University of Southern California Dissertations and Theses

Entry and intracellular trafficking of measles virus glycoprotein pseudotyping lentivector for transduction in target cells

(USC Thesis Other)

Entry and intracellular trafficking of measles virus glycoprotein pseudotyping lentivector for transduction in target cells

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

ENTRY
AND
INTRACELLULAR
TRAFFICKING
OF

MEASLES
VIRUS
GLYCOPROTEIN
PSEUDOTYPING

LENTIVECTOR
FOR
TRANSDUCTION
IN
TARGET
CELLS

by

MAN
JI

A
thesis
submitted
in
partial
fulfillment
of
the

requirements
for
the
degree

MASTER
OF
SCIENCE

in

BIOCHEMISTRY
AND
MOLECULAR
BIOLOGY

ADVISOR:
DR.
PIN
WANG,
ASSOCIATE
PROFESSOR

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

DECEMBER
2013

2

ACKNOWLEDGEMENTS
Without the guidance of my committee members, help and support from coworkers
and family, I would never have been able to finish my dissertation along this long but
fulfilling road.
First and foremost I would like to offer my sincere gratitude to my research advisor,
Dr. Pin Wang, who patiently provided help, encouragement and support for me to
proceed and complete my dissertation. His immense knowledge and insightful advice
helped going through all stages of this thesis.
I would also like to thank Yarong Liu, who has been a role model to me, serving as a
strong and supportive adviser throughout my lab life. I am also grateful to Dr. Kye Il Joo,
who was always willing to help and give his best suggestions.
Special thanks to my committee, Dr. Qilong Ying and Dr. Zoltan Tokes who were
willing to serve being my committee members. Their guidance and suggestions have
helped me a lot and I owe them my heartfelt appreciation.
Many thanks to all members in Dr. Pin Wang’s lab for their assistance and
friendship. I could not complete my work without Chupei Zhang and Jinxu Fang’s
guidance on experimental skills, Dr. Paul Bryson’s help on analysis, Xiaolu Han and Dr.
Biliang Hu’s support on molecular cloning, and the insightful discussions and assistance
from all lab members.
At last, I wish to thank my family, for them being my inspiration and driving force.
They were always supporting and encouraging me with their best wishes.

3

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ........................................................................................................... 2
LIST OF FIGURES ........................................................................................................................ 4
ABSTRACT ..................................................................................................................................... 5
I. INTRODUCTION .................................................................................................................... 6
II. MATERIALS AND METHODS ......................................................................................... 10
1. CELL LINES AND REAGENTS ................................................................................................ 10
2. ANTIBODIES ........................................................................................................................ 10
3. PLASMIDS ............................................................................................................................ 11
4. TRANSFECTION AND VECTOR PRODUCTION ........................................................................ 11
5. VIRAL TRANSDUCTION ........................................................................................................ 12
6. QUANTIFICATION OF LENTIVECTOR GENOME COPIES BY RT-PCR ..................................... 13
7. DYE LABELING OF PSEUDOTYPING LENTIVECTOR .............................................................. 13
8. DRUG TREATMENT .............................................................................................................. 13
9. TRANSDUCTION OF SIRNA, WILD-TYPE OR DOMINANT-NEGATIVE MUTANT PLASMIDS .... 14
10. FLOW CYTOMETRY ........................................................................................................... 15
11. CONFOCAL MICROSCOPY .................................................................................................. 15
12. STATISTICAL ANALYSIS .................................................................................................... 16
III. RESULTS ............................................................................................................................. 17
1. CLATHRIN MEDIATED ENTRY FOR H/F-LV ......................................................................... 17
2. INVOLVEMENT OF ENDOSOME COMPARTMENTS FOR H/F-LV INFECTION .......................... 21
3. EFFECTS OF MICROTUBULE AND ACTINS IN H/F-LV INTRACELLULAR TRAFFICKING ........ 26
IV. DISCUSSION ....................................................................................................................... 30
REFERENCES ............................................................................................................................. 35

4

LIST OF FIGURES
Figure 1. Clathrin mediated entry of H/F-LV…………………………………………...…….18
A. The effect of a panel of chemical drugs on H/F-LV transduction…………………………….18
B, C. Colocalization of H/F-LVs and clathrin or caveolin signals…………………….…………18
D. Quantification of dye-labeled H/F-LV co-localization with clathrin signals………………....18
E. Dynamin dependent clathrin pathway in H/F-LV internalization…………………………….18
Figure 2. Co-localization of H/F-LVs with either early or recycling endosomes………..…..22
A. The effect BAF on H/F-LV transduction………………………………………………..……22
B, C. Co-localization of H/F-LVs with either early or recycling endosomes…………………….22
D. Quantification of H/F-LVs co-localized with early and recycling endosomes……………….22
Figure 3. Different functional endosomes involvement and visualization of late endosomes
and H/F-LVs …………………………………………………………………………………….25
A. Visualization of late endosomes and H/F-LVs ……………………………….………………25
B. Quantification of H/F-LVs overlapping with late endosomes…………………………….….25
C. Analysis using dominant negative mutants of different endosomal proteins………….…...…25
Figure 4. H/F-LVs transport mediated by microtubule and actin network…………………27
A, B. Visualization of microtubules or actins and H/F-LVs…………………………………...…27
C. The effect of inhibitory drugs on H/F-LV transduction. …………………….……………..…27
D. The effect of α-tubulin siRNA knockdown on H/F-LV transduction…………..………..……27

5

ABSTRACT
Measles virus, enveloped RNA virus, infects lungs, airways, lymphocytes and
multiple organs, causing respiratory disease and immune suppression. Due to the
specificity of measles virus structure, recent improvements have been made on
combining lentivectors, potent gene transfer vehicles, with measles virus envelope
glycoproteins, allowing targeted cell entry. However, the detailed mechanism underlying
either measles virus or measles virus glycoprotein pseudotyping lentivectors transduction
remained unclear. In this study, we examined the transduction of lentivectors carrying
measles virus glycoprotein (H/F-LV) to Vero/hSLAM cells to understand the entry and
intracellular trafficking process. Results showed that H/F-LVs used clathrin mediated
internalization as the major entry route before trafficking through different endosomal
compartments. It was also demonstrated that microtubule network and actin filaments
played active roles for H/F-LV transduction. Collectively, our findings have shed some
light on the entry and intracellular pathway of measles virus pseudotyping lentivectors,
which may facilitate designing antiviral targets and efficient viral vector for use in gene
therapy.

6

I. INTRODUCTION
Measles is an extremely contagious and one of the most devastating infectious
diseases that still cause many childhood infections in developing countries despite the
availability of safe and effective vaccines against measles virus (MV)
1
. MV infection is
usually characterized by cough, high fever, a running nose and a maculopapular rash
which is the hallmark of measles after a latent period of ten to fourteen days
2
. In addition,
the virus can infect other organs besides lungs and airways along with rising of
complications resulting from secondary infections and neurological complications in
some rare cases
3
.
MV, the causative agent of measles, belongs to the genus Morbillivirus in the family
Paramyxoviridae
4
. It’s an enveloped virus with a nonsegmented negative-sense RNA
which encodes six tandemly linked genes, NP (nucleocapsid protein), P/V/C
(phosphoprotein/ virulence factors), M (matrix protein), F (membrane fusion protein), H
(hemagglutinin/receptor-binding protein) and L (RNA polymerase large protein)
5, 7
. The
nucleocapsid is surrounded by viral envelope membrane consisting two types of
glycoproteins: the hemagglutinin (H) and fusion (F) proteins. The H protein (H) is
responsible for virus attaching to the host-cell receptor, while the F protein (H) mediates
fusion of the viral and host cellular membranes
6
. Inside the envelope, the phosphoprotein
(P) and the large protein (L) comprised the viral RNA polymerase that associates with the
RNA genome to form the ribonucleoprotein (RNP) complex. The matrix protein (M)
helps virus assembly by linking with the cytoplasmic tails of the H, F proteins, and the
RNP complex as well
8, 9
. It is well known about the measles virus structure, but the
detailed internalization process for each step is still poorly elucidated.

7

Generally speaking, it involved many steps to for the virus trafficking from the
outside of target cell to inside to optimize the chances of replication. To begin, viruses
first bind to target cell membrane proteins, lipids or carbohydrates to interact with
specific receptors or attachment factors, followed by lateral movement of the virus-
receptor complexes
11, 12, 14
. The interactions, usually multivalent, lead to the activation of
cellular signaling pathways, resulting in the endocytic internalization of the virus
particle
13, 15
. There are different mechanisms for internalization pathways. Clathrin
mediated method is one way that many viruses are internalized into clathrin coated pits
that are quickly lost inside and the vesicles mature to endosomal compartments
16, 17
.
While for caveolin mediated pathway, the internalization of caveolae is cholesterol
dependent and much slower as compared to clathrin mediated internalization
18
. Besides
that, phagocytosis which is the engulfment of material by formation of phagosome
12, 19
,
and macropinocytosis that is primarily driven by actin are also important internalization
pathways
12, 19
. After internalization, either the viral genome alone or membrane vesicles
containing viral DNA/RNA are transported along microtubules to specific sites in the
cytoplasm or nucleus for replication and expression. During the process, the endosome
network plays indispensible role containing the early endosomes (EEs), maturing
endosomes (MEs), late endosomes (LEs), recycling endosomes (REs), and lysosomes
10
.
EEs, distributed at the peripherial cytoplasm, mature to MEs that contains both early and
late endosome markers (Rab5 and Rab7). Subsequently, MEs perform further
acidification and convert to LEs, fusing with lysosomes eventually. At last, viruses
assemble and bud either directly through the plasma membrane or through intracellular

8

membranes and leave the cell through exocytosis
12, 14
. Therefore, it is a good practice for
us to study the trafficking route step by step.
Moreover, recently new lentivector carrying the glycoprotein of measles virus at its
surface has been designed to facilitate targeted delivery using the unique characteristics
of MVs
20, 21
. Lentiviral vectors have been reported as ideal gene delivery vehicles for
research and therapeutics because of the ability to allow stable long-term transgene
expression in both dividing and non-dividing cells
26, 27
. However, the strategy has faced
many hurdles associated with needing higher titers and more targeted delivery to many
specific cell types. MV glycoproteins pseudotyping lentivectors were reported to
overcome vector restrictions in both quiescent T and B cells
29, 30, 32
. Another novel
lentiviral cell entry targeting system based on the MV glycoproteins also showed its high
selectivity retargeting EGFR and CD20 with higher titers and more targeting specificity
31
.
It was also reported that using measles virus glycoproteins displaying lentiviral vectors
performed efficient and stable transduction of resting B lymphocytes and primary chronic
lymphocyte leukemia cells and facilitated antibody production
28
. Studies involving the
intracellular trafficking ways of MV glycoproteins pseudotyping lentiviral tools may help
basic biological studies on oncogenic onset of lymphocytes and pave the way to
improved genetic vaccination strategies against cancer, infectious or autoimmune
diseases as well as genetic diseases.
In addition, the characteristics of MV can be used in many other aspects. Oncolytic
virotherapy has been reported as a less toxic and more targeted therapy that employed
specific viruses to selectively infect and destroy cancer cells
25
. Among various oncolytic
virus families, engineered attenuated MV strains were recently introduced as an oncolytic

9

platform for cancer treatment
23, 24
. The idea of using MV originated from the findings that
spontaneous hematological tumor regressed after treatment of natural MV infection
33, 34
.
Recently, attenuated MV vaccine strains also showed remarkable oncolytic efficacy and
specificity against cancers with good safety records in a variety of clinical trials. In
addition, genetic modifications of MV strains can be engineered to insert reporter and
therapeutic transgenes as well as tumor specific targeting genes
22
. The study of
intracellular trafficking of measles virus glycoprotein pseudotyping lentivectors may shed
some light for safer and more efficient design of MV based oncolytic vectors.
Therefore, the present study focused on revealing the intracellular trafficking
pathways steps for measles virus glycoprotein pseudotyping lentivectors (H/F-LVs). This
study investigated different internalization mechanisms, involvement of diverse
endosome compartments and microtubules of H/F-LVs infection to propose a more
detailed understanding for measles virus study.

10

II. MATERIALS AND METHODS
1. Cell lines and reagents
HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
(Hyclone Laboratories, Inc., Omaha, NE, USA) supplemented with 10% fetal bovine
serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 2 mM of L-glutamine (Hyclone
Laboratories, Inc., Omaha, NE, USA). Vero/hSLAM cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) (Hyclone Laboratories, Inc., Omaha, NE, USA)
containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA), 2 mM
of L-glutamine (Hyclone Laboratories, Inc., Omaha, NE) and 0.4 mg mL
-1
G418 disulfate
salt solution (Sigma-Aldrich, St. Louis, MO). G418 disulfate salt solid powder was
obtained from Sigma-Aldrich, and was dissolved in sterile water with the concentration
of 50 mg mL
-1
before filtration through a 0.22-µm filter (Corning) for instant use.
Chloropromazine (CPZ), Bafilomycin A1 (BAF), MG132, Amiloride, Nocodazol and
Filipin were purchased from Sigma-Aldrich, and used at appropriate conditions and
concentrations according to the manufacturer's protocols. Cytochalasin D (Cyto D) was
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

2. Antibodies
The mouse monoclonal antibody against EEA1 (early endosome antigen 1) and the
rabbit polyclonal antibody against Rab11 were obtained from Santa Cruz Biotechnology,
Inc.. The mouse monoclonal antibodies (MAbs) against clathrin and caveolin-1, and the
rabbit polyclonal antibody raised against CI-MPR (cation-independent mannose 6-
phosphate receptor) were purchased from Abcam (Cambridge, MA, USA). The mouse

11

monoclonal anti-α-tubulin antibody was purchased from Sigma-Aldrich. The Alexa647-
conjugated goat anti-rabbit IgG antibody, Texas red-conjugated and Alexa488-
conjugated goat anti-mouse IgG (immunoglobulin G) antibodies as well as Rhodamine-
conjugated phalloidin were obtained from Invitrogen (Carlsbad, CA, USA).

3. Plasmids
In this study, the FUGW lentiviral transfer vectors which are the third generation
HIV- based lentivectors were used as backbone vectors. Most of U3 region of the 3’ LTR
of FUGW was deleted, leading to a self-inactivating 3’ LTR, known as self-inactivating
vector (SIN). The packaging plasmids (pMDLg/pRRE and pRSV-Rev) and the envelope
plasmids in this case wild-type measles virus glycoprotein H and F genes plasmids were
also used for this study. The dominant negative form of DsRed-dynamin (DynK44A)
construct was offered by Dr. Okamoto (Department of Pharmacology and Pharmaceutical
Sciences, University of Southern California). Wild-type and dominant-negative forms of
DsRed-Rab5, DsRed-Rab7 and DsRed-Rab11 constructs were purchased from Addgene
(Cambridge, MA, USA). The α-tubulin siRNA and the related negative control siRNA
were obtained from Santa Cruz Biotechnology Inc..

4. Transfection and vector production
Measles virus glycoprotein pseudotyping lentivectors were produced by transient
transfection to HEK293T cells using calcium phosphate precipitation protocol. All
solutions needed for the calcium phosphate precipitation method were made in distilled
waster and sterilized by filtration through 0.22-µm filters (Corning). Sixteen to eighteen

12

hours prior to transfection, 2.6 × 10
6
or 18 × 10
6
HEK 293T cells were plated on 35mm
or 150mm dishes (BD Biosciences), respectively. During transfection for 35mm dish,
293T cells were transfected with 5µg of lentiviral transfer vector plasmid FUGW, along
with 2.5 µg of envelope plasmid H, F, the packaging plasmids pMDLg/pRRE and pRSV-
Rev, respectively. Four hours after transfection, the transfection mixture was removed
and the cells were washed and added with fresh media. The viral supernatants were
harvested forty-eight hours post-transfection and filtered though 0.45-µm filters
(Corning). To produce concentrated viral vectors for imaging study, the filtered viral
supernatants were ultracentrifugated at 25000 r.p.m and 4 °C for 90 minutes (Optima L-
90K Ultracentrifuge, SW-28 rotor, Beckman Coulter, Brea, CA, USA). Viral particles
were resuspended with sterilized phosphate-buffered saline (PBS) solution for use.

5. Viral transduction
Vero/hSLAM cells were seeded in a 96-well culture dish (BD Biosciences) at the
density of 0.02 × 10
6
cells per well and spin-infected with the viral supernatants or
concentrated viral particles PBS solution at 2500 r.p.m and 25 °C for 90 minutes using a
Sorvall Legend centrifuge (Newport Pagnell, England). Subsequently, the supernatants
were replaced with fresh culture media and incubated at 37 °C with 5% CO
2
for seventy-
two hours. The percentage of GFP positive cells was then detected by flow cytometry.
The transduction titer was determined by the dilution factors that exhibited a linear
response.

13

6. Quantification of lentivector genome copies by RT-PCR
Lentivector viral RNA was extracted using the ZR viral RNA kit (Zymo Research
Corporation, Irvine, CA, USA), according to the manufacturer’s provided protocol.
Quantitative RT-PCR was performed using Lenti-X qRT-PCR Titration Kit (Clontech
Laboratories Inc., Mountain View, CA, USA) according the manufacturer’s manual and
applied on the Bio-Rad MyiQ real-time system (Bio-Rad, Hercules, CA, USA).

7. Dye labeling of pseudotyping lentivector
Concentrated measles virus glycoprotein pseudotyping lentivectors were incubated
for one hour with 50nmol of Alexa488-TFP ester or Cy5 NHS ester (Invitrogen, Life
Technologies, Grand Island, NY, USA) in 0.1 M Sodium Bicarbonate buffer solution
(pH=9.3) to label the virus with organic dyes. After the incubation, the reaction was
stopped using 10mM Tris Buffer (pH= 8.0). Subsequently, a 7K MWCO Zeba desalting
spin column (Thermo Fisher Scientific) was used to remove the unbound dye molecules
by several times of buffer exchange into PBS (pH=7.4).

8. Drug treatment
Vero/hSLAM cells were trypsinized and diluted to 0.2 × 10
6
cells mL
-1
in fresh
media before incubation with CPZ (25 µg mL
-1
), filipin (5 µg mL
-1
), BAF (50nM),
Nocodazole (30 µM) and Cyto D (25 µM) at 37 °C with 5% CO
2
for 30 minutes. The
cells treated by Cyto D were centrifugated at 1200 r.p.m for 5 min after incubation and
subsequently suspended with fresh media to avoid further cytotoxicity. Then the drug-
treated Vero/hSLAM cells were seeded in a 96-well culture dish (BD Biosciences) at the

14

density of 0.02 × 10
6
cells per well and spin-infected as described before in the Viral
transduction part. The percentage of GFP positive cells was detected seventy-two hours
post-infection.

9. Transduction of siRNA, wild type or dominant-negative mutant plasmids
For viral transduction of siRNA, Vero/hSLAM cells were plated in a 24-well dish at
the density of 0.3 × 10
6
cells per well overnight at 37 °C with 5% CO
2
. The seeded cells
were transiently transfected with a negative control or α-tubulin siRNA according to the
manufacture’s guide (Santa Cruz Biotechnology Inc.). Twenty-four hour post-
transfection, the treated cells were spin-infected as describe above by the viral
supernatants. The percentage of GFP positive cells was measured by flow cytometry
seventy-two hours after infection.
For viral transduction of dominant-negative mutant plasmids, Vero/hSLAM cells
were seeded in a 24-well plate at the density of 0.5 × 10
6
cells per well sixteen to
eighteen hours prior to transfection and incubated at 37 °C with 5% CO
2
. Subsequently,
the cells were transiently transfected with either wild type or dominant-negative mutants
of Dynamin, DsRed-Rab5, DsRed-Rab7 and DsRed-Rab11 according to standard
calcium phosphate precipitation protocol. The transfection mixture was replaced with
fresh media four hours post-transfection. Twenty-four hours after transfection, the
transfected cells were spin-infected with viral supernatants at 2500 r.p.m for 90 minutes.
The GFP expression was analyzed by flow cytometry seventy-two hours post-infection.

15

10. Flow Cytometry
After infection with measles virus glycoprotein pseudotyping lentivectors,
Vero/hSLAM cells were washed and detached by trypsinization. After centrifugation, the
cells were resuspended and analyzed by FACScan (BD Biosciences). Vero/hSLAM cells
were gated to exclude cell debris based on their large side scatter (SSC) and large
forward scatter (FSC) profiles. Data analysis was conducted with FlowJo version 9.0.2
(Tree Star Inc., San Carlos, CA, USA).

11. Confocal microscopy
Vero/hSLAM cells were seeded on the poly-lysine coated glass-based dish (MatTek
Corporation, Ashland, MA, USA) at the density of 0.1 × 10
6
cells per dish and incubated
at 37 °C with 5% CO
2
for eighteen to twenty hours. After the incubation, the cells were
moved out and incubated with dye labeled measles virus pseudotyping lentivetors
(around 5000 viral genome copies per cell) at 4 °C for 30 minutes to synchronize
infection staring point. The cells were then moved to 37 °C for different incubation
durations to initiate infection before washing with PBS for two times to get rid of
unbounded viruses. Subsequently, the cells were rinsed with PBS for two times, fixed
with PBS containing 4% paraformaldehyde for ten minutes, washed again and
permeabilized with PBS containing 0.1% Triton X-100. Then the cells were washed with
PBS and incubated with a primary antibody (corresponding antibodies specific to
microtubules, actins, EEA-1, CI-MPR, Rab11, clathrin and caveolin-1) for two hours
followed by staining with a secondary antibody conjugated with Alexa Fluor 488, 594,

16

647 or Texas Red Goat anti-mouse antibody for one hour. For counterstaining of the
nuclei, the cells were treated with DAPI and incubated for fifteen minutes.
After the fluorescence staining, treated Vero/hSLAM cells were observed using a
Nikon eclipse Ti-E microscope (Nikon, Melville, NY, USA) provided with a 60 ×/1.49
Apo TIRF oil objective and an acousto-optical tunable filter (AOTF) commanded laser-
merge system was applied to offer illumination power (50mW) for 491, 561, and 640nm
lasers. In addition, the fluorescence images were acquired using a Yokogawa spinning
disk confocal scanner system (Solamere Technology Group) equipped with a Cascade II:
512 EMCCD camera (Photometrics, Tucson, AZ, USA). Data analysis was performed
using Nikon NIS-Elements software (Nikon Instrument Inc., Melville, NY, USA).

12. Statistical analysis
Assays were performed in duplicate or triplicate and replicated in separate
experiments. Data for simple pairwise comparison were analyzed using the
Student's t test. A p value of <0.05 was considered significant.

17

III. RESULTS
1. Clathrin mediated entry for H/F-LV
A variety of viruses use different entry routes to enter into the host cells. To
elucidate the entry sites for measles virus glycoprotein pseudotyping lentivector, we first
compared the effects of different membrane surface-accessible inhibitors that may block
respectively diverse entry pathways. Among all the routes, clathrin or caveolin mediated
pathways have been believed to be main category for many viruses
12
. To determine the
role of clathrin or caveolin mediated pathway in the entry of measles virus glycoprotein
pseudotyping lentivector into Vero/hSLAM cells, we pre-incubated the cells with a panel
of chemical inhibitors including Chloropromazine (CPZ), amiloride and filipin over a
time course. CPZ is a drug that is believed to disrupt clathrin pathway by dissociating
clathrin and adaptor proteins from the plasma membrane, inhibiting the formation of
clathrin coated pits and affecting receptor recycling
35
, while fillipin interferes with
caveolin mediated pathway by specifically binding to cholesterol that is a major
formation of part of caveolae
36
. In addition, amiloride affects macropinocytosis by
inhibiting Na
+
/H
+
ion exchange and membrane ruffling
37
. As depicted in Figure 1A, the
transduction of H/F-LV in CPZ (25 µg mL
-1
) pretreated Vero/hSLAM cells was
significantly decreased by about 70%, relative to control values. Also it was shown that
amiloride slightly diminished the transduction of H/F-LV to the target cells by around
35%, while no significant degree of inhibition was observed for the transduction of filipin
treated cells.
To further examine the role of clathrin or caveolin dependent pathway in the entry of
H/F-LV to Vero/hSLAM cells, we incubated the virus with corresponding dye to

18

Figure 1

Figure 1. Clathrin mediated entry of H/F-LVs. (A) The effect of a panel of chemical
drugs on H/F-LV transduction. Vero/hSLAM cells were trypsinized and pre-incubated
**"
A
0"
20"
40"
60"
80"
100"
120"
No#drug## CPZ# Filipin# Amiloride#
GFP+#cells#(rela9ve#%) *
0"
20"
40"
60"
80"
100"
120"
Dyn$WT$ Dyn$K44A$
GFP+$cells$(rela5ve$%) **"
E
Merged& H/F*LV& Clathrin&
10&min&
Merged& H/F*LV& Clathrin&
5&min&
Merged& H/F*LV& Clathrin&
15&min&
B
Merged& H/F*LV& Caveolin&
C
0"
0.2"
0.4"
0.6"
0.8"
0" 5" 10" 15"
Overlap(Coefficient Incuba3on(3me((min) D

19

with CPZ (25 µg mL
-1
), filipin (5 µg mL
-1
), Amiloride (5 mM) for 30 minutes. Then the
drug-treated Vero/hSLAM cells were seeded at the density of 0.02 × 10
6
cells per well
and spin-infected at 2500 r.p.m and 37 °C for 90 minutes. The media was replaced and
the percentage of GFP positive cells was detected and analyzed seventy-two hours post-
infection. (B, C) The cells were incubated with Alexa488-TFP ester dye labeled measles
virus pseudotyping lentivetors (green) at 4 °C for 30 minutes to synchronize infection
staring point. The cells were then moved to 37 °C for different incubation durations (5,
10 or 15 minutes) to initiate infection. Then the cells were fixed, permeabilized and
incubated with an antibody against clathrin (red, in figure 1B) and caveolin-1 (red, in
figure 1C). The boxed regions are enlarged in the right panels. Bars represent 5 µm. (D)
Quantification of dye-labeled H/F-LV co-localization with clathrin signals after 5, 10, 20-
minute of incubation. Histogram shows overlap coefficients of co-localization over time.
The overlap coefficients were represented by Mander’s overlap coefficients and analyzed
using Nikon-Elements software via analysis of more than ten images of cells at each time
point. Values are given as the mean ± SD. (E) Dynamin dependent clathrin pathway in
H/F-LV internalization. Vero/hSLAM cells were transiently transfected with either wild
type or dominant-negative mutants of Dynamin (Dyn-K44A). Twenty-four hours after
transfection, the transfected cells were spin-infected with H/F-LVs at 2500 r.p.m for 90
minutes. The GFP expression was analyzed by flow cytometry seventy-two hours post-
infection. The data are presented as the mean ± SD.

visualize the entry process. H/F-LV viruses labeled with ester-based reagents and
endocytic structures (either clathrin or caveolin) were visualized within Vero/hSLAM
cells after incubation for different periods (5, 10, 15 minutes) by confocal fluorescence
microscopy. As shown in Figure 1B, a significant colocalization of H/F-LV with the
clathrin signals was observed at the time point of 5-minute incubation. The confocal
images in Figure 1B also demonstrated significantly strong colocalization signals of the
virus and clathrin at 10 minutes after treatment and marginal signals at 15 minutes of
incubation that were assumed to diminish at a specific time point. On the clear contrast,

20

no significant overlap between H/F-LV and caveolin signals was observed at the time
point. Figure 1D also presented the quantification of viral particles co-localization with
clathrin for variant incubation time periods. It was shown that the co-localization signals
between clathrin and H/F-LV viruses peaked at 10-minute incubation time point.
Employing the same strategy previously used to identify the overlap between clathrin and
H/F-LV, it was indicated in Figure 1C that there was no significant difference for the
overlapping signals between caveolin and viruses at the corresponding time point. These
results might point to clathrin mediated internalization as one of the major mechanisms
involved in the transduction of H/F-LV to Vero/hSLAM and the lack of inhibition by
filipin suggest that there might be no involvement of caveolin in early stage viral entry.
Besides, for further confirmation of clathrin dependence and to investigate the
involvement of key proteins in the clathrin dependent pathway for measles virus
pseudotyping lentivector entry into Vero/hSLAM cells, we used dominant-negative (DN)
mutants that potently interfere with the normal functions of primary proteins of the
clathrin mediated pathway. It was reported that dynamin is specifically required for
endocytic coated vesicle formation and clathrin mediated pathway was potently inhibited
in cells overexpressing mutant dynamin because of failure to form coated pits and for
coated vesicles to bud
38
. Accordingly, Vero/hSLAM cells were transfected with either
wild type or dominant-negative mutant of dynamin (Dyn-K44A) followed by infection
with H/F-LV. As seen in Figure 1E, the transduction of H/F-LV was significantly
diminished by about 50% in cells overexpressing Dyn-K44A compared to that in cells
with the expression of wild type dynamin. Collectively, these results suggest that the
predominant route of measles virus pseudotyping lentivector entry into Vero/hSLAM

21

cells might be clathrin mediated pathway with the functional involvement of dynamin
and caveolin dependent pathway that involved the cholesterol on the cell membrane
might not be directly participated in the infection process of H/F-LVs.

2. Involvement of endosome compartments for H/F-LV infection
Upon internalization, viruses exploit different components of the cellular membrane
sorting machinery for their assembly, budding and release. It was not clear that whether
measles virus pseudotyping lentivectors are delivered to acidified endosomes where pH-
dependent fusion between the viral and the cellular membrane occurs. It was reported that
BAF at nanomolar concentrations prevents acidification of endosomes and inhibits
transport from early to late endosomes by specifically inhibiting the vacuolar proton
ATPase
45, 46, 47
. Accordingly, another drug treatment experiment was performed using
bafilomycin A1 (BAF) to investigate the role of endosomal membrane fusion. As shown
in Figure 2A, the pre-incubation with BAF significantly reduced the transduction of H/F-
LVs to Vero/hSLAM cells compared to the control group. This data might suggest that
endosomal membrane fusion might be functional involved in the H/F-LV intracellular
trafficking.
To further visualize the participation of endosomes in H/F-LVs transduction, the co-
localization experiments were performed using confocal microscopy. EEA1 (early
endosome antigen 1), CI-MPR (cation-independent mannose 6-phosphate receptor) and
Rab11 were applied as early, late and recycling endosome markers, respectively
43, 44
. As
depicted in Figure 2B, around 50% (minus the background signals) co-localization

22

signals between H/F-LV particles and EEA1 positive endosomes were detected after 30-
minute incubation. However, the acquired images showed no significant overlapping dots
Figure
2

60min&
30min&
45min&
EEA1+&
B&
EEA1+&
EEA1+&
0&
20&
40&
60&
80&
100&
120&
No#drug## BAF#
GFP+#cells#(rela4ve#%)#
#
A&
**&
60min&
0"
0.1"
0.2"
0.3"
0.4"
0.5"
0.6"
0.7"
0.8"
EEA1" Rab11"
Overlap"Coefficient"
30min"
45min"
60min"
C&
30min& 45min& Rab11+&
Rab11+&
Rab11+&
D&

23

Figure 2. Co-localization of H/F-LVs with either early or recycling endosomes. (A) The
effect BAF on H/F-LV transduction. Vero/hSLAM cells were trypsinized and pre-
incubated with BAF for 30 minutes. Then the drug-treated Vero/hSLAM cells were
seeded at the density of 0.02 × 106 cells per well and spin-infected at 2500 r.p.m and
37 °C for 90 minutes. The media was replaced and the percentage of GFP positive cells
was detected and analyzed seventy-two hours post-infection. (B, C) Vero/hSLAM cells
were incubated with H/F-LVs (green) at 4 °C for 30 minutes to synchronize infection
staring point. The cells were then moved to 37 °C for different incubation durations (30,
45 or 60 minutes) to initiate infection. Then the cells were fixed, permeabilized and
incubated with antibodies (red) against early endosome (EEA1, in Figure 2B) and
recycling endosome (Rab11, in figure 2C). The boxed regions are enlarged in the right
panels. Bars represent 5 µm. (D) Quantification of H/F-LVs co-localized with early and
recycling endosomes after 30, 45, 60-minute incubation. Histogram shows overlap
coefficients of co-localization over time. The overlap coefficients were represented by
Mander’s overlap coefficients and analyzed using Nikon-Elements software via analysis
of more than ten images of cells at each time point. Values are given as the mean ± SD.

between viral particles and either late endosomes (CI-MPR) or recycling endosomal
membranes (Rab11) 30 minutes after incubation. Subsequently, the location of H/F-LV
viral particles in Vero/hSLAM cells after 45- and 60-minute incubation was examined
using the same procedures above. Figure 2C showed that around 16% of viral particles
were observed in endosomes positive for EEA1 (early endosomes) while around 45% of
viral particles overlapped in their distributions with endosomes positive for Rab11
(recycling endosomes) after 45-minute incubation. For the 60-minute incubation point,
the analysis revealed slight decrease in overlapping between early endosomes and viral
particles compared to that of 45-minute point, but significant reduction on the contrast to
that of 30-minute incubation. In parallel experiments, the change of co-localization
signals of H/F-LV particles and Rab11 positive endosomes was not significant between

24

45-minute and 60 minute groups. The accumulation of overlapping of Rab11 positive
endosomes and H/F-LV particles, together with a similar amount of decrease of co-
localization of early endosomes (EEA1 positive) and viral particles, suggested that early
and recycling endosomes are essential for efficient trafficking of measles virus
pseudotyping lentivectors in Vero/hSLAM cells. However, no significant change of
overlapping of H/F-LV particles with CI-MPR positive endosomes was observed
comparing three incubation periods (30, 45, 60 minute) except scattering few dots in all
images acquired at all time points (Figure 3A).
To further decipher the role of late endosomes in H/F-LV trafficking, we tested the
co-localization of CI-MPR positive endosomes and viral particles after incubation of 15
minutes to determine the small overlapping signal of late endosomes and H/F-LV was
background one. As shown in Figure 3A and 3B, there was no significant difference of
co-localization signals among all four incubation time points, suggesting that most H/F-
LVs traffic might not through late endosomes.
To further confirm the confocal microscopy results, we used the dominant-negative
mutants of Rab proteins to disrupt the functional endosomal compartments. Many studies
have established that Rab proteins are distributed to different intracellular compartments
and regulate transport between organelles
42, 43
. It was also reported that distinct
compartments in the trafficking pathways contain diverse Rab GTPases on their surfaces:
Rab5 is present on early endosomes, Rab7 on late endosomes, and Rab11 on recycling
endosomes
44
. Vero/hSLAM cells were transiently transfected with either wild type or
dominant negative form of Rab5, Rab7 or Rab11 followed by infection with measles
virus glycoprotein pseudotyping virus H/F-LVs. As depicted in Figure 3C, the

25

transduction of H/F-LV to Vero/hSLAM cells overexpressing dominant negative Rab5
was significantly abolished by around 54% compared to that in cells with expression of
wild type Rab5, suggesting that the depletion of function of early endosomes may disrupt
the H/F-LV infection pathway. Similarly, expression of dominant negative Rab11
remarkably reduced the transduction of H/F-LVs to Vero/hSLAM cells by around 35%,
Figure 3

Figure 3. Different functional endosomes involvement and visualization of late
endosomes and H/F-LVs. (A) Vero/hSLAM cells were incubated with H/F-LVs (green)
at 4 °C for 30 minutes to synchronize infection and then shift to 37 °C for different
30min&
60min&
15min&
45min&
A
Merged& H/F4LV& CI4MPR& Merged& H/F4LV& CI4MPR&
Merged& H/F4LV& CI4MPR&
Merged& H/F4LV& CI4MPR&
0"
20"
40"
60"
80"
100"
120"
Rab5% Rab7% Rab11%
GFP+%cells%(rela2ve%%) WT"
DN"
C
**"
*"
0.2"
0.25"
0.3"
0.35"
0.4"
0.45"
0.5"
0.55"
15min" 30min" 45min" 60min"
Overlap%Coefficient%
Incuba2on%2me%(min)%
B

26

incubation durations (15, 30, 45 or 60 minutes) to initiate infection. Then the cells were
fixed, permeabilized and incubated with antibodies (red) against late endosome (CI-
MPR). The boxed regions are enlarged in the right panels. Scale bars represent 5 µm. (B)
Quantification of H/F-LVs co-localized with late endosomes after 15, 30, 45, 60-minute
incubation. Histogram shows overlap coefficients of co-localization over time. The
overlap coefficients were represented by Mander’s overlap coefficients and analyzed
using Nikon-Elements software via analysis of more than ten images of cells at each time
point. Values are given as the mean ± SD. (C) Analysis using dominant negative mutants
of different endosomal proteins. Vero/hSLAM cells were transiently transfected with
either wild type or dominant-negative mutants of Rab5, Rab7 or Rab11. Twenty-four
hours after transfection, the transfected cells were spin-infected with H/F-LVs at 2500
r.p.m for 90 minutes. The GFP expression was analyzed by flow cytometry seventy-two
hours post-infection. The data are presented as the mean ± SD.

as comparing to that of wild type form of Rab 11 overexpressing cells. Consistent with
the observation in the confocal imaging, however, no significant decrease was observed
for the transduction in the cells expressing dominant negative Rab7 in contrast to that in
cells with expression of wile type form of Rab7. Taken together, the early endosomes and
recycling endosomes are essential to H/F-LV viral particles trafficking while late
endosomes might not be involved in the pathway of H/F-LV infection.

3. Effects of microtubule and actins in H/F-LV intracellular trafficking
During the viruses being transported through cytosol, the cell’s own microtubule
intracellular transport machinery was employed to facilitate viral capsid’s movement to
the nucleus. Microtubules are consisted of polarized cytoskeletal filament and their
polarity is utilized to transport different membrane compartments to specific regions
39, 42
.
To investigate the role of microtubule in the migration of H/F-LVs in Vero/hSLAM cells,

27

the confocal microscopy co-localization experiment was performed to visualize the
trafficking. Microtubules were detected using MAbs specific for α-tubulin that is a major
component of microtubules. As seen in Figure 4A, the acquired image showed the
intracellular overlapping of numerous viral particles along the microtubule network. To
further clarify the overall trend, a number of antibody stained viral dots were shown in
the more zoomed pictures.
Figure
4

Figure 4. H/F-LVs transport mediated by microtubule and actin network. (A, B)
Vero/hSLAM cells were incubated with H/F-LVs (green) at 4 °C for 30 minutes to
synchronize infection and then shift to 37 °C for 60 minutes to initiate infection. Then the
cells were fixed, permeabilized, stained for microtubules (red, in Figure 4A) with anti-α-
tubulin antibody and actins (red) with rhodamine-conjugated phalloidin (red, in Figure
A
microtubule,
0,
20,
40,
60,
80,
100,
120,
No#Drug# Cyto#D# Nocodazol#
GFP+#cells#(rela7ve#%) **,
**,
C,
B"
*"
0"
20"
40"
60"
80"
100"
120"
control'siRNA' microtubule'
siRNA'
GFP+'cells'(rela7ve'%)'
D"
*"
Ac-n"filament"

28

4B). The boxed regions are enlarged in the right panels. Scale bars represent 5 µm. (C)
The effect of inhibitory drugs on H/F-LV transduction. Vero/hSLAM cells were
trypsinized and pre-incubated with Nocodazole (30 µM) or Cyto D (25 µM) for 30
minutes. Then the drug-treated Vero/hSLAM cells were seeded at the density of 0.02 ×
106 cells per well and spin-infected at 2500 r.p.m and 37 °C for 90 minutes. The media
was replaced and the percentage of GFP positive cells was detected and analyzed
seventy-two hours post-infection. Values are given as the mean ± SD. (D) The effect of
α-tubulin siRNA knockdown on H/F-LV transduction. Vero/hSLAM cells were
transiently transfected with either α-tubulin siRNA or control siRNA. Twenty-four hours
after transfection, the transfected cells were spin-infected with measles virus glycoprotein
pseudotyping lentivectors at 2500 r.p.m for 90 minutes. The GFP expression was
analyzed by flow cytometry seventy-two hours post-infection. Values are given as the
mean ± SD.

It was suggested that new actin filaments formation is involved in viral assembly
and budding and the actin cytoskeleton has been indicated to have a role in transporting
viral genomes or proteins to specific cytoplasmic sites
41
. Even though, the importance of
actin dynamics for measles virus assembly and budding steps is not known yet. To
analyze the importance of actin skeleton for H/F-LVs intracellular trafficking, confocal
imaging was performed using rhodamine-conjugated phalloidin to stain actin filaments.
As shown in Figure 4B, numerous viral particles co-localized along the actin filament.
And the intracellular overlapping of the viral particles and actin filaments was further
confirmed by altering to different regions of the imaging cells in the confocal image.
To further demonstrate the transduction dependency on microtubules and actins,
drug treatment experiment was conducted using either cytochalasin D (Cyto D) or
nocodazol. Cytochalasins D has also been reported to inhibit actin polymerization by
binding with high affinity to growing ends of actin nuclei and filaments (F-actin) and

29

preventing addition of monomers (G-actin) to these sites
40
. Moreover, nocodazole is also
used as microtubule depolymerizing agent to disrupt cell microtubules
39
. For the drug
treatment experiment, Vero/hSLAM cells were pre-treated with 30 µM of nocodazole, or
25 µM of Cyto D, or left untreated as a control before infection with H/F-LVs. The result
showed that significant reduction by about 50% for Cyto D treated group and remarkable
decrease by about 60% for nocodazole treated group in the contrast to the control one
(Figure 4C). In addition, the findings were further validated by small-interfering RNA
knocking-down experiment. Vero/hSLAM cells were transiently transfected with α-
tubulin siRNA followed by measles virus glycoprotein pseudotyping lentivectors
infections. In agreement with the results above, remarkable decrease of transduction was
observed in Vero/hSLAM cells transfected with α-tubulin siRNA compared to that cells
with control siRNA (Figure 4D). Therefore, the results of these experiments strongly
support the data derived from confocal microscopy co-localization and drug treatment,
indicating microtubule networks and actin filaments have principle roles in the infection
process of H/F-LVs.

30

IV. DISCUSSION
Measles, which can induce transient but severe immune suppression, is an airborne
disease. It evidently results from that measles virus is one of the most transmissible
pathogens
2
. And for airborne virus, efficient entry and shedding is critical for
transmission. MV has evolved many mechanisms to enter and bud from the target cells in
the most efficient way. Many efforts have been made to examine the important factors
that might improve the efficacy of MV infection, and the H and F glycoproteins have
been revealed to play most critical role among all the potential factors
8
. Although detailed
structure studies have been performed for H and F proteins, the comprehensive entry
process and distinct intracellular trafficking still persist unveiled for MV infection study.
Besides, the measles glycoprotein pseudotyping lentivectors have been reported to be
efficient genetic tools for transmission to resting T and B cells
32
. However, the entry
pathway and intracellular events for trafficking of measles glycoprotein pseudotyping
lentivectors remain largely unknown. In this study, we conducted a series of experiments
to investigate the entry and intracellular complex process of H/F-LVs, aiming to reveal
the possible biology mechanism contributing to the properties of measles glycoprotein
pseudotyping lentivectors or MV.
We have demonstrated that internalization of H/F-LVs particles from target
Vero/hSLAM cell membrane occurred rapidly via clathrin-coated pits in a dynamin-
dependent manner. This was shown by transduction reduction using inhibitory drug
treatment, by the confocal microscopy co-localization of viral particles and clathrin
positive signals, and also by the observation that transduction efficiency was largely
inhibited by overexpression of dynamin K44A mutant in Vero/hSLAM cells.

31

Interestingly, dynamin might also be involved in caveolin dependent pathway from the
plasma membrane
52
. However, rapid uptake of ligands is characteristic of clathrin-
mediated endocytosis. In contrast, caveolin mediated pathway is much slower involving
cholesterol
19
. This study showed that the uptake of H/F-LVs exhibited a rapid kinetics,
indicating that infectious entry involves clathrin-mediated pathways, while caveolae were
not involved. In addition, it was found that treatment of cells with filipin, an inhibitor of
caveola formation, did not affect H/F-LV infection. Therefore, the effect of expression of
dominant negative dynamin K44A on H/F-LV entry and infection is most likely due to
inhibition of clathrin-mediated internalization. It was known that for viruses that fused at
neutral pH at the plasma membrane virion endocytosis is not required and receptor
mediated engagement is sufficient
12
. And it was also known that binding of H protein to
the host-cell receptor triggers and activates the F protein to induce fusion between virus
and host-cell membranes
6, 8
. Therefore it was suggested MV internalization is
endocytosis independent, which is inconsistent with the clathrin-mediated endocytosis for
H/F-LV. However, it is understandable considering H/F-LVs are lentiviral vector that are
reported using endocytosis as internalization mechanism
49, 50, 59
. Moreover, CD46,
important human cell surface receptor for measles virus, was shown to be constitutively
internalized via clathrin-coated pits and traffic to multivesicular bodies, indicating the
possibility of MV using endocytosis as internalization pathway
60
.
Vectors using clathrin-mediated endocytosis have been believed to require a
subsequent reduction in pH to undergo membrane fusion in early endosomes. Fifty
nanomolar BAF inhibited H/F-LVs infection in Vero/hSLAM cells by around 50% when
added to cells prior to virus addition, confirming the functional involvement of

32

endosomes of H/F-LV intracellular trafficking. Also by using co-localization of distinct
endosomal markers positive signals and viral particles, we have demonstrated that H/F-
LVs requires both the early and recycling endosomes to efficiently infect target
Vero/hSLAM cells. This finding was further validated by the study employing dominant-
negative mutant constructs of Rab proteins. The confocal imaging experiment, performed
for different durations, also suggest that although H/F-LV viral particles rapidly enter
target cells within vesicles, the majority of particles remain within endosomal
compartments for around 60 min after uptake.
Although the detailed molecular mechanism for the intracellular trafficking events
are not yet to be elucidated, much progress has been made about the location of cellular
and viral proteins for MV infection. MV RNP complex was shown to transport through
Rab11 containing recycling endosomes using live cell imaging, which is consistent with
our study result
48
. In addition, the study about Nipah virus (NiV) also indirectly
supported our data above. NiV, also an infectious paramyxovirus, exhibited similar
properties with measles virus
56
. The co-localization study showed that NiV transported
through endosomes with EEA-1 and Rab11 positive markers (early and recycling
endosomes) and the late endosomal compartment was not needed for NiV trafficking
56
.
Generally, the early endosome is believed to serve as a cargo sorting station where
sorting events initiated and the subsequent fate was determined. It is determined by
formation and assembly of different sorting machinery whether the cargo is recycled back
to the plasma directly or via recycling endosomes, or degraded in lysosomes
51, 53
. After
that, the early endosomes may convert to late endosome. Although the through
mechanism of endosomal trafficking was still not clear, it is possible that some

33

microdomain change or conformational change triggered by H/F-LV virion fusion
affected the coordinate recruitment of the sorting machinery, resulting in different
pathways in which late endosomes might not be involved.
To be continued with the trafficking through distinct endosome membrane vesicles,
we examined the role of microtubule and actin by performing the confocal imaging study,
drug treatment using microtubule and actin inhibitory drug, and by transfection of siRNA
to knock down α-tubulin. Viruses may interact with microtubule and actin during their
replication cycle. Upon infection, virions or nucleoprotein complex are transported from
cell surface to specific site for viral transcription and replication
57
. Also in the same way,
progeny viral particles need move from the site of synthesis to the plasma membrane for
assembly and egress. But since it’s hard for large viral molecule to diffuse, they need the
help from microtubule transport machinery to penetrate layers of microfilaments
41, 55
.
This study revealed that H/F-LVs viral particles might travel along the microtubules and
inhibition by nocodazole strongly decreased the transduction of H/F-LVs. Importantly,
this drug treatment observation may, in part, be inconsistent with the previous study by
Nakatsu et al
48
. that the disruption of the microtubule by nocodazole did not remarkably
affect MV production in Vero cells. However, considering nocodazole may affect
intracellular signal transduction, the influence of nocodazole on H/F-LV infection might
be affected by subtle differences in the cell conditions. Moreover, other findings, study
by Berghall et al
58
. for example, showed similar results with our study that the infection
of MV was significantly affected due to destruction of microtubules by nocodazole.
Employing the same strategy, our data also showed the active role of actin for H/F-
LV infection and remarkable diminish in transduction efficiency by using Cyto D, a actin

34

disruptive agent. In addition, it was also reported that disruption of the actin cytoskeleton
reduced release and viral infectivity of MV
57
. However, even though actin dynamics was
reported play a role in budding of mature virions at the plasma membrane, actin
disruption may facilitate cell fusion as reported by Dietzel et al
54
. The MV-infected cells
showed an increased fusion activity after Cyto D treatment. This could be explained by
that the dependence of viruses on actin differs dramatically respect to their distinct actin
contexts with various underlying mechanisms involved different factors such as
difference of M or F proteins.
In conclusion, this study has shed some light onto the entry and intracellular
trafficking pathway for H/F-LV infection, thus improving our understanding the measles
viral pathogenesis and mechanisms that might facilitate designing antiviral targets and
efficient viral vector for use in gene therapy.

35

REFERENCES
1. Cutts FT, Lessler J, Metcalf CJE. Measles elimination: Progress, challenges and
implications for rubella control. Expert Review of Vaccines 2013 AUG;12(8):917-32.
2. Betakova T, Svetlikova D, Gocnik M. Overview of measles and mumps vaccine:
Origin, present, and future of vaccine production. Acta Virologica 2013;57(2):91-6.
3. Buchanan R, Bonthius DJ. Measles virus and associated central nervous system
sequelae. Semin Pediatr Neurol 2012 SEP;19(3):107-14.
4. Griffin DE, Lin W, Pan C. Measles virus, immune control, and persistence. FEMS
Microbiol Rev 2012 MAY;36(3):649-62.
5. Dardis MR. A review of measles. Journal of School Nursing 2012 FEB;28(1):9-12.
6. Dhiman N, Jacobson R, Poland G. Measles virus receptors: SLAM and CD46. Rev
Med Virol 2004 JUL-AUG;14(4):217-29.
7. HORIKAMI S, MOYER S. Structure, transcription, and replication of measles-virus.
Measles Virus 1995;191:35-50.
8. Navaratnarajah CK, Leonard VHJ, Cattaneo R. Measles virus glycoprotein complex
assembly, receptor attachment, and cell entry. Measles: History and Basic Biology
2009;329:59-76.
9. Rima BK, Duprex WP. The measles virus replication cycle. Measles: History and
Basic Biology 2009;329:77-102.
10. Bonifacino J, Traub L. Signals for sorting of transmembrane proteins to endosomes
and lysosomes. Annu Rev Biochem 2003;72:395-447.
11. Conner S, Schmid S. Regulated portals of entry into the cell. Nature 2003 MAR
6;422(6927):37-44.
12. Doherty GJ, McMahon HT. Mechanisms of endocytosis. Annu Rev Biochem
2009;78:857-902.
13. Maxfield F, McGraw T. Endocytic recycling. Nature Reviews Molecular Cell
Biology 2004 FEB;5(2):121-32.
14. McMahon H, Gallop J. Membrane curvature and mechanisms of dynamic cell
membrane remodelling. Nature 2005 DEC 1;438(7068):590-6.
15. Mellman I. Endocytosis and molecular sorting. Annu Rev Cell Dev Biol
1996;12:575-625.
16. Praefcke G, McMahon H. The dynamin superfamily: Universal membrane tubulation
and fission molecules? Nature Reviews Molecular Cell Biology 2004 FEB;5(2):133-
47.
17. Schmid S. Clathrin-coated vesicle formation and protein sorting: An integrated
process. Annu Rev Biochem 1997;66:511-48.
18. Nabi I, Le P. Caveolae/raft-dependent endocytosis. J Cell Biol 2003 MAY
26;161(4):673-7.

36

19. Sieczkarski S, Whittaker G. Dissecting virus entry via endocytosis. J Gen Virol 2002
JUL;83:1535-45.
20. Humbert J-, Frecha C, Bouafia FA, N'Guyen TH, Boni S, Cosset F-, Verhoeyen E,
Halary F. Measles virus glycoprotein-pseudotyped lentiviral vectors are highly
superior to vesicular stomatitis virus G pseudotypes for genetic modification of
monocyte-derived dendritic cells. J Virol 2012 MAY;86(9):5192-203.
21. Li F, Ryu BY, Krueger RL, Heldt SA, Albritton LM. Targeted entry via somatostatin
receptors using a novel modified retrovirus glycoprotein that delivers genes at levels
comparable to those of wild-type viral glycoproteins. J Virol 2012 JAN;86(1):373-81.
22. Cattaneo R. Paramyxovirus entry and targeted vectors for cancer therapy. Plos
Pathogens 2010 JUN;6(6):e1000973.
23. Peng K, Donovan K, Schneider U, Cattaneo R, Lust J, Russell S. Oncolytic measles
viruses displaying a single-chain antibody against CD38, a myeloma cell marker.
Blood 2003 APR 1;101(7):2557-62.
24. Peng K, TenEyck C, Galanis E, Kalli K, Hartmann L, Russell S. Intraperitoneal
therapy of ovarian cancer using an engineered measles virus. Cancer Res 2002 AUG
15;62(16):4656-62.
25. Patel MR, Kratzke RA. Oncolytic virus therapy for cancer: The first wave of
translational clinical trials. Translational Research 2013 APR;161(4):355-64.
26. Mercedes Segura M, Mangion M, Gaillet B, Garnier A. New developments in
lentiviral vector design, production and purification. Expert Opinion on Biological
Therapy 2013 JUL;13(7):987-1011.
27. Frecha C, Levy C, Cosset F, Verhoeyen E. Advances in the field of lentivector-based
transduction of T and B lymphocytes for gene therapy. Molecular Therapy 2010
OCT;18(10):1748-57.
28. Frecha C, Costa C, Levy C, Negre D, Russell SJ, Maisner A, Salles G, Peng K,
Cosset F, Verhoeyen E. Efficient and stable transduction of resting B lymphocytes
and primary chronic lymphocyte leukemia cells using measles virus gp displaying
lentiviral vectors. Blood 2009 OCT 8;114(15):3173-80.
29. Buchholz CJ, Muehlebach MD, Cichutek K. Lentiviral vectors with measles virus
glycoproteins - dream team for gene transfer? Trends Biotechnol 2009
MAY;27(5):259-65.
30. Funke S, Schneider IC, Glaser S, Muehlebach MD, Moritz T, Cattaneo R, Cichutek
K, Buchholz CJ. Pseudotyping lentiviral vectors with the wild-type measles virus
glycoproteins improves titer and selectivity. Gene Ther 2009 MAY;16(5):700-5.
31. Funke S, Maisner A, Muehlebach MD, Koehl U, Grez M, Cattaneo R, Cichutek K,
Buchholz CJ. Targeted cell entry of lentiviral vectors. Molecular Therapy 2008
AUG;16(8):1427-36.
32. Frecha C, Costa C, Negre D, Gauthier E, Russell SJ, Cosset F, Verhoeyen E. Stable
transduction of quiescent T cells without induction of cycle progression by a novel

37

lentiviral vector pseudotyped with measles virus glycoproteins. Blood 2008 DEC
15;112(13):4843-52.
33. Liu Y, Deisseroth A. Tumor vascular targeting therapy with viral vectors. Blood 2006
APR 15;107(8):3027-33.
34. Msaouel P, Opyrchal M, Musibay ED, Galanis E. Oncolytic measles virus strains as
novel anticancer agents. Expert Opinion on Biological Therapy 2013 APR;13(4):483-
502.
35. Callegari F, Rossini GP. Yessotoxin inhibits the complete degradation of E-cadherin.
Toxicology 2008 FEB 28;244(2-3):133-44.
36. Orlandi P, Fishman P. Filipin-dependent inhibition of cholera toxin: Evidence for
toxin internalization and activation through caveolae-like domains. J Cell
Biol 1998 MAY 18;141(4):905-15.
37. WEST M, BRETSCHER M, WATTS C. Distinct endocytotic pathways in epidermal
growth factor-stimulated human carcinoma A431 cells. J Cell Biol 1989
DEC;109(6):2731-9.
38. DAMKE H, BABA T, WARNOCK D, SCHMID S. Induction of mutant dynamin
specifically blocks endocytic coated vesicle formation. J Cell Biol 1994
NOV;127(4):915-34.
39. Kiley S, Parker P. Defective microtubule reorganization in phorbol ester-resistant
U937 variants: Reconstitution of the normal cell phenotype with nocodazole
treatment. Cell Growth & Differentiation 1997 FEB;8(2): 231-42.
40. CASELLA J, FLANAGAN M, LIN S. Cytochalasin-D inhibits actin polymerization
and induces depolymerization of actin-filaments formed during platelet shape change.
Nature 1981;293(5830):302-5.
41. Taylor MP, Koyuncu OO, Enquist LW. Subversion of the actin cytoskeleton during
viral infection. Nature Reviews Microbiology 2011 JUN;9(6):427-39.
42. Clague M. Molecular aspects of the endocytic pathway. Biochem J 1998 DEC
1;336:271-82.
43. Novick P, Zerial M. The diversity of rab proteins in vesicle transport. Curr Opin Cell
Biol 1997 AUG;9(4):496-504.
44. Zerial M, McBride H. Rab proteins as membrane organizers. Nature Reviews
Molecular Cell Biology 2001 FEB;2(2):107-17.
45. BOWMAN E, SIEBERS A, ALTENDORF K. Bafilomycins - a class of inhibitors of
membrane atpases from microorganisms, animal-cells, and plant-cells. Proc Natl
Acad Sci U S A 1988 NOV;85(21):7972-6.
46. Bayer N, Schober D, Prchla E, Murphy R, Blaas D, Fuchs R. Effect of bafilomycin
A1 and nocodazole on endocytic transport in HeLa cells: Implications for viral
uncoating and infection. J Virol 1998 DEC;72(12):9645-55.
47. VANDYKE R. Acidification of rat-liver lysosomes - quantitation and comparison
with endosomes. Am J Physiol 1993 OCT;265(4):C901-17.

38

48. Nakatsu Y, Ma X, Seki F, Suzuki T, Iwasaki M, Yanagi Y, Komase K, Takeda M.
Intracellular transport of the measles virus ribonucleoprotein complex is mediated by
Rab11A-positive recycling endosomes and drives virus release from the apical
membrane of polarized epithelial cells. J Virol 2013 APR;87(8):4683-93.
49. Lakadamyali M, Rust M, Zhuang X. Ligands for clathrin-mediated endocytosis are
differentially sorted into distinct populations of early endosomes. Cell 2006 MAR
10;124(5):997-1009.
50. Miyauchi K, Kim Y, Latinovic O, Morozov V, Melikyan GB. HIV enters cells via
endocytosis and dynamin-dependent fusion with endosomes. Cell 2009 MAY
1;137(3):433-44.
51. Amorim MJ, Kao RY, Digard P. Nucleozin targets cytoplasmic trafficking of viral
ribonucleoprotein-Rab11 complexes in influenza A virus infection. J Virol 2013
APR;87(8):4694-703.
52. Henley J, Krueger E, Oswald B, McNiven M. Dynamin-mediated internalization of
caveolae. J Cell Biol 1998 APR 6;141(1):85-99.
53. Caillet M, Janvier K, Pelchen-Matthews A, Delcroix-Genete D, Camus G, Marsh M,
Berlioz-Torrent C. Rab7A is required for efficient production of infectious HIV-1.
Plos Pathogens 2011 NOV;7(11):e1002347.
54. Dietzel E, Kolesnikova L, Maisner A. Actin filaments disruption and stabilization
affect measles virus maturation by different mechanisms. Virology Journal 2013
AUG 2;10:249.
55. Lemon K, de Vries RD, Mesman AW, McQuaid S, van Amerongen G, Yuksel S,
Ludlow M, Rennick LJ, Kuiken T, Rima BK, Geijtenbeek TBH, Osterhaus ADME,
Duprex WP, de Swart RL. Early target cells of measles virus after aerosol infection of
non-human primates. Plos Pathogens 2011 JAN;7(1):e1001263.
56. Diederich S, Sauerhering L, Weis M, Altmeppen H, Schaschke N, Reinheckel T,
Erbar S, Maisner A. Activation of the nipah virus fusion protein in MDCK cells is
mediated by cathepsin B within the endosome-recycling compartment. J Virol 2012
APR;86(7):3736-45.
57. Desfarges S, Salin B, Calmels C, Andreola ML, Parissi V, Fournier M. HIV-1
integrase trafficking in S. cerevisiae: A useful model to dissect the microtubule
network involvement of viral protein nuclear import. Yeast 2009 JAN;26(1):39-54.
58. Berghall H, Wallen C, Hyypia T, Vainionpaa R. Role of cytoskeleton components in
measles virus replication. Arch Virol 2004 MAY;149(5):891-901.
59. Marsh M, PelchenMatthews A, Hoxie J. Roles for endocytosis in lentiviral
replication. Trends Cell Biol 1997 JAN;7(1):1-4.
60. Crimeen-Irwin B, Ellis S, Christiansen D, Ludford-Menting M, Milland J, Lanteri M,
Loveland B, Gerlier D, Russell S. Ligand binding determines whether CD46 is
internalized by clathrin-coated pits or macropinocytosis. J Biol Chem 2003 NOV
21;278(47):46927-37.

Abstract (if available)

Abstract Measles virus, enveloped RNA virus, infects lungs, airways, lymphocytes and multiple organs, causing respiratory disease and immune suppression. Due to the specificity of measles virus structure, recent improvements have been made on combining lentivectors, potent gene transfer vehicles, with measles virus envelope glycoproteins, allowing targeted cell entry. However, the detailed mechanism underlying either measles virus or measles virus glycoprotein pseudotyping lentivectors transduction remained unclear. In this study, we examined the transduction of lentivectors carrying measles virus glycoprotein (H/F-LV) to Vero/hSLAM cells to understand the entry and intracellular trafficking process. Results showed that H/F-LVs used clathrin mediated internalization as the major entry route before trafficking through different endosomal compartments. It was also demonstrated that microtubule network and actin filaments played active roles for H/F-LV transduction. Collectively, our findings have shed some light on the entry and intracellular pathway of measles virus pseudotyping lentivectors, which may facilitate designing antiviral targets and efficient viral vector for use in gene therapy.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Dissecting the entry mechanism of targeting lentiviral vectors in living cells and developing quantum dot labeling of viruses for single virus tracking

PDF

Targeting Ryk-signaling to attenuate multiple sclerosis by promoting oligodendrocyte differentiation and myelination

PDF

Study of dendritic cell targeting by engineered lentivectors

PDF

Engineering lentiviral vectors for gene delivery

PDF

Asymmetric cell division during neurogenesis, and the mechanisms behind GABAergic cortical interneuron development and specification

PDF

Mixed lineage leukemia proteins (MLLs), their effect as coregulators on target gene expression and global histone methylation

PDF

Engineering viral vectors for gene and cell targeting

PDF

Wnt/β-catenin/p300 induced transcription is critical for the differentiation and maintenance of Paneth cells

PDF

Engineering lentiviral vectors for gene delivery and immunization

PDF

Pseudotyped viral vectors: HIV gene therapy applications and basic studies of SARS-COV-2

PDF

UVRAG protects cells from UV-induced DNA damage by regulating global genomic nucleotide excision repair pathway

PDF

Phosphorylation of Synaptojanin differentially regulates synaptic vesicle endocytosis of distinct vesicle pools

PDF

Characterization of the retromer complex of proteins in gastric parietal cells

PDF

Pten deletion in adult pancreatic beta-cells induces cell proliferation and G1/S cell cycle progression

PDF

Derivation and characterization of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells in clinical grade conditions

PDF

Cultured neuronal cells derived from the olfactory neuroepithelium growing in three dimensions as a model system for schizophrenia

PDF

The role of CD1d transmembrane and cytoplasmic tail domain in CD1d trafficking pathway

PDF

Neuroendocrine regulation of the transcription factor SKN-1/Nrf2 in oxidative stress response

PDF

Minibrain kinase enhances synaptojanin activity to facilitate endocytosis during synaptic activity

PDF

The relationship between DNA methylation and transcription factor binding in colon cancer cells

Asset Metadata

Creator Ji, Man (author)

Core Title Entry and intracellular trafficking of measles virus glycoprotein pseudotyping lentivector for transduction in target cells

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 12/05/2013

Defense Date 10/23/2013

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

Tag endocytosis,lentiviral vectors,measles,microtubules,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Pin (committee chair), Tokes, Zoltan A. (committee member), Ying, Qi-Long (committee member)

Creator Email mandy.manji@gmail.com,manji@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-356286

Unique identifier UC11297002

Identifier etd-JiMan-2209.pdf (filename),usctheses-c3-356286 (legacy record id)

Legacy Identifier etd-JiMan-2209-0.pdf

Dmrecord 356286

Document Type Thesis

Format application/pdf (imt)

Rights Ji, Man

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA