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Gene Therapy and Molecular Biology Vol 8, page 103
Gene Ther Mol Biol Vol 8, 103-114, 2004
Myxoma virus tropism in human tumor cells
Research Article
Joanna Sypula1, Fuan Wang1, Yiyue Ma1, John Bell2 and Grant McFadden1*
1
Robarts Research Institute and Department of Microbiology and Immunology, University of Western Ontario, London,
ON; 2Ottawa Regional Cancer Centre, Ottawa, ON
__________________________________________________________________________________
*Correspondence: Grant McFadden, PhD., BioTherapeutics Research Group, Robarts Research Institute and Department of
Microbiology and Immunology, University of Western Ontario, London, ON, N6G 2V4; Tel: 519-663-3184; Fax: 519-663-3847; e-mail:
[email protected]
Key words: Myxoma virus tropism, X-gal staining, β-galactosidase, vMyxlac and vMylacT5- replication, viral gene expression,
myxoma infection
Abbreviations: baby monkey kidney fibroblasts, (BGMK); Dulbecco’s modified Eagle medium, (DMEM); fetal bovine serum, (FBS);
newborn calf serum, (NCS); o-Nitrophenyl-b-D-Galactopyranoside, (ONGP); open reading frame, (ORF)
Received: 5 April 2004; Accepted: 15 April 2004; electronically published: April 2004
Summary
Myxoma virus is a species-specific poxvirus that causes myxomatosis in European rabbits but is nonpathogenic in
other vertebrate species, including man. We show here that myxoma virus productively infects the majority (15/21)
of human tumor cell lines tested from the NCI-60 reference collection. To assess for the potential involvement of
virus host range genes, we screened several candidate gene knockout mutants of myxoma virus for permissiveness
in these tumor cells. We observed that one particular myxoma virus variant, deleted in the ankyrin- repeat host
range gene M-T5, was uniquely defective for replication in most of the human tumor cells that were permissive for
the wild-type virus. Myxoma virus, therefore, exhibits specific tropism in a broad spectrum of human tumor cells
and thus has the potential to be exploited as a novel oncolytic virus candidate.
DNA genome that ranges in size from 130 to 300 kilobase
pairs, and encode a wide spectrum of immunomodulatory
proteins to help evade the host immune response (Seet et
al, 2003). Poxviruses replicate exclusively in the
cytoplasm of the host cell, and therefore must encode their
own transcription and replication machinery (Moss, 2001).
A number of poxviruses have been exploited as cancer
vaccine vectors, including vaccinia virus, fowlpox and
canarypox (Zeh et al, 2002; Menon et al, 2003). The
poxvirus large genome size allows for as much as 25
kilobases of contiguous DNA to be inserted, therefore
enabling the expression of large eukaryotic genes and/or
gene clusters (Kaufman, 2003; Vanderplasschen et al,
2003). This feature of poxviruses has been extensively
exploited as numerous tumor antigens have been
expressed from poxvirus vectors, including CEA
(Marshall et al, 2000) and prostate-specific antigen (Horig
et al, 2002). Poxviruses have also been used to deliver
cytokines (Kaufman et al, 2002) or co-stimulatory
molecules (Horig et al, 2000) to activate dendritic cells
and thereby increase the effectiveness of the vaccine
(Tsang et al, 2001). Human clinical trials are currently in
progress, utilizing novel and unique antigens expressed by
poxviruses to combat a wide range of cancers (Hermiston
I. Introduction
Oncolytic viruses are traditionally defined by their
capacity to selectively infect and kill cancer cells while
sparing non-transformed somatic cells (Bell et al, 2002;
Chiocca, 2002; Hawkins et al, 2002; Mullen et al, 2002;
Vile et al, 2002). Although the idea of using viruses to
treat cancer is not a new one, alternative viral vectors to
specifically target transformed cells continue to be
developed as more is learned about the specifics of
individual virus tropisms (Stojdl et al, 2003; Balachandran
et al, 2004; Tseng et al, 2004). For example, many
candidate oncolytic viruses specifically utilize host cell
signaling pathways, such as p53 or Ras, that are activated
or altered in neoplastic cells (Kirn et al, 2001; Nemunaitis
et al, 2002).
Poxviruses are among a group of viruses that have
been used to kill tumor cells. For example, certain vaccinia
virus variants have been shown to replicate to a greater
extent in transformed cells, however no poxvirus has been
described which exhibits a restrictive replication pattern
only in human tumor cells (Mastrangelo et al, 2002; Zeh et
al, 2002). Poxviruses are a family of large eukaryotic
DNA viruses that infect a wide range of vertebrates and
arthropods (Moss, 2001). They contain a double-stranded
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Sypula et al: Myxoma virus tropism in human tumor cells
et al, 2002). However, the ability of poxviruses to infect
and kill tumor cells specifically has been relatively
unexplored.
Myxoma virus is a rabbit specific virus, which
causes a lethal disease termed myxomatosis in the
European rabbit, Oryctolagus cuniculus. In the early 1950s
myxoma virus became the first example of a biological
pest control strategy, when it was used in an attempt to
control the disastrous feral rabbit population situation in
Australia (Fenner et al, 1994). To date, myxoma virus
infection of the European rabbit is one of the best models
available for the study of pathogen-host interactions, and
has allowed for the detailed investigation of viral antiimmune mechanisms as well as host immune responses
(Kerr et al, 2002). One of the notable features of myxoma
virus is its species specific ability to cause disease only in
rabbits while being nonpathogenic for all other vertebrate
species tested, including humans (Fenner et al, 1994).
Despite this extremely narrow species host range myxoma
virus can productively infect certain non-rabbit cells in
vitro, such as immortalized baby monkey kidney
fibroblasts (BGMK). Recent experiments from our lab
indicate that myxoma virus can also infect primary murine
cells genetically deficient in interferon responses
(unpublished data), which prompted us to investigate the
ability of myxoma virus to replicate in different classes of
human tumor cells. Here we report that myxoma virus can
productively infect the majority of human tumor cells
tested from a wide spectrum of tissue types. Although the
basis for this human cancer cell tropism of myxoma virus
remains to be determined, we show that one particular
viral host range gene (M-T5, an ankyrin-repeat protein
previously shown to be required for myxoma replication in
rabbit lymphocytes) is critical for virus replication in the
majority of human tumor cells tested.
II. Materials and methods
A. Cell culture and medium
Dulbecco’s modified Eagle medium, DMEM, (GibcoBRL)
was used to grow all of the cell lines. Baby green monkey kidney
cells (BGMK) were grown in DMEM supplemented with 10%
newborn calf serum (NCS) (GibcoBRL). All of the human tumor
cell lines (from the NCI-60 reference collection) were propagated
using DMEM supplemented with 10% fetal bovine serum (FBS)
(Sigma). All cell lines were grown in medium containing 100
units/mL penicillin, 100µg/mL streptomycin at 37°C in 5% CO2.
The cells used in this study are indicated in Table 1.
Table 1. Screening of Human tumor cell lines for permissiveness to infection with myxoma virus
Cell line
Cell origin
Species
BGMK
RK-13
RL5
HOS
PC3
Caki-1
HCT116
786-0
SK-OV-3
ACHN
HOP92
SK-MEL3
SK-MEL28
OVCAR4
OVCAR5
DU145
A498
T47D
Colo205
HT29
MDAMB435
M14
MCF7
SK-MEL5
Kidney
Kidney
T-Lymphocyte
Osteosarcoma
Prostate cancer
Renal cancer
Colon cancer
Renal cancer
Ovarian cancer
Renal cancer
Lung cancer
Melanoma
Melanoma
Ovarian cancer
Ovarian cancer
Prostate cancer
Renal cancer
Breast cancer
Colon cancer
Colon cancer
Breast cancer
Melanoma
Breast cancer
Melanoma
Monkey
Rabbit
Rabbit
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
104
Permissive
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Non-permissive
6
6
6
6
6
6
Gene Therapy and Molecular Biology Vol 8, page 105
E. Western blot analysis to detect early and
late viral gene expression
B. Recombinant viruses
The parental myxoma virus used in this study was
designated vMyxlac, a version of myxoma virus, strain Lausanne
(ATCC), containing the E.coli lacZ gene (under the control of the
vaccinia late p11 promoter) inserted at an innocuous site between
open reading frame (ORF) M010L and ORF M011L in the
myxoma virus genome (Opgenorth et al, 1992). Three
recombinant derivatives of myxoma virus were also used.
vMyxlacT5-, a M-T5 knockout myxoma virus, had both copies
of M-T5 replaced by lacZ (Mossman et al, 1996). Myxoma
knockout viruses vMyxlacT2- and vMyxlacM11L- were
constructed in the same manner as the vMyxlacT5-, with a lacZ
gene replacing both copies of M-T2 (Upton et al, 1991) or the
single copy of M11L (Opgenorth et al, 1992). The T5-, T2 - and
M11L- knockout viruses are all unable to replicate in rabbit T
lymphocytes (Macen et al, 1996; Mossman et al, 1996).
Western blot analysis was used to assess the expression
levels of M-T7 and Serp–1, as prototypical early and late
myxoma genes, respectively, in BGMK, HOS and 786-0 cells
following infection with vMyxlac or vMyxlacT5-. Each cell line
was infected at a moi of 5 and cells and supernatants were
collected at 2 hours and 16 hours post infection. Supernatants
were collected and concentrated 10 fold using 10K Omega
centifugal concentrators (PALL Life Sciences). Cells were
harvested by spinning at 3000rpm followed by three rounds of
freeze/thaw for cell lysis. Proteins were extracted by spinning the
lysed cells at 10000 rpm for 10 minutes and resuspending in
50µL of 1xPBS. The protein concentrations were determined by
the Bradford assay, using a spectrophotometer (Beckman
DU640). Equal amounts of protein (20µg) from cell supernatants
and cell lysates were loaded in each lane and run on a 12% SDSPAGE gel to resolve M-T7 and 10% SDS-PAGE gel for the
Serp1 blot. The separated proteins were transferred onto a
nitrocellulose membrane (Amersham Biosciences) by a semi-dry
transfer apparatus (BioRad) and probed with corresponding
antibodies according to indicated conditions. The presence of MT7 and Serp1 proteins was visualized with horseradish
peroxidase-conjugated second antibody using the enhanced
chemiluminescence detection system (Amersham Pharmacia
Biotech).
C. Infection of cell lines with myxoma virus
and X-gal staining
Cells were infected at 90-95% confluency at a multiplicity
of infection (moi) of 10, 1, 0.1, 0.01 or as otherwise indicated.
Appropriate amounts of virus corresponding to the indicated moi
was added to the cells, adsorbed for 1 hour and then the infection
allowed to proceed in DMEM with 10% FBS. Infected cells were
incubated in a CO2 incubator at 37°C for 48 hours. Cells were
stained with X-gal (100mg/mL X-gal, 500mM Kferricyanide,
500mM Kferrocyanide, 100mM MgCl2 and PBS) for 4-8 hours
after being fixed in neutral buffered formalin (NBF, [10%
formaldehyde, PBS]) for 5 minutes.
F. Micro β-galactosidase assay
Cells were seeded in 96-well plates and allowed to grow to
95% confluency, corresponding to approximately 3.3x104cells in
each well. Each cell line was infected at a moi of either 0.01 or 5.
Virus inoculum was adsorbed for 1 hour and infected cells were
transferred to a -80° freezer at 24, 48, 72 and 96 hours pi and
stored until further analysis. After the collection of all required
time points, plates were subjected to three rounds of freeze/thaw.
50µL of lysed cells containing virus from each sample (well) was
transferred into a new dish. To each sample well, 110µL of
buffer A-β-mercaptoethanol mixture pH7.5 (100mM NaH2PO4,
10mM KCL, 1mM MgSO4, 50mM β-Mercaptoethanol) was
added giving a final volume of 160µL. The components were
mixed by inversion and the plate was incubated at 37°C for 5
minutes. 50µL of ο-Nitrophenyl-β-D-Galactopyranoside
(ONGP) substrate was added to each well, the plate was covered
with a lid and incubated at 37°C. The β-galactosidase activity, as
measured by cleavage of the ONGP substrate, was read by a
plate reader at 415nm at 10 and 15 minutes after the addition of
ONGP. The results were graphed as average with corresponding
standard deviation bars.
D. Virus growth curves
For single step growth analysis, the appropriate virus at a
moi of 5 was added to a cell monolayer at 95% confluency. The
inoculum was allowed to adsorb for 1 hour, virus was removed
and each well was washed three times with 1xPBS.
Supplemented DMEM was added to the cells, which were then
incubated at 37°C. Cells were collected by scraping following
infection at the indicated time points: 1, 4, 8, 12, 24 and 48
hours. Following a 5 minute spin at 1500rpm the cells were
resuspended in 100µL of hypotonic swelling buffer. To release
virus from infected cells, each Eppendorf tube containing
infected cells was frozen at -80°C and subsequently thawed at
37°C, this freeze-thaw cycle was repeated twice more. The lysed
cells were sonicated in a cup sonicator for 1 minute to
disaggregate virus complexes and then spun at 1500rpm for 5
minutes.
For multi-step virus growth curves, cells were infected at a
moi of 0.01 and collected at the following time points: 12, 24, 48,
72 and 96 hours after infection.
Infectious virus at each time point was titrated on BGMK
cells. Virus was diluted 1:20 in DMEM supplemented with
serum and further serial dilutions were performed for each time
point of each growth curve. The appropriately diluted virus was
added to BGMK cells and allowed to adsorb for 1 hour, the virus
was removed and DMEM supplemented with serum was added
to each well. The infections were allowed to proceed for 48
hours, at which point the cells were fixed using NBF and stained
with X-gal. Blue foci, indicating virus replication and spread,
were counted and viral production per 105 cells was determined.
Titration of each time point was done in triplicate and graphed as
average with corresponding standard deviation bars.
III. Results
A. Myxoma virus has the ability to
productively infect a wide spectrum of human
tumor cells
To investigate the tropism of myxoma virus, a wide
spectrum of human tumor cell lines from the NCI-60
reference collection were screened for productive infection
with vMyxlac, wild type myxoma virus that expresses βgalactosidase. The results are summarized in Table 1 and
Figure 1. The majority of the cell lines screened (15 of
21) were permissive to infection with myxoma virus, the
exception being six cell lines that did not exhibit
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Sypula et al: Myxoma virus tropism in human tumor cells
Figure 1. Myxoma virus infects human tumor cells. Selected human tumor cells were screened for permissiveness to infection with
myxoma virus. Cells were infected with vMyxlac at indicated multiplicities of infection and infection was allowed to proceed for 48
hours. Cells were then fixed and stained with X-gal. Foci were visualized using light microscopy. See Table 1 for a list of all the cells
screened in this study.
permissivity to infection with myxoma virus and cancer
tissue origin or cell type.
significant focus formation 48 hours following infection.
A cell line was defined permissive to myxoma virus
infection when visible foci were formed, that were
comparable to those seen in the permissive control
reference cell line, BGMK cells (Figure 1 upper row). A
focus can be defined as a localized aggregate of myxoma
infected cells and indicates a productive infection. In other
words, the myxoma virus has infected a cell, efficiently
replicated and spread to neighboring cells, thereby
forming a distinct focus. Cells that were designated nonpermissive (e.g. Colo205, HT29) failed to form
discernable viral foci (Figure 1). Potential cytopathology
in the nonpermissive cells was not investigated in this
study. No direct correlation could be made between
B. M-T5 is critical for myxoma infection
To determine whether any specific virally encoded
factor might be critical for the myxoma tropism observed,
three of the known myxoma host range knockout viruses
were selected for screening. They were vMyxlacT5-,
vMyxlacT2- and vMyxlacM11L-, and the results of this
screen are summarized in Table 2. The myxoma knockout
viruses tested represent previously defined myxoma
encoded host range genes, M-T2, M-T5 and M11L that
were shown to replicate in rabbit fibroblasts or BGMK
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Gene Therapy and Molecular Biology Vol 8, page 107
Table 2. Permissiveness of selected cell lines to infection with myxoma virus
controls
supportive
abortive
restrictive
Cell line
BGMK
RK-13
RL5
HOS
PC3
Caki-1
M14
MCF7
COLO 205
HCT116
786-0
SK-OV-3
ACHN
Cell origin
Kidney
Kidney
T-Lymphocyte
Osteosarcoma
Prostate cancer
Renal cancer
Melanoma
Breast cancer
Colon cancer
Colon cancer
Renal cancer
Ovarian cancer
Renal cancer
Species
Monkey
Rabbit
Rabbit
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
VMyxLac
+
+
+
+
+
+
+
+
+
+
MT5+
+
+
+
+
-
MT2+
+
+
+
+
+
+
+
+
M11L+
+
+
+
+
+
+
+
+
+ represents positive staining for X-gal and indicates formation of foci
- represents lack of X-gal staining
cells but are non-permissive in rabbit T-cells (Macen et al,
1996; Mossman et al, 1996). Ten representative human
tumor cell lines were screened, originating from a wide
variety of tissue types, including prostate, kidney, colon,
ovarian, breast, bone and skin (Table 2). Cell
permissiveness was determined based on the presence of
detectable X-gal stained foci 48 hours pi. Cell lines where
only isolated individual blue cells were observed were
scored as non-permissive, however, it should be noted that
in these cases myxoma virus still entered the cell and
expressed β-galactosidase but was unable to spread to
neighboring cells. Upon closer analysis of the data in
Table 2, it was noted that the human tumor cells could be
operationally divided into three groups based on the
permissiveness to infection with vMyxlac and
vMyxlacT5-. The first group (designated as supportive)
was cells permissive to infection with both vMyxlac and
vMyxlacT5- and included HOS, PC3 and Caki-1 cells.
The second group (abortive) appeared to be completely
non-permissive to infection with wild type myxoma virus
as well as any of the knockout viruses and included M14,
MCF7 and COLO205 cells. The last group of human
tumor cells (called restrictive) which included HCT116,
786-0, SK-OV-3 and ACHN cells were permissive to
infection with wild type myxoma virus, however, they
were non-permissive to infection with the M-T5 knockout
virus. M-T5 was the only host range gene tested that was
shown to be required for myxoma virus infection of any
group of the human tumor cells. This observation was the
first indication of the significance of M-T5 in the host
range of myxoma virus outside the rabbit system. In
contrast to M-T5, the absence of either M-T2 or M11L had
no effect on the ability of myxoma virus to infect any of
the human tumor cells tested.
Three cell lines were chosen to best represent the
three phenotypes observed that were differentially affected
by M-T5 (Table 2). BGMK cells were utilized as a
positive control, HOS cells represented fully permissive
cell lines regardless of the presence or absence of M-T5,
and 786-0 cells represented cell lines that specifically did
not support infection with vMyxlacT5-. The results
obtained in Table 2 were expanded by visualization of
foci under a light microscope (Figure 2A) and
quantification of virus titers obtained on indicated cell
lines (Figure 2B). The virus titers of wild type myxoma
virus obtained from 786-0 and HOS cells infected with
vMyxlac were significantly lower than BGMK cells,
emphasizing the infection efficiency differences between
individual cell lines. It was also evident from the light
microscope images that the lack of M-T5 was responsible
for a decrease in focus size even in BGMK and HOS cells,
and resulted in the complete absence of foci in 786-0 cells
(Figure 2A). In 786-0 cells, the vMyxlacT5- virus titer
was undetectable, therefore confirming the inability of
vMyxlacT5- to productively infect this cell line. Thus, MT5 affects the replication efficiency in all the cells tested,
but in some cases the defect was severe enough to
completely prevent cell-cell spread of the virus.
C. M-T5 does not affect viral gene
expression following high multiplicity
infection
A standard approach to assess the nature of a block
to poxvirus replication is to quantify early or late viral
gene expression following infection. The three cell lines
were infected with vMyxlac and vMyxlacT5- at a moi of 5
and samples were collected at 2 and 16 hours pi.
Supernatants and lysates of infected cells were prepared
for western blot analysis of early or late gene expression.
To examine early gene expression, western blots were
analyzed for the expression of M-T7, a 35kDa myxoma
virus protein expressed and secreted early during
infection. In BGMK, HOS and 786-0 cells M-T7
expression began as early as 2 hours and was readily
detected at 16 hours pi with vMyxlac and vMyxlacT5infection, indicating that early viral gene expression
occurred in all three cell lines infected with both viruses
(Figure 3C). The M-T7 protein slightly differed in size
when detected in cell lysates and cell supernatants because
it is a secreted protein and undergoes glycosylation
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Sypula et al: Myxoma virus tropism in human tumor cells
Figure 2. Myxoma virus replication in BGMK, HOS and 786-0 cells. All three cell lines were infected at serial dilutions ranging from
10-2 to 10-8 of vMyxlac and vMyxlacT5-. At 48 hours post infection, cells were fixed and stained with X-gal. Foci were visualized by
light microscopy (A) and counted to determine the viral titers in each cell line (B).Virus titers were calculated as focus forming units
(ffu)/mL.
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Gene Therapy and Molecular Biology Vol 8, page 109
Figure 3. Demonstration of early and late viral gene expression following infection of three selected cell lines. Myxoma virus replication
over the period of one replication cycle was investigated using high moi infection single-step growth curves in BGMK, HOS and 786-0
cells (A). Infectious virus progeny produced during the 48 hours time course was determined by titration on BGMK cells. Early (C) and
late (D) viral gene expression following infection was investigated by resolving infected cell lysates or supernatants on SDS-PAGE gel
and staining with corresponding antibodies. Early viral gene expression was determined by the expression of M-T7 (C) at 2 and 16 hours
pi in the supernatant (lanes A) and cell lysate (lanes B). Late viral gene expression was determined by the expression of Serp-1 (D) at 16
hours pi in cell supernatants. The expression of beta-galactosidase over a 4 day timecourse following a high moi infection with vMyxlac
or vMyxlac-T5 was determined to assess the ability of the two viruses to sustain late viral gene expression replication in all three cell
lines (B).
.
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Sypula et al: Myxoma virus tropism in human tumor cells
modifications during egress through the ER and Golgi.
Nevertheless, the expression of M-T7 in all infections
indicated that both viruses underwent successful viral
binding, entry, uncoating and early gene expression in all
three cell lines. Therefore, the non-permissive phenotype
of the M-T5 knockout virus was attributed to an event
following entry and early viral gene expression.
To complete the analysis of viral gene expression,
cells infected with vMyxlac and vMyxlacT5- were
analyzed in the same manner using the expression level of
Serp1, a secreted late myxoma virus protein, as an
indicator for late viral gene expression. In all three cell
lines, Serp1 expression could be detected at 16 hours pi in
both the vMyxlac and vMyxlacT5- infected cells (Figure
3D), indicating that both viruses reached the late gene
expression stage. The lower amounts of Serp-1 secretion
from HOS cells is not indicative of grossly lower levels of
overall gene expression but may indicate a lower
efficiency of the secretory pathway in HOS cells. This
observation led to the conclusion that the block leading to
the non-permissive phenotype in vMyxlacT5- infected
786-0 cells was after late gene expression.
To quantify the amount of late viral gene expression
following infection, a micro β-galactosidase plate assay
was performed. In this particular assay the activity of βgalactosidase, an enzyme engineered to be expressed by
the virus under a late promoter, was assessed over a four
day time period. BGMK, HOS and 786-0 cells were
infected with vMyxlac and vMyxlacT5- at a moi of 0.01
(Figure 4B) or 5 (Figure 3B). The infections were
allowed to proceed for 24, 48, 72 and 96 hours and ONGP,
a substrate cleaved by the β-galactosidase enzyme, was
added. Cleavage of ONGP could be quantified by
measuring absorbance at a wavelength of 415nm. In
control BGMK cells, both vMyxlac and vMyxlacT5behaved similarly to one another. At a moi of 5, both
viruses exhibited progressively increasing β-galactosidase
activity up until at least 96 hours pi (Figure 3B).
Figure 4. The ability of myxoma virus to replicate and spread following a low moi infection of BGMK, HOS and 786-0 cells. A low moi
infection multiple step growth curve was performed in BGMK, HOS and 786-0 cells using both vMyxlac and vMyxlacT5- to investigate
the ability of both viruses to infect and spread through the cell monolayer (A). Expression of beta-galactosidase was determined in the
three cell lines infected with both viruses to quantify late viral gene expression over a 4 day time course (B).
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Gene Therapy and Molecular Biology Vol 8, page 111
At the lower moi of 0.01, the β-galactosidase activity was
significantly lower during the first 48 hours pi when
compared to a moi of 5 (Figure 4B). However, at 72 and
96 hours pi cells infected at a moi of 0.01 with both
vMyxlac and vMyxlacT5- exhibited levels of βgalactosidase comparable to the higher moi infections
(Figure 3B). In HOS and 786-0 cells, the induced βgalactosidase levels following infection with the two
viruses at both moi of 5 and 0.01 appeared to be much
lower than that observed in BGMK cells (Figure 3B,
Figure 4B). Notably, β-galactosidase levels obtained for
vMyxlacT5- infected HOS or 786-0 cells at a moi of 0.01
were extremely low, with little or no increase over the
96hour time course. This indicated that at a low moi, the
vMyxlacT5- virus appeared to be markedly defective in
both the “permissive” HOS and “nonpermissive” 786-0
cells.
D. Comparison of vMyxlac
vMylacT5- replication and spread
rounds of viral replication and cell to cell spread. All three
cell lines were infected with vMyxlac and vMyxlacT5- at
moi of 0.01. The samples were collected and harvested for
infectious virus at 12, 24, 48, 72 and 96 hours pi, then
titrated on BGMK cells. Wild-type myxoma virus
underwent several rounds of replication in all three cell
lines, evident by the progressive cell-cell spread over time
(Figure 4A). A comparison of viral titers indicated that
although all three cell lines were permissive to infection,
the amount and efficiency of virus replication was
approximately 1.5 logs higher in BGMK cells than in both
of the human tumor cell lines (Figure 4A). BGMK cells
appeared to be fully permissive to vMyxlacT5- infection
as the titers of the knockout virus were comparable to wild
type virus at 96 hours pi. HOS cells behaved in a similar
manner to BGMK cells, with vMyxlacT5- producing viral
titers approximately 1 log lower throughout the growth
curve duration. However, in the 786-0 human tumor cell
line, a dramatic difference in viral titers (approximately 2
logs) between vMyxlacT5- and vMyxlac was observed
even as late as 96 hours pi (Figure 4A), indicating a
significant defect in virus growth and/or spread in the
absence of M-T5 expression. We conclude that M-T5 is
required for optimal virus replication and generation of
infectious progeny in all the cell lines tested, but the
requirement is particularly stringent in the human tumor
cells.
and
In order to quantitatively assess the ability of both
viruses to infect and spread in each cell line, single step
and multi step growth curves were performed in BGMK,
HOS and 786-0 cells. Single step growth curves are
performed at a high moi and indicate infectious virus
progeny produced during a single replication cycle of the
virus. Cells were infected with vMyxlac and vMyxlacT5at a moi of 5 and samples were harvested for infectious
virus particles at 1, 4, 8, 12, 24 and 48 hours pi. All time
point samples were titrated on BGMK cells by serial
dilutions and stained with X-gal 48 hours pi to visualize
foci. Infection of all three cell lines with vMyxlac
produced growth curves closely resembling a classical
poxvirus replication curve, reaching a minimum at
approximately 8 hours pi followed by a continuous
increase until 48 hours pi, at which point the virus yield
had reached maximal levels (Figure 3A). Comparison of
vMyxlac and vMyxlacT5- growth curves in the three cell
lines revealed an interesting trend. Although BGMK and
HOS cells were scored as permissive to infection with the
vMyxlacT5- knockout virus, it was clear that the T5minus myxoma virus was to some degree defective in
growth in both cells. The titers obtained at all time points
were lower than for the vMyxlac titers, indicating a role
for M-T5 in optimal virus replication (Figure 3A).
Unexpectedly, the samples harvested in the nonpermissive
786-0 cells at all time points after infection with
vMyxlacT5- contained detectable infectious virus particles
that could be titrated on BGMK cells. As well, the growth
curve of vMyxlacT5- infection of 786-0 cells followed the
same classical poxvirus replication curve. Although the
titers at 48 hours pi were considerably lower in the
vMyxlacT5- infections than wild type myxoma infections
(Figure 3A), infectious virus could nevertheless still be
detected, therefore indicating some minimal level of virus
replication following high multiplicity infection of 786-0
cells with the T5- knockout virus.
Multiple step growth curves are performed at a lower
moi and for longer periods of time to measure multiple
IV. Discussion
Myxoma virus has always been regarded as a rabbit
specific poxvirus because it causes clinical disease only in
rabbits; however it is not known which virally encoded
genes or host determinants are responsible for this species
specificity (Kerr et al, 2002). All attempts to demonstrate
cell or species-specific receptors for poxviruses have been
negative, and it is now presumed that intracellular events
following binding and entry determine the tropism
characteristics of a given poxvirus (Johnston et al, 2003).
In this study we investigated myxoma virus tropism in the
context of human tumor cells. To date, there are no reports
of myxoma virus infection in primary human cells. A
general survey of a panel of human tumor cells from the
NCI reference panel was performed to determine
permissiveness to infection with wild type myxoma virus
(Table 1) as well as selected gene knockout viruses
(Table 2). The knockout viruses that were tested represent
three mechanistically distinct host range factors encoded
by myxoma virus, namely, M-T2 (a TNF receptor
homolog), M11L (a mitochondrially-located apoptosis
inhibitor) and M-T5 (an ankyrin repeat protein) (Macen et
al, 1996; Mossman et al, 1996). Of these, M-T5 appeared
to be uniquely critical for the ability of the virus to
optimally infect the human tumor cells tested (Table 2).
Three of the permissive cell lines were chosen to
facilitate an investigation of the role of M-T5: BGMK
cells, as a positive control cell line, HOS cells,
representing the fully permissive group of human tumor
cells that supported both wild-type myxoma and T5knockout infection and 786-0 cells, representing cells for
which the presence of M-T5 was essential for myxoma
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Sypula et al: Myxoma virus tropism in human tumor cells
virus infection and spread. Closer investigation of the
three cell lines demonstrated pronounced differences in the
ability of myxoma virus to productively infect and
generate progeny virus. Foci size and density differed in
all three cases, with BGMK cells forming classic myxoma
virus foci, whereas HOS and 786-0 cell foci appeared
much smaller in size (Figure 2A). ACHN and 786-0
(renal), HCT116 (colon), SK-OV-3 (ovarian) cells
infected with vMyxlacT5- produced no observable blue
foci or blue cells, indicating a complete absence of virus
replication at 48 hours pi. Analysis of viral replication and
production of infectious virions in 786-0 was performed
by conducting single and multi-step growth curves
(Figures 3A, 4A). In BGMK cells there appeared to be no
difference in virus replication levels regardless of the
presence or absence of M-T5. In the two human tumor cell
lines, however, differences between vMyxlac and
vMyxlacT5- were much more evident. For these cells, the
absence of M-T5 caused a delay in virus replication and
less infectious progeny was produced, yielding much
lower virus titers than in cells infected with vMyxlac
(Figure 3A, 4A). It was evident that in both HOS and 7860 cells, M-T5 was required for optimal myxoma virus
infection and the production of any substantial levels of
infectious progeny. However, it was still unclear at which
stage during the replication cycle the vMyxlacT5- virus
was hindered. Early and late gene expression was
monitored in BGMK, HOS and 786-0 cells using western
blot analysis, probing for M-T7 and Serp1, early and late
myxoma virus genes respectively (Figure 3C, 3D). Both
early and late gene expression appeared unaffected by the
absence of M-T5 following high multiplicity infection of
all three cell lines, leading to the conclusion that the block
in virus replication must occur at some point following
late viral gene expression. To investigate this further, a
colorimetric assay measuring the expression of the LacZ
gene, under a late promoter, was performed. This assay
allowed us to quantify the amount of late viral gene
expression that accumulated following single and multiple
cycle growth. Consistent with data obtained from the
growth curves, in BGMK cells both wild type and T5minus viruses appeared to behave the same, implicating
similar amounts of late viral gene expression. However, in
HOS and 786-0 cells the absence of M-T5 caused viral
replication to significantly decrease over multiple
replication cycles. Therefore, although late viral gene
expression appeared to be normal in the initially infected
cells when investigated at 16 hours pi following high
multiplicity infection (Figure 3D), the colorimetric assay
indicated that the absence of M-T5 indeed progressively
hindered myxoma virus late gene expression at later times
irrespective of the multiplicity. It may be that the function
of M-T5 becomes increasingly critical at a later point in
the replication cycle, during viral assembly or virus
spread, and therefore is less obvious at 2 and 16 hours pi
following high multiplicity infection. Although we
conclude that M-T5 plays a critical role during myxoma
virus infection of human tumor cells, it is also required for
optimal virus replication even in permissive BGMK cells.
At present, the known structural features of M-T5 (Figure
5) do not provide clues as to the precise mechanism of
action of this viral host range factor.
Although the molecular basis for myxoma tropism in
human tumor cells remains to be determined, the available
evidence suggests that differences in intracellular
Figure 5. Schematic representation of the location and features of M-T5.
The 483 amino acid protein is present in two copies and located within the inverted terminal repeat regions of the myxoma virus
genome. M-T5 contains 4 predicted palmitoylation sites (✳) as well as well as 7 predicted ankyrin repeats (lined bars).
The predicted ankyrin repeats are situated at amino acids 32-63, 67-104, 105-140, 141-175, 177-213, 250-282 and 283-315.
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signaling within the infected cell are critical for
distinguishing the permissive vs. restrictive phenotype
(Johnston et al, 2003). Oncolytic virus candidates have
been selected to optimally replicate within transformed
cells, and this work indicates that myxoma virus exhibits
pronounced tropism for productive replication in the
majority of human tumor cells tested. Further analysis of
the determinants at this tropism should yield insights into
the unique phenotype of human tumor cells, as well as the
ability of myxoma virus to induce cell death of
transformed cells. More importantly, the ability of
myxoma to infect human tumor cells, the lack of preexisting immunity in humans and a large genome allowing
for insertion of therapeutic genes all suggests significant
potential for the exploitation of myxoma virus as a novel
oncolytic virus platform.
Acknowledgments
We would like to thank Xuijuan Gao for technical
assistance, John Barrett for reviewing the manuscript and
Doris Hall for help with corrections. GM holds a Canada
Research Chair in Molecular Virology. The lab is
supported by the CIHR and NCI of Canada.
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From left to right, first row Dr. Grant McFadden, Dr. Joanna
Sypula
From left to right second row, Dr. Yiyue Ma, Dr. Fuan Wang
114