Note: Descriptions are shown in the official language in which they were submitted.
CA 02352439 2001-07-17
Canadian Patent Application
File: 45928.2
ENGINEERING ONCOLYTIC VIRUSES
FIELD OF THE INVENTION
The present invention relates to a method for engineering viruses to be anti-
cancer
agents.
BACKGROUND OF INVENTION
Considerable effort has recently been directed at the potential use of
viruses,
including wild-type reovirus and attenuated mutants of herpes viruses, as anti-
cancer agents
by direct lysis of cancer cells while sparing normal cells [1-3]. Attenuated
herpes simplex
viruses (HSV's) have been shown to selectively infect cancer cells while
sparing normal
cells. One such mutant of HSV-1 (designated R3616), which has the
neurovirulence gene
134.5 deleted from both loci, is effective in the treatment of experimental
brain tumors [2,
4,5]. Derivatives of this mutant (e.g. G207) are effective against some human
tumors
implanted in mice [6-10], and are currently being used in clinical trials.
Reovirus has also
been found to be effective in the treatment of neoplasms [11].
Overactivation of the proto-oncogene ras and its signaling pathway is believed
to
contribute to approximately 30% of all human tumors [12,13]. The role that Ras
plays in the
pathogenesis of human tumors is specific to the type of tumor. Activating
mutations in ras
are found in most types of human malignancies, and are highly represented in
pancreatic
cancer (80%), sporadic colorectal carcinomas (40-50%), human lung
adenocarcinomas (15-
24%), thyroid tumors (50%) and myeloid leukemia (30%) [14-16].
Double-stranded RNA-activated protein kinase (PKR) in its phosphorylated state
has
been hypothesized to lead to an inhibition of viral RNA translation and
thereby make cells
non-permissive to viral infection. Activation of ras is correlated with a
decreased level of
phosphorylated-PKR in cells that are infected with wild-type reovirus [ 11 ].
The
permissiveness of cells expressing activated ras to infection by wild-type
reovirus, is found
to be related to the decreased level of PKR-phosphorylation in these cells [
11 ].
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CA 02352439 2001-07-17
The mechanism of oncolysis using attenuated HSV-1 mutants has yet to be
elucidated. In view of the potential use of HSV as an anti-cancer therapeutic,
it is imperative
that the mechanism of HSV oncolysis be defined in relatively precise terms. By
understanding how these attenuated HSV-1 viruses mediate oncolysis, we can
transmit this
knowledge to other viruses that, as of yet, have not been shown to have
potential in anti-
cancer therapeutics.
SUMMARY OF THE INVENTION
The applicants have discovered that mammalian cells that are transformed with
oncogenes that activate the Ras signaling pathway are more permissive to HSV-1
infection than
untransformed cells, and this permissiveness is linked to the inhibition of
virus-induced
activation (phosphorylation) of double-stranded RNA-activated protein kinase
(PKR). By
inhibiting components of the Ras pathway, the applicants were able to show
that PKR
phosphorylation was restored and viral replication was inhibited. In addition
to HSV-1 and other
herpes viruses other viruses such as reovirus and influenza viruses also
utilize the Ras signaling
pathway. Cells which have an activated Ras pathway may be more permissive to
all such
viruses.
It is thus one object of this invention to provide a method for engineering
viruses to
be anti-cancer agents based upon the alteration or elimination of an inherent
viral anti-PKR
activity.
In one embodiment this method comprises the following steps, which are used
for
viruses that are not known to possess an inherent anti-PKR activity:
(A) determination of whether a virus has inherent anti-PKR activity;
(B) identification of the viral gene or genes responsible for the viral anti-
PKR
activity;
(C) alteration of the viral genes or genes responsible for the viral anti-PKR
activity,
such that a mutant virus strain with reduced or eliminated anti-PKR activity
is
created,
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CA 02352439 2001-07-17
(D) testing the mutant virus strain in culture to determine whether it
preferentially
lyses cancer cells over normal cells, and
(E) optionally, modification of the level of anti-PKR activity in the virus,
by
alteration of the viral genes or genes that are responsible for the anti-PKR
activity,
to create a mutant virus strain with a level of viral anti-PKR activity that
optimizes it's utility as an anti-cancer agent.
In another embodiment this method comprises the following steps, which are
used for
viruses that are known to possess an inherent anti-PKR activity:
(A) identification of the viral gene or genes responsible for the viral anti-
PKR
activity;
(B) alteration of the viral genes or genes responsible for the viral anti-PKR
activity,
such that a mutant virus strain with reduced or eliminated anti-PKR activity
is
created,
(C) testing the mutant virus strain in culture to determine whether it
preferentially
lyses cancer cells over normal cells, and
(D) optionally, modification of the level of anti-PKR activity in the virus,
by
alteration of the viral genes or genes that are responsible for the anti-PKR
activity,
to create a mutant virus strain with a level of viral anti-PKR activity that
optimizes it's utility as an anti-cancer agent.
In yet another embodiment this method comprises the following steps, which are
used
for viruses for which the inherent anti-PKR activity is known to result from
the activity of a
particular viral gene or genes:
(A) alteration of the viral genes or genes responsible for the viral anti-PKR
activity,
such that a mutant virus strain with reduced or eliminated anti-PKR activity
is
created,
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CA 02352439 2001-07-17
(B) testing the mutant virus strain in culture to determine whether it
preferentially
lyses cancer cells over normal cells, and
(C) optionally, modification of the level of anti-PKR activity in the virus,
by
alteration of the viral genes or genes that are responsible for the anti-PKR
activity, to create a mutant virus strain with a level of viral anti-PKR
activity that
optimizes it's utility as an anti-cancer agent.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: A comparison, by immunoflourescence, of host cell permissiveness to
HSV-1
infection of NIH-3T3 cells and NIH-3T3 cells that are transformed with various
oncogenes.
Figure 2: A comparison, by immunoblotting of viral proteins, of permissiveness
to HSV-1
infection of NIH-3T3 cells and NIH-3T3 cells that are transformed with various
oncogenes.
Figure 3: A comparison, by plaque titration, of HSV-1 virus yield from HSV-1
infected NIH-
3T3 cells and NIH-3T3 cells that are transformed with various oncogenes. The
upper panel (A)
shows the results using a MOI of 0.5 PFU/cell, whereas the lower panel (B)
shows the results
using a MOI of 5.0 PFU/cell.
Figure 4: Panel A shows a comparison, by immunoblotting of viral proteins, of
the effect of
FTI-1, an ERK pathway inhibitor (PD98059) and a p38 pathway inhibitor
(SB203580) on the
ability of HSV-1 to infect H ras transformed cell lines. Panel B shows the
effect of FTI-1 and
PD98059 on Erk42/44 phosphorylation, and the effect of SB203580 on ATF2
phosphorylation as
an activity control for these chemicals.
Figure 5: Panel A shows the effect of three different inhibitors, FPTI-1, FPTI-
2 and FTI-4 on
HSV-1 viral protein synthesis, as compared to control (H ras transformed)
cells that have not
been exposed to inhibitors. Panel B shows the effect of 100 M FTI-1 on HSV-1-
infected A549
cells (human lung carcinoma, a standard cell line for HSV growth), using
immunofluorescence.
Figure 6: HSV-1 virus yield from H ras transformed cell lines in the presence
of FTI- I in two
different concentrations, PD98059 and SB203580 in two different
concentrations.
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CA 02352439 2001-07-17
Figure 7: The left panel shows a comparison, by immunoblotting of viral
proteins, of the effect
of Wortmannin, an inhibitor of PI3-kinase on the ability of HSV-1 to infect H
ras transformed
cell lines, and the effect of Wortmannin on Akt phosphorylation. The right
panel shows the
ability of HSV-1 to infect NIH-3T3 cells that express Ras effector domain
mutants. The mutant
cell lines are V12C40 (C40), V12G37(G37) and V12S35(S35). PDCR represents NIH-
3T3 cells
which have been transfected with control vector and exert no Ras overactivity.
Figure 8: Panel A shows quantitative RT-PCR of early, middle and late HSV-1
gene expression
in HSV-1 infected NIH 3T3 cells and H ras transformed cells. Panel B shows a
comparison, by
immunoblotting, of the level of ICP27, ICP8 and gC, in HSV-1 infected NIH-3T3
cells and H
ras transformed NIH-3T3 cells.
Figure 9: Panel A shows an immunoblot comparison showing the early detection
of HSV-1
infection in THC-11 and H ras transformed cell lines as compared to A549 cell
line. Panel B
shows that infection of H-ras cells by HSV-1 can be detected using
immunofluorescence with
anti-gC antibody as early as 8 hours after the cells have been exposed to the
virus.
Figure 10: Panel A shows a comparison, by immunoblotting, of the
phosphorylation state of
PKR and eIF-2 in HSV-1 infected and uninfected cell lines (NIH-3T3 and
oncogene-
transformed NIH-3T3 cell lines). Panel B shows a comparison, by
immunoblotting, of the
phosphorylation state of PKR in HSV-1 infected and uninfected cell lines (NIH-
3T3 and
oncogene-transformed NIH-3T3 cell lines) after exposure to FTI-1 and PD98059
(left panel). A
comparison, by immunoblotting, of phosphorylation state of PKR in HSV-1
infected NIH-3T3
cells and MEF cells (right panel).
Figure 11: A comparison, by immunoblotting, of the level of viral protein
synthesis in NIH-3T3
cells and oncogene-transformed cell lines, after infection with 83616.
Figure 12: A comparison, by immunoblotting, of the level of viral protein
synthesis after
infection of PKR+~+ and PKR-~- fibroblasts with HSV-l and 83616.
Figure 13: A comparison, by immunoblotting, of the level of viral protein
synthesis after
infection of PKR+~+ and PKR-~- fibroblasts with HSV-l and exposure to FTI-1.
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CA 02352439 2001-07-17
Figure 14: Model demonstrating how the host cell and viral anti-PKR mechanisms
can influence
the infection of normal and transformed cells by wild-type and mutant HSV-1
virus.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise indicated, all terms used herein have the same meaning as is
commonly
understood by one skilled in the art of the present invention. Practitioners
are particularly
directed to Maniatis et al., in Molecular Cloning (Cold Spring Harbor, N.Y.,
Cold Spring Harbor
Laboratory), and Ausubel et al., in Current Protocols in Molecular Biology
(John Wiley and
Sons, Inc.), the contents of which are incorporated herein by reference, for
terms of the art.
As used herein the following terms have the following meanings:
"anti-cancer agent" means an agent which kills or interferes with the
viability or replication of
cancer cells. Ideally, such an agent has a reduced or no effect on normal or
non-cancerous cells.
"anti-PKR activity" includes any activity which has the consequence of
opposing, countering
or acting contrary to, the PKR system, or the effect of the PKR system. The
anti-PKR activity
may originate from a cellular or viral element and can be directed against PKR
itself, or elements
upstream or downstream of PKR, such as, for example eIF-2a.
"cancer" or "cancerous" as used herein are synonymous with tumor or tumorous
and neoplasm
or neoplastic, and includes cultured cells of cancerous, tumorous or
neoplastic tissues, in the
appropriate context.
"herpes or herpes virus" includes herpes simplex virus type 1 (HSV-1); herpes
simplex virus
type 2 (HSV-2); varicella-zoster virus (VZV); cytomegalovirus (CMV); Epstein-
Barr virus
(EBV) and various other human herpes viruses (HHV) such as HHV-6, HHV-7 and
HHV-8.
"HSV" means herpes simplex virus, and includes both type-l and type-2.
"HSV-1 infected" refers to cells that are exposed to HSV-1 virus, and does not
incorporate any
reference as to whether the cells are permissive or non-permissive to the
virus. In the
appropriate context, HSV-1 infected may mean cells that are exhibiting signs
of active infection.
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CA 02352439 2001-07-17
"oncogene-transformed cell lines" means cell lines that are transformed with
an oncogene.
"oncolytic" refers to an agent which kills cancerous cells.
"Ras pathway" includes signal transduction pathways that are downstream of
receptor tyrosine
kinases (RTK's) such as epidermal growth factor receptors or non-receptor
kinases (nRTK's)
such as the src family kinases, which can lead to the activation of Ras and
its downstream
elements. As used herein, "Ras pathway" includes other biochemical pathways
which lead or
can lead to the activation of Ras, or its upstream or downstream elements
(e.g. through activation
of any G proteins, RAL, RAP, PI3 kinase, PKC, Calcium, FAK etc.). Pathways
downstream of
Ras which are included in this definition include the MAPK cascade consisting
of Raf isoforms,
MEK 1 /2, and ERK 1 /2.
"non-permissive cells" refers to cells that do not support virus growth as
demonstrated by the
substantial lack of cytopathic effects, viral protein synthesis or virus
output after exposure to a
virus.
"permissive cells" refers to cells that support virus growth as demonstrated
by the induction of
substantial cytopathic effects, substantial viral protein synthesis or virus
output after exposure to
a virus.
As described herein, the applicants have discovered that HSV-1 exploits an
activated
tyrosine receptor kinase/Ras pathway for infection. NIH-3T3 cells, which are
non-permissive to
HSV-1 infection, become permissive when transformed by the oncogenes v-erb B,
Sos, or H
Ras. These oncogenes are all activators of the Ras signaling pathway.
Permissiveness of these
cells to HSV-1 infection is defined by the induction of cytopathic effects,
enhanced viral protein
synthesis, and/or production of progeny HSV-1 virus. The applicants have
demonstrated that
cells non-permissive to HSV-1 infection inhibit viral replication at the
protein translational level
by phosphorylated PKR. Cells permissive to HSV-1 infection have an activated
Ras pathway
that dephosphorylates or prevents the phosphorylation of PKR (or its down
stream elements like
eIF2a), which allows viral protein translation to proceed.
The applicants have shown that the farnesyl transferase inhibitors FTI-1 and
FPTI-II
effectively block HSV-1 infection in H ras transformed NIH-3T3 cells, which
otherwise is
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CA 02352439 2001-07-17
permissive to HSV-1 infection. Posttranslational farnesylation of Ras is
necessary for
association of Ras with the plasma membrane, and is known to be important for
the initiation of
downstream events, including three distinct MAPK cascades, (eg. ERK, JNK) [17-
21]. The
enzyme farnesyl transferase covalently links a farnesyl group (15 carbon
isoprenoid) to a
cysteine residue located in the carboxy terminal CAAX motif of Ras, allowing
the latter to be
anchored to the plasma membrane. Farnesyl transferase inhibitors have been
developed as
potential anti-cancer agents that block farnesylation and thus inhibit the
function of oncogenic
Ras. Without being limited to a theory, it appears that the inhibition of HSV-
1 replication by
farnesyl transferase inhibitors means that HSV-1 infection requires an
activated Ras pathway of
the host cell.
Extracellular signals received by cell surface receptors are transformed into
intracellular
instructions that coordinate the appropriate cellular responses [20]. Nearly
all cells use one or
more MAPK (mitogen-activated protein kinase) cascades to accomplish this. The
ERK pathway
(extracellular signal regulated kinase) is one such cascade, which acts
downstream of Ras to
regulate cellular growth [20-22]. Ras regulates the activity of Raf, a serine-
threonine kinase in
this pathway. Raf activates MEK1/2 [MAPK/ERK kinase], which activates ERK1/2,
one of the
latter members in a pathway that plays a role in cellular proliferation and
differentiation.
The applicants have further shown that MEK1/2 activity is required for HSV-1
infection.
PD98059 [24], an inhibitor of MEK1/2, partially blocked HSV-1 infection in H
ras transformed
NIH-3T3 cells. Combined, these results show that the ERK pathway is involved
in HSV-1
infection. The applicants have further shown that neither SB203580 [25] a
specific inhibitor of
p38 kinase, or Wortmannin [26] a specific inhibitor of PI3-kinase, had any
measurable effect on
HSV-1 infectivity.
The applicants have studied the effect of three ras effector mutant cell lines
V 1 X40, V 12637
and V12S35 [27-29] on the ability of ras to increase the infectivity of NIH-
3T3 cells to HSV-1.
All three cell lines have a common activating G12V mutation as well as one
other unique
mutation in Ras, causing them to activate distinct pathways downstream of Ras.
Mutant
V 12637 is unable to signal via the RAF/ERK and the PI3-kinase pathway, but
allows signaling
via the RAL-GDS pathway. The V 12C40 mutation disrupts signaling via the
RAF/ERK and the
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CA 02352439 2001-07-17
RAL-GDS pathways, but does not affect the PI3-kinase pathway. The V 12535
mutant cannot
signal through the RAL-GDS and the PI3-kinase pathway, but can do so via the
RAF/ERK
pathway. PDCR represents NIH-3T3 cells which have been transfected with
control vector and
exert no Ras overactivity.
Our results show that the V12S35 mutant is considerably more permissive to HSV-
1
infection than the other two mutants, suggesting a more significant role of
the RAF/ERK
pathway (as compared with the PI3-kinase or the RAL-GDS pathway) in the
infection process.
The role of the RAL/GDS pathway in HSV infection deserves further attention,
as G37 cells are
found to be susceptible to reovirus infection (not shown).
The applicants have demonstrated that non-permissiveness to HSV-1 infection in
cells is
predominantly at the level of a gene translation. The viral immediate early
transcript a27
accumulates to comparable levels in both non-permissive and permissive cell
lines. However,
the viral /3- and y-class genes, whose transcription is dependent upon the
presence of a gene
products, are much less abundant in non-permissive cells. The a27 gene
product, ICP27, is also
much less abundant in non-permissive cells than in permissive cells. Without
being limited to a
theory, it appears that the a-gene transcripts (for example: a27) are not
translated in non-
permissive cells, therefore downstream events, such as (3 and y gene
expression, do not occur,
resulting in abortive HSV-1 infection.
The mechanism of host cell non-permissiveness to viral infection is correlated
with viral-
transcript induced phosphorylation of an approximately 65 kDa cellular
protein, determined to be
a double-stranded RNA-activated protein kinase (PKR) [11]. Phosphorylated PKR
will, in turn,
phosphorylate eIF-2a, a translation initiation factor that efficiently
inhibits viral gene translation.
Permissiveness to viral infection then, is correlated to lack of
phosphorylation of PKR, which
means that eIF-2a is not phosphorylated, and therefore translation of viral
genes can proceed.
The applicants have discovered that permissiveness to HSV-1 infection is
correlated to
the lack of PKR phosphorylation in transformed cells. These cells, which are
permissive to
HSV-1, have a lower capability of inducing PKR/eIF2a phosphorylation following
viral infection
as compared with non-permissive cells. It appears that HSV-1 permissive cells
either lack the
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CA 02352439 2001-07-17
ability to phosphorylate PKR, or have an enhanced ability to dephosphorylate
PKR, or its
downstream elements, for example eIF-2 .
Attenuated mutants of HSV-1 are being tested as anti-cancer agents in clinical
trials, but
the exact mechanism of their anti-cancer effect is unknown. HSV-1 mutant 83616
[2] contains
deletions of both copies of the viral y134.5 gene. The gene product of y134.5
(called ICP34.5)
presumably forms a complex with protein phosphatase 1, and redirects its
activity to
dephosphorylate eIF-2a [30-32]. Since PKR phosphorylates eIF-2a, ICP34.5
therefore plays an
antagonistic role to PKR- i.e. it acts "anti-PKR", and mutant 83616 is a virus
that has lost its
inherent anti-PKR mechanism.
The applicants have discovered that the reason these mutant HSV-1 viruses
selectively
kill cancer cells is that elements of the Ras pathway inactivate PKR or
inhibit the
phosphorylation of PKR. If inactivated, PKR is unable to phosphorylate eIF-2a,
and viral
infection proceeds to kill the cancer cells. In normal cells, the Ras pathway
is not up-regulated
and therefore anti-PKR activity is not manifested. As a result PKR is active
and phosphorylates
eIF-2a, which blocks translation of viral transcripts thereby inhibiting viral
infection.
Therefore, HSV-1 exploits both the host cell and/or viral anti-PKR mechanism
for
infection (see Figure 14). When the viral intrinsic anti-PKR mechanism is
weakened or
destroyed, as is the case with 83616, a stronger host cell anti-PKR "arm" is
required to
compensate for this loss, such that productive infection can result. Host
cells with an activated
Ras pathway (e.g. certain cancer cells) would therefore support viral growth,
whereas normal
cells, whose Ras pathway activity does not normally reach the threshold level
to inactivate PKR,
will not be able to support viral growth. Thus, viruses whose intrinsic anti-
PKR mechanism is
rendered ineffective would be "attenuated", in that they are incapable of
infecting normal cells,
but become cytolytic in cells with a strong anti-PKR activity, as would be
found in cancer cells.
Based upon these discoveries Applicants have developed methods for creating
attenuated viruses for use in anti-cancer therapeutics.
Modification of Viruses to Create Attenuated Viruses for use in Anti-Cancer
Therapeutics
CA 02352439 2001-07-17
Based upon the observations that wild-type HSV 1 can infect cells that are
transformed
with oncogenes; that inhibitors of the Ras signaling pathway suppress HSV-1
infection; that
activation of the Ras signaling pathway dephosphorylates or prevents the
phosphorylation of
PKR; that HSV-1 mutants which have a reduced ability to suppress PKR function
can infect
PKR-deficient, but not PKR-containing cells, and that infectivity is generally
associated with a
decreased PKR or eIF2 phosphorylation, the Applicants have devised a method
for altering
viruses that have an inherent anti-PKR mechanism, such that they become
suitable for use as
anti-cancer agents.
The method of engineering viruses for use as anti-cancer agents is based upon
the theory,
which has been shown by the inventors to be true for HSV 1 that a virus which
has lost its
inherent anti-PKR activity will selectively infect neoplastic cells over
normal cells [47]. Anti-
PKR activity, which can originate from the host cell or the virus, is required
for viral infection.
If the viral anti-PKR activity is reduced or eliminated, the host cell must
compensate with a
higher level of anti-PKR activity, if viral infection is to occur. Since some
neoplastic cells, (i.e.
ras-transformed cells) have elevated anti-PKR activity, they can be infected
by a mutant virus
that lacks anti-PKR activity. On the other hand, the level of anti-PKR
activity in normal cells is
not sufficient to compensate for the loss of viral anti-PKR activity in the
mutant virus, and
infection cannot occur. The method of engineering viruses with an inherent
anti-PKR activity
for use as anti-cancer agents comprises the following steps:
(A) determination of whether a virus has inherent anti-PKR activity;
(B) identification of the viral gene or genes responsible for the viral anti-
PKR activity;
(C) alteration of the viral genes or genes responsible for the viral anti-PKR
activity, such
that a mutant virus strain with reduced or eliminated anti-PKR activity is
created,
(D)testing the mutant virus strain in culture to determine whether it
preferentially lyses
cancer cells over normal cells, and
(E) optionally, modification of the level of anti-PKR activity in the virus,
by alteration of
the viral genes or genes that are responsible for the anti-PKR activity, to
create a
CA 02352439 2001-07-17
mutant virus strain with a level of viral anti-PKR activity that optimizes
it's utility as
an anti-cancer agent.
A variety of strategies are employed by viruses in an attempt to avoid
activation of the
cellular PKR system, which would lead to the inhibition of viral protein
synthesis. This inherent
viral anti-PKR activity has been identified for example: during poliovirus
infection, where PKR
itself is degraded [33]; during influenza virus infection, where the viral NS
1 protein blocks the
binding of dsRNA to PKR [34]; during adenovirus infection, where short dsRNA
(VAI RNA) is
produced which binds to PKR but does not activate it [35]; during Vaccinia
virus infection,
where the viral K3L protein inhibits phosphorylation of eIF-2 by competing
with it for binding
to PKR [36]; and during HSV-1 infection, where ICP34.5 (a viral protein) binds
to phosphatase-
A and redirects its activity to eIF2 [38].
In accordance with step (A) of this method, viruses that are not known to have
an anti-
PKR activity can be investigated for the presence of such an activity using a
variety of different
methods known to those in the art. In a preferred method, lysates of host
cells that are infected
with the virus of interest, lysates from mock-infected host cells, and lysates
from host cells that
are treated with interferon (IFN; up to 1000U/ml for 24 hrs) are collected at
various times post
infection with the virus. Host cells that are treated with interferon provide
a source of PKR.
The preparation of the lysates is described in [37].
Lysates of mock-infected or virus-infected cells are added to an equal amount
of lysate
from IFN-treated cells. In addition, an activator of PKR (for example dsRNA)
is added in
varying amounts to different samples of mixed lysates. The level of
phosphorylation of PKR is
measured in each of these samples by using in vitro-phosphorylation/
immunoprecipitation
methods which are described in the Examples contained herein. If the virus has
an anti-PKR
mechanism, the observed level of PKR phosphorylation will be lower in virus
infected cells than
mock infected cells. If such an anti-PKR activity exists, an increase in the
amount of activator
may be required to induce the PKR phosphorylation in the samples containing
extracts from
virus-infected cells as compared with mock-infected cells (this increase can
be as much as 100-
fold). The same procedure can be conducted for detecting eIF2a-phosphorylation
as some viral
products might act at the level of eIF2a and not PKR.
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As mentioned above, some viruses are already known to have an inherent anti-
PKR
activity, and therefore step (A) of this method would be unnecessary.
Once it is known that a virus has an anti-PKR activity, the identity of the
anti-PKR
element is ascertained, in accordance with step (B) of this method. In some
instances, it may be
desirable to know the mechanism of the anti-PKR activity, as it will aid in
the identification of
the responsible gene and/or protein. For instance, if the anti-PKR mechanism
involves
competition with PKR for double stranded RNA, then the anti-PKR activity may
be purified by
affinity chromatography. Whether or not an anti-PKR activity is the result of
competition with
PKR for double-stranded RNA, should be ascertainable by determining whether
the addition of
excess double-stranded RNA to the lysates of host cells that are infected with
the virus of
interest, restores the PKR activity in these lysates.
Affinity chromatography using dsRNA has been described in [37]. To perform
affinity
chromatography, cellular lysates of virus- and mock-infected cells (as
controls) are incubated
with Poly (rI).Poly (rC)-Sepharose beads. The beads are then washed to remove
proteins that do
not specifically bind to the beads. Proteins which remain bound to the beads
throughout the
washes are released from the beads with elution buffer. By electrophoresing
the eluted proteins)
from both virus- and mock-infected cells on an acrylamide gel, proteins that
specifically bound
to the Poly (rI).Poly (rC)-Sepharose beads, and which are present in the virus-
infected, but not
the mock-infected samples are identified. Once the viral protein which
competes with PKR for
dsRNA is identified, the viral gene for this protein can be identified using
any one of a number
of techniques, such as for instance protein sequencing followed by the design
of degenerate PCR
primers to use for amplification of viral DNA. Alternatively, depending upon
how much of the
viral DNA has been sequenced already, the protein sequencing results may lead
to an immediate
identification of the viral gene that codes for the protein. The entire coding
sequence of the viral
gene can be determined using methods known to those skilled in the art. A
search in the gene
bank can also be used to find potentially similar sequences in the genome of
other viruses.
The anti-PKR mechanism may be degradation of PKR, as described in [33]. To
identify
this type of anti-PKR activity, extracts are prepared from both virus- and
mock-infected cells and
tested against [35S]methionine-labelled PKR, to determine whether the extracts
degrade PKR.
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Briefly, cells are washed with ice-cold PBS and lysed in lysis buffer. As a
source of radiolabeled
PKR, cellular extracts are prepared from HeLa cells labeled with
[35S]methionine and treated
with beta- and alpha-Interferon (IFN). The [35S]-labeled extracts are mixed
with either mock- or
virus-infected cell lysates for a short period of time. Radiolabeled PKR is
then
immunoprecipitated from the mixture by use of appropriate antibody and
subjected to SDS-
PAGE analysis to determine whether there is a difference in the level of PKR
between the
samples, or a difference in the amount of degredation of PKR between the
samples (as observed
by streaks, rather than distinct bands of protein).
In order to identify the protein that is responsible for the PKR degradation,
routine
protein purification techniques that are known to those skilled in the art can
be used and
degradation activity can be followed by assaying fractions using the in vitro
assay for PKR-
degradation. An example of the use of this technique is in [33]. For example,
virus-infected
cells can be lysed in cold lysis buffer, nuclei and membranes removed by
centrifugation and
lysates ammonium sulfate fractionated. The pellets are resuspended and, after
dialysis against
lysis buffer containing glycerol, assayed for their ability to degrade PKR as
described above.
The active fraction is then subjected to further purification procedures such
as gel-exclusion
and/or ion exchange chromatograpy and other techniques known to those skilled
in the art. At
this point, the protein responsible for the PKR degradation may be
identifiable on a SDS-
poylacrylamide gel. Once the protein is identified, the viral gene for the
protein can be identified
using any one of a number of techniques, such as for instance protein
sequencing followed by the
design of degenerate PCR primers to use for amplification of viral DNA.
Alternatively,
depending upon how much of the viral DNA has been sequenced already, the
protein sequencing
results may lead to an immediate identification of the viral gene. The entire
coding sequence of
the viral gene can be determined using methods known to those skilled in the
art.
Some viruses produce a viral protein that acts as a pseudosubstrate for PKR,
for example
by competing with eIF-2a for phosphorylation by PKR. Examples include the tat
protein of HIV
and the K3L protein of vaccinia virus. Detection of such viral
pseudosubstrates can be carried
out using in vitro kinase assay containing various combinations of dsRNA, PKR,
and the
(putative) viral protein. An example of how this method has been used is
described in [40].
14
CA 02352439 2001-07-17
Reactions containing [y-32P]ATP and PKR purified to the mono-S stage [46] can
be conducted as
described [39] in the presence of dsRNA derived from reovirus (which activates
PKR).
Substrate competition assays containing eIF2a and the putative viral
protein(s), derived from the
cellular lysates of virus-infected cells, can be carried out to determine if a
viral proteins) acts as
a pseudosubstrate, as described in [40]. For example, in addition to the
purified eIF2a in the
reaction mix, increasing amounts of the viral proteins are added. To each
tube, the kinase assay
containing activated PKR (as described above) is added, reactions are
incubated for a period of
time, and stopped by the addition of Laemmli sample buffer. Proteins are
resolved in an SDS-
polyacrylamide gel and autoradiographed. The levels of phosphorylated eIF2a
between the
samples will be compared. If increasing the amount of viral proteins causes a
decrease in eIF2a
phosphorylation and a concomitant increase in the phosphorylation of another
protein, a
competition probably exists between these two substrates for phosphorylation
by PKR. The
experiment can be modified by keeping the concentration of viral protein
constant while
increasing the concentration of eIF2a. The psuedosubstrate can then be
purified using
techniques known to those skilled in the art, however the process of
identifying the protein is
greatly aided by knowing what size it migrates to in an SDS-polyacrylamide
gel. Once the
protein is identified, the viral gene for the protein can be identified using
any one of a number of
techniques, such as for instance protein sequencing followed by the design of
degenerate PCR
primers to use for amplification of viral DNA. Alternatively, depending upon
how much of the
viral DNA has been sequenced already, the protein sequencing results may lead
to an immediate
identification of the viral anti-PKR gene. The entire coding sequence of the
viral anti-PKR gene
can be determined using methods known to those skilled in the art.
The above describes only some of the methods used by viruses as an anti-PKR
mechanism. Other anti-PKR mechanisms, including the redirection of
phosphatases against eIF-
2a, eIF-2a phosphorylation bypass (e.g. SV40 T antigen) and PKR dimerization
inhibition (e.g.,
influenza virus activated p58, and HCV NSSA), can also be used for engineering
oncolytic
versions of previously known or novel viruses [43].
The above methods describe identification of the anti-PKR protein and gene by
using its
mechanism of action as a starting point. However, it is not necessary to know
how the anti-PKR
CA 02352439 2001-07-17
mechanism works in order to purify the protein, and in some instances it may
not be possible to
determine this at this stage, in any event. Therefore, the protein responsible
for the anti-PKR
activity may be identified by using routine protein purification techniques,
known to those
skilled in the art, and described in such manuals as Maniatis et al, in
Molecular Cloning (Cold
Spring Harbor, N.Y., Cold Spring Harbor Laboratory), and Ausubel et al., in
Current Protocols
in Molecular Biology (John Wiley and Sons, Inc.). Throughout each step of the
purification, the
anti-PKR activity is tracked in the various samples and fractions by using in
vitro-
phosphorylation/immunoprecipitation methods which are described in the
Examples contained
herein. Steps of protein purification that may prove to be useful include
ammonium sulphate,
urea or other salt precipitation; ion exchange, gel exclusion or affinity
chromatography;
electrophoresis, FPLC and the like. With each step of the purification, the
protein of interest is
selectively separated from the other proteins in the cellular extract of
virally-infected cells, until
such a point is reached where sufficiently pure protein can be recovered in
order to perform
protein sequencing. Once the protein sequence is obtained, the gene sequence
can be
determined, for example by designing degenerate PCR primers to amplify viral
DNA, after
which it can be cloned and sequenced. Alternatively, depending upon how much
of the viral
DNA has been sequenced already, the protein sequencing results may lead to an
immediate
identification of the viral gene. The entire coding sequence of the viral gene
can be determined
using methods known to those skilled in the art.
Once it is determined that a virus contains an inherent anti-PKR activity, the
viral genes)
responsible for the anti-PKR activity could also be identified by genetic,
rather than protein-
based methods. This may be accomplished by screening available mutant strains
of the virus, to
determine whether they still have the anti-PKR activity that was identified in
step (A) above, or
which they were known to have. Alternatively, new viral mutant strains can be
generated and
screened for anti-PKR activity. For instance, if the anti-PKR activity of a
virus is suspected to
be the result of the activity of a particular gene, perhaps based upon
homology to another known
genes that have an anti-PKR activity, the viral gene of interest can be
mutated using any one of a
number of techniques referenced below.
It is logical to expect to find viral anti-PKR genes amongst viral genes which
are
"indispensable" for viral growth, and these genes therefore represent obvious
targets for
16
CA 02352439 2001-07-17
manipulation. However, one must view this statement with caution, as viral
genes may be
indispensable in one host cell, but not in another, according to the
biochemical properties of
these cells. For example, as Example 11 and 12 herein show, if the host cell
line has low ras
activity, such as MEF cells, mutant 83616 which has a mutation in the y, 34.5
gene would be
considered to be "indispensable", as the virus does not grow in these cells.
However, H-ras cells
are permissive to viral mutant 83616, suggesting that the y134.5 gene is
"dispensable".
Therefore it is important to use a variety of host cell lines (including
transformed cells) to
explore the oncolytic activity of mutants of indispensable genes. This
approach should also be
utilized for any genes that are altered according to the methods of this
invention, in order to
ensure that a useful viral mutant is not missed.
For the purpose of determining which gene in a virus provides the anti-PKR
activity of
that virus, the present invention provides methods for sequentially
constructing and testing
mutant viral strains for the loss of their anti-PKR activity. Mutants of viral
genes can be created
by a number of methods. These methods can disrupt the expression of a gene,
for instance by
interfering with the promoter region, or other regulatory elements. Viral
genes can also be
inactivated by insertion of a DNA sequence, such as an oligonucleotide or
reporter gene into the
viral coding region [44]. Viral genes can be altered by insertions, deletions
and/or base changes
[45]. One common method of deleting specific genes from the genomes of viruses
is described
in [46], and involves two steps:
1. Insertion of the HSV thymidine kinase (HSV-tk) gene into the genome at a
specific site
2. Deletion of the HSV-tk gene and desired sequence flanking the insertion
site.
In both steps, genomes are recombined with cloned chimeric fragments and
selected for/against
thymidine kinase to select the desired genomes.
As explained above, in the first step, the objective is to insert the HSV-tk
gene at a desired site
within the sequence of the gene to be deleted (e.g., a viral anti-PKR gene).
In order to do so, the
tk-expressing progeny virus is selected from the cells which have been
transfected with intact
DNA carrying a deletion in the tk gene and a cloned chimeric DNA fragment
consisting of tk
inserted into a desired location within another DNA fragment.
17
CA 02352439 2001-07-17
In the next step, the tk gene and the sequence flanking its insertion location
will be deleted by
transfecting cells with the intact DNA of chimeric virus produced in the first
phase and a cloned
DNA fragment containing a deletion encompassing the site of the tk gene
insertion [46,47].
Following the production of the anti-PKR mutant, the complete sequence of the
altered gene is
determined to confirm that mutation of the desired gene has been accomplished.
Laboratory manuals such as Maniatis et al., Molecular Cloning (Cold Spring
Harbor, N.Y., Cold
Spring Harbor Laboratory), and Ausubel et al., Current Protocols in Molecular
Biology (John
Wiley and Sons, Inc.), provide general methods in recombinant DNA technology
needed for this
and other procedures. A variety of kits are also available for isolation of
DNA and RNA, for
PCR, RT-PCR, transfection (calcium phosphate, lipid and polymer based methods)
and other
techniques that may be required. Kits are avialable from different sources,
including Qiagen,
Gibco-BRL, Ambion, Amersham, Stratagene, and Promega. If the target anti-PKR
gene has to
be mutated [see 47], a variety of mutagenesis tools are available from
different sources as
mentioned above.
After a viral strain with the desired mutation has been created, it is tested
for loss of the
anti-PKR activity as described above for step (A) of this process. If the anti-
PKR activity is still
present in the lysates of virus-infected cells, there are several possible
explanations, which are
listed here to be informative and not limiting. It is possible that the gene
that was mutated is not
responsible for the anti-PKR activity; that the mutation does not result in
the actual inactivation
of that gene; that the mutation does not result in actual
elimination/attenutation of the protein
activity associated with that gene; that there are other genes which
contribute to the anti-PKR
activity. Therefore, it may be necessary to make a different mutation in the
gene that is
suspected to be responsible for the anti-PKR activity, or to identify other
viral genes that have a
role in the anti-PKR activity of the virus, using the techniques outlined
above.
Once a viral mutant strain lacking, or having substantially reduced anti-PKR
activity has
been created or identified by the above methods, it is tested for its ability
to selectively infect
cancer cell lines over normal cell lines. Mutated and wild-type viral strains
are tested for
infectivity using normal, transformed or cancer cell lines. Transformed cell
lines are cell lines
that are transformed with an oncogene that is an activator of the Ras
signaling pathway, such as
18
CA 02352439 2001-07-17
v-erbB, Sos, or H ras or any other oncogene that leads to activation of the
Ras signallying
pathway. Cancer cell lines are cell lines that were originally derived from
cancer or tumor tissue.
Normal (untransformed or non-cancerous) cell lines are used as controls. The
cell line used
depends upon the cell specificity of the particular virus in question and more
than one cell line
may be used to complete this analysis. For example, in the case of vaccinia
virus and
adenovirus, a number of human cancer cell lines, including breast cancer cell
lines, colorectal
cell lines and prostate cancer cell lines can be used in this analysis.
Cell lines are usually infected with an MOI of between 0.01-100 PFU/cell.
Generally, a
range of MOTs would be tested to determine the amount of inoculum to use for
both the mutated
and wild-type virus strains in both normal and transformed or cancer cell
lines. Cells are grown
in culture medium until cytopathic effects are observed, usually up to 4 days
after infection, but
potentially longer than that. The period of time needed to establish infection
is dependent upon
the virus, the cell line and other factors, but typically infection is
established between 10-100
hours post-inoculation. Indicators of permissiveness to infection include
cytopathic effects, as
observed microscopically; viral protein production, as determined by [35S]
methionine labeling,
Western blotting or immunofluorescence; and progeny virus production, as
determined by plaque
titration. These techniques are know to those skilled in the art or described
in the Examples
herein.
If the cells being tested do not demonstrate the induction of cytopathic
effects, viral
protein synthesis or virus output after inoculation with MOTs that are usual
for that virus and
cell type, and within the normal time period for infection to be established,
then the cells will be
considered to be non-permissive to the virus. If the cells being tested
demonstrate the induction
of cytopathic effects, viral protein synthesis or virus output after
inoculation with MOTs that are
usual for that virus and cell type, and within the normal time period for
infection to be
established then the cells will be considered to be sensitive to the virus.
Mutant viral strains that lack an inherent anti-PKR activity are likely to
infect
transformed or cancer cells, but not normal cells. This is because transformed
or cancer cells
have strong anti-PKR activity and can therefore compensate for the lack of
this activity in the
viral mutants. Wild-type viral strains may infect both normal and transformed,
or cancer cell
19
CA 02352439 2001-07-17
lines, since these viruses have their own anti-PKR activity which may
compensate for the
relatively weak anti-PKR activity in normal cells.
A virus lacking anti-PKR activity, that will be useful as an anti-cancer
agent, will be
defined by one that specifically replicates in, and lyses cancer cells while
sparing normal cells.
For example, such a virus will not infect untransformed NIH-3T3 cells. Such a
virus should also
induce regression of tumors implanted in immune compromised mice wile causing
no major side
effects. Such a virus should also result in an enhanced survival rate of mice
with implanted
tumors.
Once identified as preferentially infecting transformed or cancer cell lines,
the anti-PKR
activity of the mutant virus strain may need to be modified, in order to
optimize it's utility as an
anti-cancer agent. For instance, it may be found that complete elimination of
a viral anti-PKR
activity is not as good as partial elimination of the viral anti-PKR activity,
when considering its
use as an anti-cancer agent. Additional or alternative mutations of the viral
anti-PKR genes can
be made by the techniques described above to fine-tune the level of anti-PKR
activity, or the
quality of the anti-PKR activity. For instance, mutations in the promoter or
regulatory regions of
these genes can be utilized to limit the levels of expression of the wild-type
anti-PKR gene of the
virus. Alternatively, it may desirable to alter the quality (i.e. specificity;
stability) of the anti-
PKR activity to provide the ideal anti-cancer agent.
CA 02352439 2001-07-17
EXAMPLES
Cells and viruses
Parental NIH-3T3 and NIH-3T3 cells transfected with the Harvey-ras (H ras)
oncogene
were a generous gift of Dr. Douglas Faller (Boston University School of
Medicine). NIH-3T3
cells along with their Sos-transformed counterparts (designated TNIH#5) were a
generous gift of
Dr. Michael Karin (University of California, San Diego). Dr. H.J. Kung (Case
Western Reserve
University) kindly donated parental NIH-3T3 cells along with NIH-3T3 cells
transfected with the
v-erbB oncogene (designated THC-11). All cell lines were grown in DMEM
containing 10%
FBS.
The PKR+/+ and PKR-r mouse embryo fibroblasts were obtained from Dr. B.R.G.
Williams (the Cleveland Clinic Foundation) and were grown in a-MEM containing
10% FBS
and antibiotics, as described [21 ].
Wild-type HSV-1 strain F [HSV-1(F)] and mutant HSV 83616 were both gifts from
B.
Roizman and have been described [17, 22-24].
Immunoflourescent analysis of HSV-1 infection
NIH-3T3, TNIH#5, THC-11, and H-ras cells were grown in 8-well slide chambers
(Falcon) and infected with HSV-1 at a MOI of 0.5 PFU/cell, or mock-infected by
application of
PBS to the cells in an identical fashion as the administration of virus to the
cells. At 20 hours
after infection, the cells were fixed in 100% acetone for 10 min and then left
at room temperature
to dry. The fixed and dried cells were then incubated with a fluorescein-
labeled mouse
monoclonal antibody to HSV-1 gC antigen (SyvaMicrotak from Behring) for 30 min
at 37°C.
The slides were then washed with distilled water, dried, mounted in 90%
glycerol containing
0.1 % phenylenediamine, and viewed with a Zeiss Axiophot microscope on which a
Carl Zeiss
camera was mounted. The magnification for all pictures was 200X.
Western blot (Immunoblot) analysis
21
CA 02352439 2001-07-17
Infected cells were lysed with the single detergent lysis buffer [50 mM Tris
(pH 8.0), 150
mM NaCI, 0.02% sodium azide, 100 p,g/ml phenylmethy-sulfonyl fluoride, 1 pg/ml
aprotinin,
and 1% Triton X-100], normalized for the amount of total protein and subjected
to SDS-PAGE,
followed by electroblotting onto nitrocellulose paper. The membrane was then
washed and
incubated with the primary antibody [rabbit antibody against all HSV-1
antigens (Dako, CA;
1:20,000); mouse anti-ICP27 or anti-gC antibody (Rumbaugh-Goodwin Institute,
FL; 1:1,000);
rabbit anti-ICP8 antibody (from Dr. Paul Olivo, Washington University, St.
Louis; 1:1,000);
mouse anti-PKR and mouse anti-eIF-2 antibody (Santa Cruz; 1:1,000); rabbit
anti-phospho-
PKR and rabbit anti-phospho-eIF-2 antibody (Biosource, CA; 1:1,000) followed
by HRP-
conjugated secondary antibody (1:2,000). After extensive washing, the blot was
exposed to
Lumigel detection solution (New England Biolabs) and subjected to
autoradiography.
Uninfected H-ras cells were used to demonstrate the effects of FTI-1, PD98059
on
ERKl/2 phosphorylation, SB203580 on ATF-2 phosphorylation, and Wortmannin on
Akt
phosphorylation. Briefly, subconfluent monolayer cultures were lysed with the
recommended
SDS-containing sample buffer, the lysate was subjected to SDS-PAGE and
electroblotted onto
nitrocellulose paper. Blots were probed with anti-ERK1/2 or anti-phospho-
ERKl/2 antibodies
(for samples treated with FTI-1 or PD98059), anti-ATF-2 or anti-phospho-ATF-2
(for samples
treated with SB203580), and anti-Akt and anti-phospho-Akt (for samples treated
with
Wortmannin). The antibody kits were purchased from New England Biolabs (MA).
FTI-l,
PD98059, SB203580 and Wortmannin were purchased from Calbiochem (CA).
Polymerase chain reaction
Cytoplasmic RNA from infected cells was isolated using the RNeasy kit by
Quiagen
(CA). Briefly, at various times post-infection, monolayers of cells
(approximately 1 x 10' cells)
were lysed using the RLN buffer (50 mM Tris-Cl pH 8.0, 140 mM NaCI, 1.5 mM
MgCl2, 0.5%
NP-40, 1 mM DTT and 1000 U/ml RNasin). After removal of the nuclei by
centrifugation, the
supernatant was mixed with buffer RLT and ethanol, and applied to an RNeasy
spin column.
The column was subsequently washed and RNA was eluted in water. Equal amounts
of
cytoplasmic RNA from each sample were then subjected to RT-PCR using random
hexanucleotide primers (Pharmacia) and reverse transcriptase (GIBCO-BRL)
according to the
22
CA 02352439 2002-10-08
manufacturers' protocol. The cDNAs from the Krf-PCR step was then subjected to
selective
amplification of cDNAs of a27, U,,29, U~.30, y,34.5, and U~_44. For a27 the
primers 5'-
CTGGAATCGGACAGCAGCCGG-3' [SEQ ID # I ] and
5'-GAGGCGCGACCACACACTGT-3' [SL;Q ID #2] were used, which produced the
predicted 222 by fragment. For U~2 the primers used were S'-GCGCCCCATGGTCGTGTT-
3' [SEQ ID #3] and 5'-CTCCGCCGCCGAGGTTC-3' [S1:Q ID #4], which produced the
predicted 206 by fragment. For U~.30, the primers S'-ATCAACTTCGACTGGCCCTTC-3'
[SEQ ID #S] and S'-CCGTACATGTCGATGTTCACC-3' [SEQ ID #6] were used, which
produced the predicted 180 by fragment. For y, 34. S, the primers used were ~'-
CTCGGAGGGCGGGACTGG-3'[SEQ ID #7] and S'-GCGGGAGGCGGGGAATAC-3'
[SEQ ID #8], which produced a predicted 282 by Fragment. For U, 44, the
primers used were
5'-GCCGCCGCCTACTACCC-3' [ SEQ ID #9 ] and S'-GCTGCCGCGACTGTGATG-3'
[SEQ ID #10], which produced a predicted 661 by fragment. As a PCR and gel
loading
control, GAPDH primer S'-CGGAGTCAACGGATTTGGTCGAT-3' [SEQ ID # I 1 ] and ~'-
AGCCTTCTCCATGGTGGTGAAGAC-3' [SEQ ID # 12] were used to amplify a predicted
306 by GAPDH fragment. Selective amplification of the various cDNAs was
performed
using HotStarTaq DNA polymerise (Quiagen) in a MiniCycler PTC-1 SO (MJ-
Research).
PCR was carried out for 30 cycles, with each cycle consisting of a denaturing
step for 1 min
at 94°C, an annealing step for 2 min at 60"C, and a polymerization step
for 2 min at 72"C.
The PCR products were separated on a 1.S% agarose gel impregnated with
ethidium bromide,
and photographed under UV illumination with Polaroid S7 film.
EXAMPLE 1: Activators of the Ras Pathway Augment HSV-1 Infection Efficiency
NIH-3T3 cells are known to be poorly infectible with HSV-1. It was of
particular
interest to determine whether NIH-3T3 cells that were transformed with
oncogenes which are
activators of the Ras pathway were equally as non-permissive to HSV infection.
Monolayers of NIH-3T3 cells, v-erhB- (THC.'-11), Sos- (TNIH#S), or H-ras-
transformed NIH-3T3 cells were exposed to HSV-1 (strain F) at a MOI of O.S PFU
per cell.
Cells were photographed 20 hours after infection in order to determine the
cytopathic effects
of the virus on these cells. Cells were then fixed, processed and reacted with
a FITC-labeled
mouse anti-HSV-1 gC antibody. As shown in Figure 1, little or no morphological
change
could be detected in NIH-3T3 cells, which exhibited a typically flattened,
spread out
morphology with marked contact
23
CA 02352439 2001-07-17
inhibition. In contrast, cells transformed with v-erbB, Sos or H ras exhibited
rounding or
clumping, which are characteristic cytopathic effects of HSV-1 infection.
In order to determine whether viral proteins were being synthesized by the HSV-
1
infected, oncogene-transformed cells, immunofluorescent microscopy of these
cells was
performed as described above. As shown in Figure 1, the results show that
virus proteins were
detected only in a very small population of NIH-3T3 cells, whereas in oncogene-
transformed cell
lines, pronounced viral protein synthesis was observed. Scale bar, 150 Vim.
The amount of HSV-1 protein synthesis in these cell lines was also determined
by
Western Blot analysis. Cells were infected as described above, or mock-
infected, and were
harvested at 10, 22 or 36 hours after infection (or mock-infection). Western
blot analysis was
performed on these samples using rabbit polyclonal antibody against all HSV-1
antigens, as
described above, and the results are shown in Figure 2. Lanes 13-16 show
uninfected NIH-3T3,
TNIH#5, H-ras, and THC-11 cells, respectively. As can be seen in Figure 2,
viral proteins were
not present in NIH-3T3 cells, but were abundant in the oncogene-transformed
NIH-3T3 cells.
As all these cell lines have identical doubling times, the observed
differences in the level of viral
protein synthesis were not due to intrinsic differences in growth rates or
translational efficiencies
for these cell lines.
Similar results to the above were obtained by metabolic labeling with [35S]-
methionine.
Briefly, at 12 hours post infection, the medium was replaced with methionine-
free medium
containing 0.1 mCi/ml [35S]-methionine. After further incubation for 36 hours,
the cells were
washed in PBS and lysed in PBS containing 1% Triton X-100, 0.5% sodium
deoxycholate and 1
mM EDTA. The nuclei were removed by low speed centrifugation and the
supernatants stored at
-70°C. Aliquots (normalized for protein content) were electrophoresed
through SDS-
polyacrylamide gels and autoradiographed. The autoradiographs demonstrated
that viral proteins
were not present in NIH-3T3 cells but were abundant in the transformed NIH-3T3
cells.
The doubling times for uninfected NIH-3T3 and uninfected transformed cells are
identical and they show very similar patterns and levels of cellular protein
synthesis. Therefore,
the observed differences in the level of viral protein synthesis could not be
due to intrinsic
differences in growth rates or translational efficiencies for these cell
lines. This was further
24
CA 02352439 2001-07-17
supported by the observation that NIH-3T3 cells could not be rendered more
permissive even
when infections were carried out at a lower cell density.
To determine whether the HSV-1 infected, oncogene-transformed NIH-3T3 cells
were
actually producing intact HSV-1 virus particles, HSV-I virus yield from these
cells was
determined by plaque titration on Vero cells. Briefly, Vero cells were grown
in 6-well multi-
well plates. Different dilutions of virus, from 10-1 to 10-6 are applied to
each well and the cells
are incubated for about 1.5 to 2 hours. Agar-DMEM ( 10%) was overlaid on the
cells, which
were then incubated for about 2 to 3 days, or until cytopathic effects are
observed. The wells
were then stained with neutral red. Areas containing plaques remained clear,
whereas viable
cells stained dark red. As shown in Figure 3, THC-I1, H-ras and TNIH#5 cells
produced
significant amounts of viral particles as early as 15 hours after infection,
with titres between 1 x
105 and 1 x 106 PFU/ml. By 35 hours post-infection, the viral titres were
between 1 x 10' and 1 x
10g pfu/ml. In contrast, the viral titres from infected NIH-3T3 cells at the
15- and 25-hr time
points were below 1x104 PFU/ml. Figure 3 shows the HSV-I virus yield from
infected NIH-
IS 3T3, THC-11, TNIH#5 and H-ras cells using two different MOTs (Upper panel:
MOI = 0.5
PFU's/cell; lower panel: MOI = 5 PFU's /cell)
This example illustrates that cell lines, once transformed with activators of
the Ras
pathway, become permissive to infection by HSV-l and exhibit not only
cytopathic effects from
infection, but synthesize viral proteins and intact viral particles. In
contrast, untransformed cell
lines are non-permissive to HSV-1 infection.
EXAMPLE 2: Ras-activated cells lines as a diagnostic tool for HSV-1 infection
Figure 3 shows that as early as 15 hours after infection with HSV-1,
measurable virus
titres are produced from cell lines that are transformed with activators of
the Ras pathway.
Figure 9, Panel "A" compares the sensitivity of detection of HSV-1 antibodies
in three
different cell lines. Cell line A549 (human lung carcinoma) is typically used
in medical
laboratories to diagnose HSV-I infection. Cell line THC-11 is transformed with
v-erbB, and H-
ras cell lines are transformed with H ras. All three cell lines were infected
with HSV-1 at a MOI
of 0.25-0.5. At 1 l, 13, 15, 17 or 21 hours post infection, cells were
harvested and Western blot
CA 02352439 2001-07-17
analysis was performed on these samples as described above. The results showed
that at 11
hours post infection, both THC-11 and H-ras cell lines exhibited significant
levels of HSV-1
protein synthesis, whereas cell line A549 did not exhibit detectable amounts
of protein until 17
hours post infection. The Panel "B" of Figure 9 shows that infection of H-ras
cells by HSV-1
can be detected using immunofluorescence with anti-gC antibody, as early as 8
hours post-
infection. Therefore, the use of transformed cell lines such as THC-I 1 or H-
ras can significantly
shorten the time for the diagnosis of herpes virus infections in clinical
samples. This would be
important in situations where life-threatening herpes virus infections require
immediate medical
attention.
EXAMPLE 3: The use of Farnesyl-Transferase Inhibitors to Reduce HSV-1
Infection Efficiency
Post-translational farnesylation of Ras is necessary for association of Ras
with the plasma
membrane, and is a crucial process for the initiation of downstream events,
including the three
distinct MAPK cascades. It was postulated that if Ras, or downstream events
initiated by Ras, is
in fact involved in HSV-1 infection, then farnesyl transferase inhibitors
should block HSV-1
replication in oncogene-transformed cells.
H ras transformed NIH-3T3 cells were exposed to the farnesyl transferase
inhibitors FTI-
I at a final concentration 50 ~M or 100 p,M in the culture medium. Control
cells were not
exposed to inhibitor. At 22 hours post-infection the cells were harvested and
Western blot
analysis was performed on these samples using rabbit anti-HSV-1 antibody, as
described above.
Figure 4, panel "A" shows that compared to the control cells, which were not
exposed to FTI-1
the production of viral proteins in cells that were exposed to FTI-1 is
drastically reduced.
The bottom part of Figure 4, Panel "B" shows the results of Western blots of
identical
samples as described in the paragraph above, except that they were probed with
mouse anti-
ERK1/2 antibody [Erkl/2] or mouse anti-phosphoERKI/2 antibody [P-Erkl/2].
These results
show that, as a result of exposure to FTI-1, there is a lack of activity of
MEKI/2 which leads to
reduced phosphorylation of ERK 1 /2 (ERK42/44).
To determine whether other inhibitors of farnesyl transferase would have the
same effect
on viral protein synthesis, three additional inhibitors were tested. H ras
transformed NIH-3T3
26
CA 02352439 2001-07-17
cells were exposed to the farnesyl transferase inhibitors FPTI-1, FPTI-2 and
FTI-4 at a final
concentration 150 ~M, 150 ~M or or 50 pM in the culture medium. Control cells
were not
exposed to inhibitor. At 22 hours post-infection the cells were harvested and
Western blot
analysis was performed on these samples using rabbit anti-HSV-1 antibody, as
described above.
Figure 5, panel "A" shows that compared to the control cells, viral protein
synthesis in cells that
were exposed to FPTI inhibitors is drastically reduced.
Panel "B" of Figure 5 shows the effect of FTI-1 on HSV-1 infection, using
immunofluorescence. As can be seen, addition of FTI-1 to the culture medium
dramatically
reduces the level of viral proteins detected and it appears that fewer cells
are actually infected by
HSV-1.
Figure 6 shows the effects of FTI-1 at two different concentrations, on the
virus yield, as
quantitated by plaque titration on Vero cells (described above). Cells were
infected at an MOI of
0.5 PFU/cell and were harvested 22 hours post infection. The inhibitors were
present for the
entire duration of the infection. As can be seen, FTI-1 treatment of cells
dramatically decreased
virus yield.
EXAMPLE 4: The use of Inhibitors of the ERK Pathway to Reduce HSV-1 Infection
Efficiency
A major pathway downstream of Ras that regulates cell growth is the ERK
pathway [6].
Stimulation of this pathway requires the phosphorylation of ERK1/2 by the
mitogen-activated
extracellular signal-regulated kinase kinase MEK1/2 which itself is activated
(phosphorylated)
by Raf, a serine-threonine kinase downstream of Ras. To determine if MEK1/2
activity is
required for HSV-1 infection, we studied the effect of the MEK1/2 inhibitor
PD98059 [10, 16]
on infected H ras-transformed cells.
HSV-1 infected H ras transformed NIH-3T3 cells were exposed to 40 ~M PD98059
in
the culture medium. Control cells (HSV-1 infected) were not exposed to
PD98059. At 22 hours
post-infection, the cells were harvested and Western blot analysis was
performed on these
samples using rabbit anti-HSV antibody, as described above. Figure 4, panel
"A" shows that
compared to the control cells, which were not exposed to PD98059, the
production of viral
proteins in cells that were exposed to PD98059 is reduced.
27
CA 02352439 2001-07-17
Figure 4 Panel "B" shows the results of Western blots of identical samples as
described
in the paragraph above, except that they were probed with mouse anti-
phosphoERKl/2 antibody
[P-Erkl/2) to demonstrate that, as a result of exposure to PD98059, there is a
lack of
phosphorylation of ERK1/2 (Erk42/44).
As seen in Figure 4, Panel "A", the p38 kinase specific inhibitor, SB203580
had no effect
on HSV-1 infection when used at effective doses, suggesting that elements
downstream of p38
kinase are likely not involved with HSV-1 infection. Panel "B" shows the
results of Western
blots of identical samples except that they were probed with mouse anti-
phospho-ATF-2
antibody [P-ATF2). These results show that, as a result of exposure to
SB203580, there is a lack
of phosphorylation of ATF-2.
Figure 6 shows the effects of PD98059 on the virus yield, as quantitated by
plaque
titration on Vero cells (described above). Cells were infected at an MOI of
0.5 PFU/cell and
were harvested 22 hours post infection. The inhibitor was present for the
entire duration of the
infection. As can be seen, PD98059 treatment of cells dramatically decreased
virus yield,
equivalent to the effect of FTI-1 on viral yield.
Figure 6 also demonstrates the effects of SB203580 at two different
concentrations, on
the virus yield, as quantitated by plaque titration on Vero cells (described
above). Cells were
infected at an MOI of 0.5 PFU/cell and were harvested 22 hours post infection.
The inhibitors
were present for the entire duration of the infection. As can be seen,
SB203580 treatment of
cells does not effect virus yield
EXAMPLE 5: P-13 Kinase Pathway is Not Involved in HSV-1 Infection
H-ras cells that were infected with HSV-1 were exposed to Wortmannin at a
final
concentration of 200 nM for the entire duration of the infection. Cells were
harvested at 22 hr.
post infection, lysed and subjected to SDS PAGE followed by immunoblotting
with a rabbit anti-
HSV-1 antibody. As the left panel of Figure 7 shows, Wortmannin has no effect
on HSV-1
infection. The bottom part of Panel "A" shows the results of Western blots of
identical samples,
except that they were probed with mouse anti-phospho-Akt antibody [P-Akt). The
inhibitory
effect of Wortmannin at this concentration is indicated by the lack of Akt
phosphorylation.
28
CA 02352439 2001-07-17
The right panel of Figure 7 shows the ability of HSV-1 to infect NIH-3T3 cells
that
express Ras effector domain mutants. The mutant cell lines V 1 X40, V 12637
and V 12535 all
have a common activating G12V mutation as well as one other unique mutation in
Ras, causing
them to activate distinct pathways downstream of Ras. Mutant V 12637 is unable
to signal via
the RAF/ERK and the PI3-kinase pathway, but allows signaling via the RAL-GDS
pathway.
The V 12C40 mutation disrupts signaling via the RAF/ERK and the RAL-GDS
pathways, but
does not affect the PI3-kinase pathway. The V12S35 mutant cannot signal
through the RAL-
GDS and the PI3-kinase pathway, but can do so via the RAF/ERK pathway. Panel B
of Figure 7
shows that the V12S35 mutant is considerably more permissive to HSV-1
infection than the
other two mutants, suggesting a more significant role of the RAF/ERK pathway
(as compared
with the PI3-kinase or the RAL-GDS pathway) in this process.
EXAMPLE 6: Viral Transcripts are Generated but not Translated in NIH-3T3 Cells
To elucidate the role of the Ras pathway in HSV-1 infection, it was important
to identify
the step at which HSV-1 infection is blocked in NIH-3T3 cells. Virus binding
and
internalization is known to be comparable between permissive and non-
permissive cells.
Therefore, the transcription of viral genes was investigated. Functional
protein products of the
immediate early viral a genes are required for the subsequent transcription of
the polypeptide
groups (3 and y.
The relative amounts of HSV-1 transcripts generated in HSV-1 infected NIH-3T3
cells
and H ras-transformed cells was compared. Cells were infected with HSV-1 at a
MOI of 0.5
PFU/cell. At 2, 5, 10, 20 and 25 hours after exposure to the virus, cells were
harvested and RNA
was extracted from them. Equal amounts of RNA from each sample were then
subjected to RT-
PCR, whereby selective amplification of specific viral cDNAs (a27, UL29, UL30,
y~34.5, and
UL44) was accomplished using the methods described in the "General Methods".
GAPDH,
which is constitutively expressed, served as a PCR and gel loading control.
Figure 8, panel "A97
shows that the immediate early transcript a27 accumulated to comparable levels
in the two cell
lines. However, the (3 and y transcripts were preferentially synthesized in
the Ras-transformed
cells and barely detectable, if at all, in the non-permissive NIH-3T3 cells.
29
CA 02352439 2001-07-17
Since transcription of these ~i and y genes requires immediate early a gene
products, the
drastic reduction in their expression in NIH-3T3 cells was likely due to the
inability of the a
transcripts to be efficiently translated in these cells. Therefore, the level
of the protein product of
the a27 gene (ICP27), the UL29 gene (ICPB) and the UL44 gene (gC) were
compared between
infected NIH-3T3 and H ras-transformed cells. Cells at 20 hours post infection
were harvested,
and subjected to Western blot analysis using mouse anti-ICP27 antibody, as
described in
"General Methods". Figure 8, panel "B" shows that ICP27 was present at a much
lower level in
NIH-3T3 cells than in H ras-transformed cells, even though the levels of a27
transcripts were
comparable between the two cell lines. Therefore, it appears that the a
transcripts were not
efficiently translated in NIH-3T3 cells, which in turn led to the lack of
progression of
downstream events as evidenced by the drastically reduced (or undetectable)
levels of both the (3
and y transcripts. Inefficient translation of these transcripts further
reduced the output of the
protein products such as gC and ICP8 to undetectable levels in NIH-3T3 cells
as compared to H
ras transformed cells.
EXAMPLE 7: PKR is Phosphorylated in HSV-1 Treated NIH-3T3 Cells, but not in
HSV-1
Infected Oncogene-Transformed Cells.
Because viral transcripts were generated but not translated in NIH-3T3 cells,
it was
investigated whether PKR is activated (phosphorylated) in these cells.
Phosphorylation of PKR
leads to inhibition of translation of viral genes presumably because activated
PKR
phosphorylates the translation initiation factor eIF-2a, which then inhibits
translation of viral
transcripts. In oncogene-transformed cells, PKR phosphorylation is prevented
or reversed by
Ras or one of its downstream elements, allowing viral gene translation to
ensue.
NIH-3T3 cells and oncogene-transformed cells were infected with HSV-1 at a MOI
of
0.5 PFU/cell, and incubated for 20 hours. Control (uninfected) cells were
incubated for the same
length of time. At this time, the media was aspirated off and the cells were
lysed in a solution of:
20 mM HEPES, pH 7.4, 120 mM KCI, 5 mM MgCl2, 1 mM DTT, 0.5% Nonidet P-40, 2
pg/ml
leupeptin, and 50 pg/ml aprotinin. The nuclei were then removed by low-speed
centrifugation
and the supernatants were stored at -70°C until use.
CA 02352439 2001-07-17
Cytoplasmic extracts were normalized for total protein concentration, using
the Bio-Rad
protein microassay method. Each in vitro kinase reaction contained 20 ~1 of
cell extract, 7.5 ~1
of reaction buffer (20 mM HEPES, pH 7.4, 120 mM KCI, 5 mM MgCl2, 1 mM DTT and
10%
glycerol) and 7.5 p,1 of ATP mixture (1.0 ~Ci [y-32P]ATP in 7 p1 of reaction
buffer), and was
incubated for 30 minutes at 37°C [25]. Aliquots were then either boiled
in Laemmli SDS
sample buffer and used for Western Blotting with mouse monoclonal anti-PKR
antibody for the
detection of total PKR or immunoprecipitated with the same antibody followed
by SDS-PAGE
and autoradiography for the detection of 32P-labelled PKR (Figure 10). The
phosphorylation
state of eIF-2 , the main substrate of PKR was also measured by Western blots
using antibodies
against total and phosphorylated forms of eIF-2 .
The results of the Western Blot showed that PKR levels were comparable in all
the four
cell lines, whether infected with HSV-1, or not (Figure 10, Panel "A").
However, PKR
phosphorylation was seen only in infected cells, and was consistently more
pronounced in NIH-
3T3 cells than in the oncogene-transformed cells. PKR phosphorylation did not
occur in
uninfected NIH-3T3 cells, but did occur in infected NIH-3T3 cells, which
suggests that it was a
virus-triggered event (compare lanes 1 and 5). The differential
phosphorylation of PKR between
untransformed and transformed cells is consistent with the observed difference
in their capacity
to promote HSV-1 protein synthesis. As shown in the bottom of Figure 10, Panel
"A", although
the levels of eIF-2 are constant, eIF-2 phosphorylation is enhanced in NIH-3T3
cells upon
HSV-1 infection, but not in transformed (HSV-1 sensitive) cells. The different
phosphorylation
of PKR and eIF-2 between untransformed and transformed cells is consistent
with the observed
difference in their capacity to promote HSV-1 protein synthesis.
If inhibition of PKR phosphorylation is due to elements of the Ras signaling
pathway
then FTI-l and PD98059 (blockers of Ras plasma membrane association and MEK
activity
respectively) which effectively inhibit HSV-1 protein synthesis and HSV-1
virus production in
transformed cells, should restore PKR phosphorylation in infected cells.
Figure 10 Panel "B"
shows the effect of FTI-1 and PD98059 on PKR phosphorylation in HSV-1 infected
H-ras cells.
H-ras cells were exposed to FTI-1 (100 M) or PD98059 (40 M) and the
phosphorylation state
of PKR was assessed using anti-PKR ("total PKR") or anti-phospho-PKR ("P-PKR)
antibodies .
As can be seen, PKR phosphorylation is restored by these inhibitors. The
difference in
31
CA 02352439 2001-07-17
phosphorylation of PKR in infected NIH-3T3 cells and in infected MEF cells is
shown in the
right side of Figure 10, Panel "B". As can be seen, MEF cells, which are
sensitive to HSV-1,
have much lower levels of phosphorylated PKR than do NIH-3T3 cells, which are
relatively non-
permissive to HSV-1 infection.
EXAMPLE 8: Oncogene-Transformed Cells are more Permissive to HSV-1 Mutant
83616 than
are NIH-3T3 Cells
The permissiveness of oncogene-transformed NIH-3T3 cell lines to attenuated
mutants of
HSV-1 was assessed by Western blot analysis. NIH-3T3 cell lines, and oncogene-
transformed
cell lines were infected with Mutant 83616 [17, 22-24]. Cells were harvested
at 20 hours after
infection and subjected to Western blotting with rabbit anti-HSV-1 antibody,
as described in the
"General Methods". As shown in Figure 11, as evidenced by the fact that they
make
substantially more viral proteins, oncogene-transformed cell lines are more
permissive to 83616
than NIH-3T3 cells.
EXAMPLE 9: PKR Deletion Enhances the Permissiveness of Cells to HSV-1
Infection
Mutant 83616 contains deletions in both copies of the y134.5 gene. The gene
product of
y134.5 (called ICP34.5) presumably forms a complex with protein phosphatase 1
and redirects its
activity to dephosphorylate eIF-2a. Once dephosphorylated, eIF-2a is
inactivated and unable to
inhibit viral transcription. The net result of ICP34.5 activity is to
dephosphorylate eIF-2a,
whereas the activity of phosphorylated PKR is to phosphorylate eIF-2a. Thus,
ICP34.5 plays an
antagonistic role to PKR - i. e. it acts "anti-PKR", and mutant 83616 can be
regarded as a virus
that has lost its inherent anti-PKR mechanism.
A direct approach to test these hypotheses, and to define the role of PKR in
HSV-1
infection, is through the use of host cells that are devoid of the PKR gene.
Host cells that have
lost the PKR gene should be more permissive to 83616 infection than host cells
that have the
PKR gene. In the absence of PKR, the absence of an anti-PKR mechanism in
mutant 83616 is
of no consequence. Conversely, wild-type HSV-1 infectivity should not differ
between host-
cells that do or do not have PKR activity. Since the anti-PKR activity is
working in wild-type
HSV-1, the presence of PKR in the host cell is of negated by the viral anti-
PKR activity.
32
CA 02352439 2001-07-17
Primary embryo fibroblasts from PKR~/~ mice and PKR+/+ mice were compared in
terms
of permissiveness to HSV-1 and 83616 infection. Cells were infected with MOI
of 0.5 PFU/cell
and 20 hours after infection were harvested and subjected to Western blotting
with rabbit anti-
HSV-1 antibody, as described in the "General Methods". Figure 12 (right two
lanes) shows that
wild-type HSV-l, armed with its own anti-PKR mechanism, was able to infect PKR-
/- and
PKR+/'~ mouse embryo fibroblasts equally well. In contrast, 83616 viral
proteins were
synthesized at a significantly higher level in the PKR-/- cells than in the
PKR+/-' cells (Figure 12,
left two lanes).
The level of infection by 83616 in PKR-/- fibroblasts is equivalent to that
seen in cells
transformed by Ras, or elements of the Ras pathway. Therefore, PKR deletion
enhances host
cell permissiveness to HSV-1 infection in the same way as does transformation
by ras or
elements of the Ras pathway. Further evidence for the Ras-PKR connection comes
from the
demonstration that FTI-1 inhibited HSV-1 infection of PKR+/'- cells, while
having no effect on
PKR-/- cells, as shown in Figure 13. In this experiment, cells were infected
with HSV-1 at a
MOI of 0.5 PFU/cell, in the presence of FTI-1. Cells were harvested at 20 hr
post-infection and
analyzed for viral proteins as above. Viral protein synthesis was reduced by
FTI-1 in only the
PKR+/+ cells.
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38
CA 02352439 2002-10-08
OncolyticvirusesCA.ST25.txt
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CA 02352439 2002-10-08
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<211> 18
<212> DNA
<213> herpes simplex virus 1
<400> 8
gcgggaggcg gggaatac 18
<210> 9
<211> 17
<212> DNA
<213> herpes simplex virus 1
<400> 9
gccgccgcct actaccc 17
<210> 10
<211> 18
<212> DNA
<213> herpes simplex virus 1
<400> 10
gctgccgcga ctgtgatg 18
<210>11
<211>23
<212>DNA
<213>homo Sapiens
<400> 11
cggagtcaac ggatttggtc gat 23
<21.0> 12
<211> 24
Page 3
CA 02352439 2002-10-08
OncolyticvirusesCA.ST25.txt
<212> DNA
<213> homo Sapiens
<400> 12
agccttctcc atggtggtga agac 24
Page 4
