
MoMLV(VSV-G) particles.
The stability of each vector was determined based on the ability of
particles in a viral vector mixture to transduce NIH 3T3 cells following
incubations at 37°C, as described in materials and methods. Each viral vector
mixture was identical to the corresponding mixture used in the NH4CI and BFLAl
titer assays. As expected, the ability of MLV-A and MLV-E to transduce target
cells decreased as the incubation time with NH4CI, H2O, BFLAl, and DMSO was
increased (Figs. 21 and 22). This reduction in titer, relative to that at t - 0
59
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minutes was approximately the same for both vectors. The reduction was the
same in H2 O, DMSO, and BFLAl after 120 and 240 minute incubations at 37°C.
MLV-A and MLV-E titers were reduced by approximately 20-30% at 120
minutes and 40-S0% at 240 minutes. Both vectors, however, appeared to be
slightly less stable in NH4 CI. The reduction of MLV-A and MLV-E titers
following incubation at 37°C with NH4 CI was approximately 30% after 120
minutes and 70% after 240 minutes.
As would be expected based on the NH4 CI and BFLAl titer data and the
proposed model, MoMLV(VSV-G) particles appear to be very unstable in the in
vitro stability assay (Fig. 23). In H2 O, DMSO, and BFLAl the titer of
MoMLV(VSV-G) virions was significantly reduced following incubations at
37°C. The titers, relative to t = 0 minutes, were reduced by approximately 80-
85% after 120 minutes and slightly more after 240 minutes. As with MLV-A and
MLV-E, MoMLV(VSV-G) particles were less stable in NH4 CI. The reduction in
titer was approximately 85% after an incubation with NH4 CI for 120 minutes and
93% after 240 minutes. These data indicate that there is a correlation between the
in vitro instability of all three viral vectors and the duration of infection in the
NH4 CI and BFLAl titer assays (Figs. 24 and 25)
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0 120 240
Time (min)
% Relative Titer
Time (min) HzO NH4CI DMSO BFLAl
0 1 0 0 too 1 0 0 1 0 0
1 2 0 73.1 ± 12.4 68.1 ±2.5 73.8 ±12.1 6 8 . 8 ±9.5
240 57.4 ±13.4 32.0 ±L3 46.3 ±5.8 47.2 ±4.8
Figure 21. Stability of MLV-A at 37°C in medium containing H2 O, S O mM
NH4 CI, 0.1% DMSO, and S O nM BFLAL Aliquots of viral vector mixtures in
medium with solvent or drug were titered as described in materials and methods.
To facilitate comparison, the titers at t =0 minutes were normalized to 100%.
The 100% titer values ranged from 8.7 x 10 3 - 2.2 x 104 CFU/ml. Each value is
the mean of two experiments with three and six replicate dishes in each, ± SEM .
61
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0 120 240
Time (min)
% Relative Titer
Time (min) H2 0 NH,C1 DMSO BFLAl
0 100 100 100 100
120 84.8 ±6.1 74.7 ±9.8 84.5 ±10.1 79.3 ±13.1
240 51.6 ±19.4 31.9 ±8.1 47.2 ±9.3 47.6 ±10.8
Figure 22. Stability of MLV-E at 37°C in medium containing H2 O, S O mM
NH»CI, 0.1 % DMSO, and 50 nM BFLAl. Aliquots of viral vector mixtures in
medium with solvent or drug were titered as described in materials and methods.
To facilitate comparison, the titers at t = 0 minutes were normalized to 100%.
The 100% titer values ranged from 7.2 x 10 3 - 2.0 x 104 CFU/ml. Each value is
the mean of three experiments with three to six replicate dishes in each, ± SEM.
62
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0 120 240
Time (min)
% Relative Titer
Time (min) H2 0 NH4CI DMSO BFLAl
0 1 0 0 1 0 0 1 0 0 1 0 0
1 2 0 23.1 ±.03 13.4 ±6.2 19.6 ±2.1 16.7 ±0.6
240 20.0 ±.04 6 . 8 ± 2 . 8 13.3 ± 1.9 14.0 ±3.4
Figure 23. Stability of MoMLV(VSV-G) at 37°C in medium containing H2 O, 50
1 1 1 MNH4 CI, 0.1% DMSO, and 50 nM BFLAl. Aliquots of viral vector mixtures
in medium with solvent or drug were titered as described in materials and
methods. To facilitate comparison, the titers att = 0 minutes were normalized to
100%. The 100% titer values ranged from 6.3 x 10 3 -1.3 x 104 CFU/ml. Each
value is the mean of two experiments with three replicate dishes in each, ± SEM.
63
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0 120 240
Time (min)
MLV-A
MLV-E
2 Hour/No Chase 2 Hour/2 Hour Chase
Figure 24. Correlation between the in vitro instability of MLV-A, MLV-E, and
MoMLV(VSV-G) viral vectors in S O mM NH4CI and the reduction in relative
titer in the NH4CI titer assays. (A) Stability of all three vectors in medium
containing S O mMMLCl at 3TC. (B) Reduction in relative titers of all three
vectors in the NH4CI titer assay with an increase in the duration of infection with
drug from 2 hours to 4 hours.
64
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0
[ I
I MLV-A
I MLV-E
120 240
Time (irin)
I MLV-A
I MLV-E
2 Hour/No Chase 2 Hour/2 Hour Chase
Figure 25. Correlation between the in vitro instability of MLV-A, MLV-E, and
MoMLV(VS V-G) viral vectors in S O nM BFLA1 and the reduction in relative
titer in the BFLA1 titer assays. (A) Stability of all three vectors in medium
containing S O nM BFLA1 at 37°C. (B) Reduction in relative titers of all three
vectors in the BFL Al titer assay with an increase in the duration of infection with
drug from 2 hours to 4 hours.
65
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Chapter 5
5.1 Viral vectors are inactivated by pH 3 citric acid buffer. The evidence
from the literature presented here indicates that NH4CI and BFLA1 function to
raise the pH in endosomes and lysosomes and that this action results in a blockage
of transport through the endosomal pathway. Importantly, a majority of those
studies reported that internalization from the plasma membrane persisted.
Additionally, studies specifically looking at target cell entry by the ecotropic
MLV C57MC and SFV in the presence of lysosomotropic agents reported that
virus internalization was not affected. Based on this data, the model presented
here suggests that, because relative viral vector titer is reduced by NH4CI and
BFLA1, and these drugs are known to have their primary effect on the endocytic
pathway, these viral particles must be using an endosomal pathway for target ceil
entry. This is an important point that must be addressed directly in order to
validate the possibility of viral vector entry by endocytosis.
In order to determine whether or not viral particles were internalized in the
presence of NH4CI and BFLA1, a method was required for distinguishing
between intracellular and extracellular viral particles. A pH 3 citric acid buffer
has been used to inactivate herpesviruses(54), as well as MLV-E(24). This
method for inactivating extracellular virus is rapid. It was appealing because such
a rapid effect meant that the target cells associated with viral particles would not
be exposed to the buffer for any significant period of time. I, therefore, tested this
66
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citric acid buffer over a pH range from 3 to 7 in order to determine the optimal pH
for inactivation in my system. Virions associated with NIH 3T3 cells were treated
on ice for 30 seconds with either PBS + Ca2 + /Mg2 + or citric acid buffer, as
described in materials and methods. Treatments were performed on ice because
there was no observed cytotoxicity as compared with treatments at room
temperature and 37°C, where there was substantial cytotoxicity and detachment of
cells from tissue culture dishes. This cytotoxic effect was more profound when
cells had been incubated with 50 mM NH4 CI prior to the citric acid treatment. All
results of citric acid inactivation are with MLV-A and MLV-E, as MoMLV(VSV-
G) particles were not affected at all by treatment with citric acid buffer at any pH
(data not shown).
There was a pH-dependence to the ability of citric acid buffer to inactivate
cell-associated MLV-A and MLV-E (Fig. 26). Using the PBS + Ca2 + /Mg2 +
treatment as 100%, pH 3 citric acid buffer was the most effective, inactivating 97-
100% of virions. This determination is limited by the sensitivity of the assay and
is based on a loss of titer over approximately 3 orders of magnitude. It is
important to note that the 100% titers here are dependent on the duration of the
virion binding step at 4°C. Viral supernatants used here were the same as those
used in the titer assays of the present study, and have titers greater than 103
CFU/ml in those assays.
67
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120-.
PBS pH 3 pH 4 pH 5 pH6 pH 7
% Resistant Viral Vector Titer
Citric Acid Buffer MLV-A MLV-E
PBS Control 100 100
pH 3 3.1 ± 1.2 0.0 ±0.0
pH 4 81.9 ±19.1 53.5 ± 16.0
pHS 69.9 ±20.9 75.3 ±6.2
pH 6 77.0 ±16.3 96.0 ±19.5
pH 7 74.3 ±24.6 75.3 ±9.2
Figure 26. Inactivation of cell-bound MLV-A and MLV-E by citric acid
treatment from pH 3 to 7. Virus adsorbed to cells at 4°C was treated with citric
acid buffer at each pH for 30 seconds, on ice as described in materials and
methods. A representative set of data from one experiment for each vector is
reported in which each MLV-A data point is the result of six replicate dishes and
each MLV-E data point is the result of duplicate dishes. To facilitate comparison,
the PBS control samples were normalized to 100%. Error bars represent die
standard deviation.
68
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Viral particles were inactivated to a lesser extent when the pH of the citric
acid buffer was higher than 3. Treatment at all other pH values resulted in a small
reduction of titer relative to the control. This reduction in the relative titer was in
the range of S to 50%, with the 50% inactivation being of MLV-E treated with pH
4 citric acid buffer.
Because there was still a small inactivation of cell associated MLV-A. and
MLV-E by citric acid buffer at pH values above 3, the possibility of a cellular
effect of citric acid that inhibited transduction of the target cells independently of
viral vector inactivation was considered. In order to determine if there was such
an effect, NIH 3T3 cells were treated with PBS + Ca2 + /Mg2 + , pH 3 citric acid
buffer, or pH 7 citric acid buffer exactly as in the viral vector inactivation assay.
Following this treatment, the cells were incubated with the viral vector mixture so
that any effect on the ability of the target cells to be transduced could be
determined. Pre-treatment of cells with citric acid buffer at either pH was not
significantly different from the PBS + Ca2 7Mg2 + treated cells, with respect to the
ability of those cells to be transduced with MLV-A and MLV-E (Fig. 27).
Therefore, it is believed that citric acid treatment did not make NIH 3T3 cells
resistant to transduction by MLV-A or MLV-E.
69
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140-1
□ MLV-A
5 ,2°- T M MLV-E
III
PBS pH 3 pH 7
% Relative Titer
Citric Add Buffer MLV-A MLV-E
PBS Control 100 100
pH 3 94.0 ±2.5 98.8 ±10.2
PH 7 96.5 ±26.6 95.0 ±13.2
Figure 27. Effect of citric acid treatment on the transducability of NIH 3T3 cells.
Cells were treated with either PBS or citric acid buffer for 30 seconds on ice, as
described in materials and methods. Any effect of treatment on the ability of the
cells to be transduced was determined by incubating with MLV-A and MLV-E for
2 hours. Each value is the mean of two experiments with four replicate dishes in
each, ±SEM.
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In addition to the observation that pH 3 citric acid buffer inactivated viral
particles, it was determined that this viral vector inactivation corresponds to
changes in the viral envelope protein. Cells that are confluently infected with
replication competent virus express the viral envelope glycoprotein on the plasma
membrane. As described in materials and methods, the envelope protein can be
detected with the mouse mAb S73 in an FITC-based immunofluorescence assay.
The epitope recognized by this antibody is in the C-terminus of the SU subunit of
the envelope protein.
NIH 3T3 cells confluently infected with either 4070A or MoMLV were
treated exactly as cell-associated virions were treated in the citric acid inactivation
assay. Following this treatment it was determined that there was no detectable
fluorescence in the viral envelope immunofluorescence assay only when the pH 3
citric acid buffer was used (Figs. 28 and 29). The amount of fluorescence was
approximately that of control dishes for both viruses at all pH values above 3.
Fluorescence slightly less than control levels was observed for the pH 4 citric acid
treatment of4070A infected cells. This data agrees nicely with the viral vector
inactivation data and suggests that the inactivation of viral particles by pH 3 citric
acid buffer is due to loss of envelope protein function. This loss of function must
either be due to the shedding of the SU subunit or a conformation change in the
envelope protein that also results in the loss of the epitope for mAb S73.
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5.2 MLV-A and MLV-E are internalized in the NH4C1 and BFLA1 titer
assays. After determining that treatment of cell-associated viral particles on ice
for 30 seconds with pH 3 citric acid buffer was an effective means of inactivating
virions on the cell surface, this method was used to determine the extent of vector
internalization following infection in the presence of NH4CI and BFL A1. Viral
vector mixtures were incubated with cells in the presence of NH4CI, H2 O,
BFLA1, or DMSO. Following the incubation at 37°C for 2 hours, cells in each
agent were treated for 30 seconds on ice with either PBS + Ca2 7Mg2 + or pH 3
citric acid buffer. Any residual titer following treatment with the pH 3 citric acid
was indicative of viral particles that had been internalized into the target cells and
were, therefore, protected from the citric acid.
There was very little effect of citric acid treatment on viral vector titer
following an infection in the presence of H2 O (Fig. 30). Approximately 74% and
88% of the MLV-A and MLV-E titers, respectively, were citric acid resistant.
Thus, after the 2 hour infection period, 74% of MLV-A and 88% of MLV-E had
already been internalized into the target cells. As a result, the particles that had
been internalized were no longer accessible to citric acid buffer. Only the 26%
and 12%, respectively, were still on the surface of the cell, which rendered them
citric acid sensitive. This is suggestive of a fairly rapid rate of internalization for
both vectors.
72
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owner. Further reproduction prohibited without permission.
Bright Field Control pH 3 pH 4
pH 5 pH 6 pH 7
Figure 28, Effect of pH 3 citric acid treatment on the immunofluorescent detection of the
MoMLV envelope protein on MoMLV infected NIH 3T3 cells.
Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission.
mmm
Bright Field Control
pH 5 pH 6 pH 7
Figure 29, Effect of pH 3 citric acid treatment on the immunofluoiescent detection of the
4070A envelope protein on 4070A infected NIH 3T3 cells.
Following an infection in the presence of S O mM NH4CI, approximately
45% of both vectors were citric acid resistant, suggesting that this portion of each
viral vector pool had also been internalized into the NIH 3T3 cells (Fig. 30).
Results similar to those with H2 O and NH4 CI were obtained with DMSO
and BFLA1. After an infection in the presence of 0.1% DMSO, 82% ofMLV-A
and 95% of MLV-E were determined to be internalized based on resistance to
treatment with citric acid buffer (Fig. 31). The small reduction in the percentage
of internalized particles in the presence ofNHtCl or BFLA1, as compared with
the extent of internalization in the presence of H2 O or DMSO, is likely due to the
possibility of viral particles being recycled back out to the plasma membrane
following internalization in the presence of the drugs. In preliminary experiments
I have observed that, following an infection in the presence of NH4 CI, a larger
percentage of viral vectors were inactivated with pH 3 citric acid treatment at
37°C for 2 minutes compared to treatment on ice for 2 minutes (data not shown).
Thus, suggesting that at the higher temperature, where membrane trafficking
would be active, viral particles were more accessible by citric acid. Such an
increase in accessibility could very well be due to the recycling of internalized
particles back out into citric acid at the plasma membrane during the treatment.
Supporting such a hypothesis is the report that the recycling of the transferrin
receptor is not significantly affected by BFLA1, despite a blockage in trafficking
through the endosomal pathway(56).
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1= 1 MLV-A
M MLV-E
l
m 4 cvc»ic
Add
% Relative Titer
MLV-A MLV-E
H20/pH 3 Citric Acid 73.6 ±3.1 87.6 ± 2.7
NH4CI/PH 3 Citric Acid 43.7 ±5.3 48.1 ±5.3
Figure 30. Viral vector internalization into NIH 3T3 cells in the presence of H2 O
and S O mM NH4CI based on resistance to pH 3 citric acid treatment. Viral vector
mixtures were incubated with cells in the presence of H2 O or NH4CI, as described
in materials and methods. Following each incubation, cells were either washed
with PBS or with pH 3 citric acid buffer to inactivate virus that had not been
internalized. To facilitate comparison, PBS washed samples from the H2 O and
NH4CI infections were normalized to 100%. The 100% titer values for the H2 O
treated, PBS washed samples ranged from 6.4 x 104 -1.2 x 10s CFU/ml for
MLV-A and 7.0 x 10 4 -1 .2 x 10 for MLV-E. Each value is the mean of three
experiments with three to four replicate dishes in each, ± SE M .
76
120-1
e 100
^ 40-
£
HjO/Otrfc
Acid
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1 2 0 - .
c m MLV-A
M MLV-E
% Relative Titer
MLV-A MLV-E
DMSO/pH 3 Citric Acid 81.9 ±0.4 94.8 ±10.1
BFLAl/pH 3 Citric Arid 61.5 ±22.7 80.2 ±15.6
Figure 3J. Viral vector internalization into NIH 3T3 cells in the presence of 0.1%
DMSO and 50 nM BFLA1 based on resistance to pH 3 citric acid treatment. Viral
vector mixtures were incubated with cells in the presence of DMSO or BFLA1, as
described in materials and methods. Following each incubation, cells were either
washed with PBS or with pH 3 citric acid buffer to inactivate virus that had not
been internalized. To facilitate comparison, PBS washed samples from the DMSO
and BFLA1 infections were normalized to 100%. The 100% titer values for the
DMSO treated, PBS washed samples ranged from 7.9 x 104 -1.3 x 10s CFU/ml
for MLV-A and 8.1 x 10 4 -1.3 x 105 for MLV-E. Each value is the mean of three
experiments with three to four replicate dishes in each, ± SEM .
77
e 100-
o
tS 80-
H 40-
DMSO/Ctric BFLAI/Ctie
Add Add
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This data is similar to that obtained with H2 O and indicates that in the presence of
either solvent both MLV-A and MLV-E particles are internalized at comparable
rates. Based on citric acid resistance following an infection in the presence of S O
nM BFLA1,61% of MLV-A and 80% of MLV-E appeared to be intracellular.
This result is in agreement with the previous reports that have been discussed
regarding the effect of BFLA1 and NH4CI on the internalization of endocytosed
ligands.
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Chapter 6: Discussion
Lysosomotropic agents have been routinely used to determine whether
viruses enter target cells through an endocytic pathway or directly through the
plasma membrane (Fig. 1X3,34,35,41). An inhibitory effect of such agents on
viral titer has been inferred to indicate that such viruses require endocytosis for
entry. Additionally, because these drugs block the acidification of endocytic
vesicles, it has been generally assumed that an inhibitory effect on viral titer
indicates the requirement for an acidic compartment during viral entry. Based on
this interpretation, such viruses have been termed “pH-dependent”. Those viruses
not displaying sensitivity to lysosomotropic drugs have been assumed to enter
target cells directly through the plasma membrane, independently of any acidic
compartment, and of endocytosis, and have been classified as “pH-independent”.
Based on such studies, MLV-A has been determined to be pH-independent, and
MLV-E and VS V have been classified as pH-dependent. The results presented
here indicate that titer assays using lysosomotropic agents are not measuring the
necessity for an acidic compartment during viral entry. Rather, these assays are
likely to be measuring the stability of viral particles. Based on these findings, I
have proposed a model (Fig. 4). This model predicts that any virion exhibiting a
reduction in relative titer following an infection in the presence of a
lysosomotropic agent is entering the target cell by an endocytic pathway. This is
because lysosomotropic drugs exert their action primarily on the endosomal
pathway and are not reported to have other effects that may influence fusion
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events at the plasma membrane. The reason for the loss of titer is because, while
the viral particles are arrested in an endosomal compartment due to the ability of
the lysosomotropic drugs to block trafficking through the endosomal pathway,
they undergo spontaneous denaturation as a direct result of their inherent
instability. Following the removal of the drug at the end of the assay, any virions
that have not been denatured are now able to resume a productive infection.
Any inhibition of viral entry, however, does not necessarily indicate the
direct involvement of endosomal pH on viral entry. It is possible that the
endosomal pathway is used for internalization regardless of endosomal
acidification. The fact that acidification takes place may be inconsequential to the
requirements for viral entry. Inhibiting the acidification of this pathway, though,
does result in a block of the maturation of early endosomes to downstream
endocytic compartments and lysosomes. Thus, transport of an endocytosed viral
particle to a vesicular compartment containing some required factor for viral entry
may be prevented. Additionally, any alterations in intracellular vesicular
trafficking by lysosomotropic drugs could also potentially prevent delivery of
some soluble or membrane-associated factor required for entry to the
compartment containing the viral particles. Such a factor could be in the form of
a co-receptor or accessory protein that may be required for envelope protein
mediated fusion. Examples of co-receptors residing on the plasma membrane, in
the case of HIV-1, are CXCR4(15) and CCR5(2).
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This model also predicts that a lack of viral particle sensitivity to
lysosomotropic drugs in titer assays cannot be interpreted to indicate a plasma
membrane route of entry into target cells. Virions that are relatively stable over
the course of a titer assay would not significantly denature during their arrest in an
endosomal compartment. The result would be no detectable reduction in relative
titer. I have shown here that the relative titers of MLV-A, MLV-E, and
MoMLV(VSV-G) are all reduced following a titer assay with either NH4 CI or
BFLA1 (Figs. 16,17,19, and 20). Additionally, I have shown that the observed
reduction of relative titer in the titer assays correlates with the in vitro instability
of each viral particle (Figs. 24 and 25). Also in agreement with the proposed
model is the fact that MLV-A and MLV-E have been shown to be internalized
following an infection in the presence of NH4 CI and BFLA1 (Figs. 30 and 31),
directly indicating that both viral particles are utilizing an endosomal pathway for
target cell entry.
The possibility exists that the reason for resistance to citrate inactivation in
the viral internalization assay is because particles have fused with the plasma
membrane and have, thereby entered the target cells independently of
endocytosis. This is not believed to be the case, however, because of the known
functions of NH4CI and BFLA1. Both of these drugs are known to exert their
effects on the endocytic pathway and are not known to have any significant effect
on events at the plasma membrane that would influence viral fusion. Any
secondary effect of NH4CI would be expected to be due to the alkalinization of
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non-endosomal or lysosomal compartments, such as the trans Golgi network,
which should not influence potential fusion events at the plasma membrane.
Likewise, any secondary effects of BFLA1 would be expected to be due to the
ability of this drug to inhibit other H*-ATPases besides the vacuolar FT -ATPase,
of which there is still no reason to believe that there would be an effect on fusion
events at the plasma membrane. However, at concentrations below 1 |iM, BFLA1
is specific for the vacuolar ET-ATPase(7). Additional evidence for the use of an
endocytic entry pathway by MLV-E comes from studies with confocal
immunofluorescence microscopy in which MLV-E envelope protein was shown
to be rapidly internalized into NIH 3T3 cells (personal communication with
Sunyoung Lee in the laboratory of W. French Anderson, MD.). This envelope
protein was co-localized with the MLV-E receptor (mCAT-l) and was observed
being internalized into cells.
I have developed a titer assay for studying the effects of NH4 CI and
BFLA1 on viral vector target cell entry. In this assay, vectors carrying two
different marker genes have been combined and treated as a mixture in order to
eliminate any variables that may exist between the two viral vector supernatants.
When the duration of infection in the presence ofNHtCl was increased, there was
a corresponding decrease in the relative titers of MLV-A, MLV-E, and
MoMLV(VSV-G) (Figs. 15-20). The same trend was observed in titer assays
containing BFLA1.
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Despite the fact that MLV-A and MLV-E titers were approximately equal
under all titer assay conditions, the MLV-A relative titer was consistently slightly
higher in all except the 5 minute infection in the presence of NH4 CI. This is an
interesting observation, especially considering the fact that in previous NH4 CI
assays, MLV-A has generally been less sensitive to NH4 CI that MLV-E. As I
have suggested with the model proposed here, the decrease in titer in the assays
with lysosomotropic drugs is due to the instability of the viral particles. So, this
result in the titer assays would imply that MLV-A is slightly more stable then
MLV-E under the conditions of the assay. In fact, the results presented here
regarding the in vitro instability of both viral particles indicates that, although
both vectors are roughly equal in their decay at 37°C, MLV-A seems to be
slightly less stable that MLV-E. However, this may not be a significant
difference. Despite the fact that I have tried to simulate the titer assay conditions
in the viral vector instability assay, it is possible that there are slight differences
between the two. The environment of an endosomal compartment most certainly
will contain some elements that are not present in tissue culture medium alone,
and it is possible that the stabilities of these two vectors may be affected slightly
differently.
The relative MoMLV(VSV-G) titer in the NH4CI and BFLA1 assays is
reduced to a much greater extent than either MLV-A or MLV-E. This rapid loss
of titer can be explained by the short half-life of MoMLV(VSV-G) particles
relative to MLV-A and MLV-E particles. As can be seen in the in vitro stability
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assay, this reduction in the relative titer correlates very will with the inherent
instability of the MoMLV(VSV-G) particles. However, it appears that this vector
also displays a slightly greater titer drop in the NH4 CI and BFLA1 titer assays
than would be expected from the in vitro stability data alone.
Because the three viral vectors used here are virtually identical with the
exception of the envelope proteins, it seems likely that this protein would be the
source of differential stability. A conformational change in the envelope protein
may be a destabilizing force in the environment of an endosomal compartment
and the possibility exists that solutes or proteins within this compartment may be
contributing factors. However, little is known about conformational changes in
the envelope protein that may accompany binding of a virus to its receptor. The
possibility of a “destabilized” pre-fusion conformation in the envelope protein
following receptor binding is an attractive hypothesis. Despite the different
receptor specificity of MLV-A and MLV-E, the envelope proteins share 80%
homology in their transmembrane (TM) subunits, with the cytoplasmic tails being
identical. Thus, the small difference between the two virions in the NH4 CI and
BFLA1 titer assays may be due to slight differences in the stability of each
envelope protein associated with its corresponding receptor. There is reason to
believe that the envelope protein of MLV-A and MLV-E is the determinant of
viral vector stability. Rationally, this is the portion of the viral particle that is
exposed to the environment outside the virion lipid bilayer. As such, it would be
the only component in direct contact with any destabilizing factors in the
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environment. Additionally, the MLV envelope is composed of two non-
covalently attached subunits that must work cooperatively in the initial stages of
viral entry(46). The precise nature of the cooperativity that is required for a
functional envelope protein in not known, but would need to include the release of
a hydrophobic fusion peptide following receptor binding. The fusion peptide
facilitates the fusion between the viral and cell lipid bilayers that needs to take
place before the vial core can be released into the cytosol. Any disruption in this
cooperativity would likely interfere with receptor binding or fusion with the cell
membrane, thus resulting in a non-viable virion with respect to target cell entry
and transduction. The notion of a retrovirus envelope protein being the primary
determinant of viral particle stability is supported by the observation that, at short
incubations times, the loss of HIV-1 infectivity correlated with spontaneous
shedding of the SU(gpl20) envelope subunit from virions(27).
The VSV-G envelope protein on the MoMLV(VSV-G) particles is quite
different from the envelope proteins of MLV-A and MLV-E. It is a single
polypeptide containing a cytoplasmic tail that is different from the cytoplasmic
tail of the MLV TM subunit. The fact that the MoMLV(VSV-G) particles are
very unstable is unexpected, considering their durability during viral vector
concentration. These particles have been able to be concentrated by
ultracentrifugation without any significant loss in titer(59), a task that has been
difficult with particles containing MLV envelope proteins because of the non-
covalently attached SU and TM subunits. It is possible that the MLV-based
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particles with the VSV-G envelope protein are lacking some interaction between
the viral core proteins and the envelope cytoplasmic tail that would normally
provide some stability in the NH4CI and BFLA1 titer assays, as well as the in
vitro stability assays.
Another possibility is that the MoMLV(VSV-G) production system used
here actually creates unstable particles. As mentioned in materials and methods,
MoMLV(VSV-G) particles were generated using a transient three plasmid
expression system in 293T cells. In this system the envelope plasmid, containing
an SV40 origin of replication, is separate from the expression plasmid for the
MLV core proteins. Therefore, not only is the VSV-G protein foreign to the
MLV core, but the stoichiometry of envelope and core proteins may be very
different from the MLV-A and MLV-E particles. Despite the apparent instability
of the MoMLV(VSV-G) particles, however, a previous study has reported that
95% of wild-type VSV infectivity was lost following a 3 hour infection in the
presence of 30 mM NH4 C1 (34). This is in agreement with the results presented
here and suggests that the MoMLV(VSV-G) viral vector is behaving similarly to
the wild-type VSV.
It is interesting that all three viral vectors exhibit decreased stability in the
presence of 50 mM NH4 CI. It is possible that excess ammonium or chloride ions
may be interfering with charge-charge interactions in the envelope proteins,
resulting in a destabilization. In the case of MLV-A and MLV-E, such an
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electrostatic disruption could be either within the SU or TM subunits, or at a site
or sites of interaction between the subunits.
Although pH 3 citric acid buffer treatment has been used merely as a tool
in the present study, the sensitivity of MLV-A and MLV-E particles to this
treatment may also be suggestive of envelope protein instability. The pH 3 citric
acid buffer inactivation of both viral vectors when associated with cells is
virtually complete (Fig. 26). This inactivation is probably due to an acid-induced
denaturation and/or shedding of SU. Because this pH is non-physiological, the
denaturation or shedding is most likely not related to normal envelope function.
The fact that relative MLV-A and MLV-E titers do not come back up to 100% of
control levels in the citric acid inactivation assay, however, is interesting. There
seems to be a diminishing sensitivity to citric acid treatment as the pH is raised
from 4 to S. Above pH S, citric acid treatment still destroys about 10-20% of
transduction by both vectors. Thus, it appears that, regardless of the pH, citric
acid buffer treatment is inactivating viral particles. One possibility is that this
solution is able to denature the viral envelope proteins as has been suggested for
the NH4 CI in the titer and stability assays. In addition to 40 mM citric acid, the
citric acid buffer contains 10 mM KC1 and 13 5 mMNaCl. It seems very possible
that these salts may interfere with electrostatic interactions in the envelope
proteins, resulting in irreversible conformational changes that destroy normal
function. Again, the pre-fusion form of the viral envelope bound to its receptor
may make it particularly sensitive to this salt-induced conformational change.
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This may explain why there is not complete inactivation of viral vectors, as not all
envelope proteins are likely to be bound to receptors on the cell surface.
It is also possible that citric acid may be complexing divalent metal ions
required for the structure and function of the MLV-A and MLV-E envelope
proteins. Citric acid has pKa values of 3.13,4.76, and 6.4. As the pH of the citric
acid solution is increased above the first pKa, each of the three carboxyl groups
will be progressively deprotonated. These deprotonated carboxyl groups are then
able to function in the chelation of divalent metal ions. Though the importance of
divalent metal ions in retroviral envelope structure and function is not known, the
receptor-binding domain of the Friend murine leukemia virus envelope protein
has been crystallized with both zinc and calcium ions(l4). If zinc, calcium, or
any other divalent metal ion was normally involved in the conformation and
integrity of the native envelope protein, citrate may be able to complex these ions,
resulting in an irreversible conformational change.
There is also the possibility that, above pH 3, the citric acid buffer is able
to inactivate viral vector components independently of the envelope protein. It is
known that the divalent manganese (Mn2 + ) and magnesium (Mg2 + ) ions are
required for the reverse transcriptase(RT) functions of HIV-1 and MoMLV(9).
Additionally, Mn2 + is required for HIV-1 integrase catalysis and appears to be
tightly bound in a ternary enzyme-metal-DNA complex upon viral DNA
integration(33). Thus, if citrate were able to chelate divalent metal ions that are
necessary for MLV reverse transcription or integration away from these enzymes,
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the result would be defective viral particles that were not able to transduce the
target cells. One would speculate that if this were the case, the replacement of
medium following the citric acid treatment would replenish the missing metal
ions. However, if the removal of the ions caused an irreversible conformational
change to take place in these enzymes, activity would still be lost. It is even
possible that the conformational changes in enzymes resulting from removal of
divalent metal ions would be irreversible only in a portion of the enzyme
population, which would explain the partial inactivation of viral particles. It is of
interest to note that, in the viral envelope immunofluorescence assay, not only
was there no envelope-dependent fluorescence after the cells were treated with pH
3 citric acid buffer, but there appeared to be fluorescence slightly less than the
PBS control in infected cells treated with citric acid buffer at pH values above 3.
This observation is in agreement with the data from the viral vector inactivation
assay.
Another noteworthy observation is that, as mentioned previously, there
appeared to be a synergistic cytotoxicity associated with the pH 3 citric acid
treatment and NH4CI treated NIH 3T3 cells when the citric acid treatment was at
room temperature or 37°C. The result was a large amount of cell death in addition
to the detachment of cells from tissue culture dishes. These effects were much
more pronounced at 37°C than at room temperature, and treatment on ice, as
performed in the assays here, resulted in no detectable cytotoxicity. The reason
for this cytotoxic effect is likely due to the secondary effect ofNFkCl on
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endosome and lysosome swelling. As discussed previously, the diffusion o f NH3
into acidic vesicles results not only in an increase in vesicular pH, but also leads
to the osmotic swelling due to a sequestration ofN H / within the vesicles. This
swelling is apparent as extensive vacuolization when cells are observed under
phase contrast bright-fleld microscopy. When cells in this state are treated with
pH 3 citric acid buffer, it is possible that the buffer is endocytosed via fluid phase
uptake into vesicles that are potentially leaky due the NHtCl-induced swelling.
The aqueous contents of the vesicles, including the citric acid buffer, would then
be able to leak into the cytosol where salt effects or pH effects might be able to
interfere with normal cellular function. This could directly interfere with
cytoskeletal elements, thus causing the cells to lose their structural integrity and
detach from the dish. In fact, cytosol acidification and hypertonicity have been
used for the inhibition of clathrin-mediated endocytosis, but it has been reported
that the mechanism of action for each is unknown and that both treatments may
have other pleiotropic effects(26). To exemplify the potential magnitude of citric
acid buffer internalization at 37°C is the report that all of the plasma membrane is
turned over every 30 minutes to 2 hours at 37°C, depending on the cell type(49).
The rapid rate of fluid-phase uptake that would accompany such membrane
turnover would presumably be internalizing a substantial volume of citric acid
buffer into the leaky endocytic vesicles of an NH4CI treated cell.
The present study has attempted to shed light on the interpretations of
assays utilizing lysosomotropic drugs, with respect to viral entry. As has been
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discussed already, a reduction in relative viral titer resulting from treatment with
lysosomotropic agents has been interpreted to indicate the requirement for
endocytosis by viral particles and has been assumed to mean that the acidification
of endosomes is necessary for viral entry. Conversely, a lack of sensitivity to
lysosomotropic drugs, with respect to viral entry, has been interpreted to indicate
entry of target cells directly through the plasma membrane, independent of
endocytosis. These interpretations fail to account for the mechanism whereby
titer is reduced by the drugs. Additionally, the effects of these drugs on target cell
physiology has not been taken into account. While it is true that, under normal
conditions lacking any lysosomotropic agents, viral particles not able to cany out
fusion and release of the viral core may end up being degraded in lysosomes, this
is an unlikely mechanism of virion degradation in the presence of such agents.
Not only would transport of particles to lysosomes be expected to be blocked, but
acid-dependent lysosomal hydrolases would not be active due to the alkalinization
of lysosomes.
The evidence presented here makes a compelling case for the use of an
endocytic target cell entry pathway by MLV-based vectors, but may extend to
other viruses and viral vectors. I have eluded to the fact that existing data, with
respect to HIV-l target cell entry, fits very nicely with the model that I have
proposed. That is, that HIV-1 may also enter cells by endocytosis, but that the
stability of these virions has led to the false assumption of a plasma membrane
entry pathway, based on titer assays using lysosomotropic agents. The fact that
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the stability of HIV-1 exceeds the duration of infection in the typical NH»C1 assay
by 6 to 10 times accounts for the lack of titer reduction. Such misinterpretations
of titer assays with lysosomotropic drugs may very well be widespread,
particularly with the characterization of entry pathways used by other
retroviruses. Prior to the present study, this has contributed to the assumption that
all mammalian retroviruses, with the exception of MLV-E, use predominantly a
plasma membrane route of target cells entry. While the results here, by no means,
prove that all viruses enter target cells by an endosomal route, they certainly
warrant the use of assays besides the lysosomotropic drug-dependent viral entry
assay for this determination. These results may even call into question the
existence of a productive viral entry pathway other than by endocytosis.
The implications of the re-determination of viral entry pathways are
potentially far-reaching and could affect both strategies for combating viral
infections as well as for constructing targeted vectors for gene therapy. The
consideration of new therapies for HIV-l infection is an obvious consequence of
new insight into retroviral entry. If HIV-1 does, in fact, enter target cells via an
endocytic pathway, this would change the reasoning behind the development of
anti-viral therapies. For example, the lysosomotropic drugs amantadine and
rimantadine have been used to potentially block infection of influenza virus. The
reason for using these drugs is because influenza is known to possess acid-
dependent fusion properties that are required for infection. Both of these drugs
raise the pH in endosomes and lysosomes, thereby inhibiting entry and, as
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determined in this study, most likely make the survivability of the infecting
virions dependent on their stability. If influenza virus was thought to infect cells
independently of endocytosis, however, drugs such as these probably would not
have been considered. Thus, for treating infections by HTV-1 and other viruses
that may use an endocytic route of target cell entry, a whole new perspective
could be given to the development of new anti-viral drugs.
The consideration of entry pathways for viral vectors plays an important
role in the design of targeted vectors for gene therapy. For example, a completely
different environment would be encountered by a vector internalized via
endocytosis versus one that entered cells through the plasma membrane. Such
factors as binding affinity of the vector envelope protein for its receptor, stability
of a chimeric viral vector envelope protein, and rates of removal from the cell
surface, all play a role in the design of such vectors. The MLV-A envelope
protein is currently used extensively for human gene therapy clinical trials. This
envelope has been thought to mediate viral entry through the plasma membrane
so, in engineering a chimeric MLV-A envelope for targeting a new receptor, it has
been assumed that this envelope would need to function in this local environment.
However, the environment of an endosome is very different from the plasma
membrane and factors such as acidic pH, the curvature of an endosomal
membrane, and possible trafficking to a sorting endosome, endosomal carrier
vesicle, late endosome, or lysosome may influence the thought process involved
in developing such a vector. We are just beginning to learn about how viral
93
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envelope proteins function to mediate viral entry. But as we decipher the
mechanism of action of these proteins, the significance of environmental factors
will become more important.
The rate of viral vector internalization is very important in the design of
vectors for successful gene delivery. The reason for this is the potential
complication of an immune response that a patient may mount to a viral vector.
This has specifically been a concern with the administration of adenoviral vectors
because the DNA from these vectors remains episomal and is lost over time,
resulting in the necessity for repeated vector administration. As a result, knowing
that a particular viral vector entered cells by endocytosis and characterizing the
rate of endocytosis could be very useful in designing such a vector. The quicker a
vector can be internalized so that it is no longer accessible to the circulation, the
less likely that vector would be to elicit an antibody mediated immune response.
Of course, these aspects of vector entry are dependent on the affinity of a vector
for its cellular receptor and the role of the targeted receptor on the endocytosis
process.
The role that the receptor plays in viral entry is still unknown and is
potentially important in the design of efficient viral vectors. One possibility is
that the receptor merely serves as an anchor for binding of a viral vector before
allowing the viral particle to actively mediate its own entry. Another is that the
viral particle binds to the receptor and remains bound until the receptor is
internalized by the normal constitutive cellular membrane recycling machinery, as
94
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is the case for the endocytosis of low density lipoprotein receptors (LDL-R) and
transferrin receptors (TfRX53). The last case would be the active triggering of
internalization induced by the binding of the vector to the receptor, as is the case
with the endocytosis of the epidermal growth factor receptor (EGF-R) and
platelet-derived growth factor receptor (PDGF-RX53). It has been shown by
confocal immunofluorescence microscopy that MLV-E does remain co-iocalized
with its receptor (mCAT-l) while it is internalized into NIH 3T3 cells (personal
communication with Sunyoung Lee in the laboratory of W. French Anderson,
M.D.), suggesting that the receptor is not merely serving as an anchor at the
plasma membrane and allowing the virion to independently enter the cell. In fact,
it will likely turn out that affinity of viral vectors for their receptors, as well as the
rate of internalization, will be dependent on each targeted receptor. Because of
this, it may not only require an extensive knowledge of viral envelope protein
function, but also of internalization rates and mechanisms for cellular proteins that
may serve as receptors for viral vectors.
95
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Creator Katen, Louis John, III (author)

Core Title Characterization of target cell entry by murine leukemia viruses

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,biology, microbiology,biology, molecular,health sciences, medicine and surgery,OAI-PMH Harvest

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Contributor Digitized by ProQuest (provenance)

Advisor Anderson, W. French (committee chair), Farley, Robert (committee member), Stallcup, Michael R. (committee member)

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