Correlation between interferon sensitivity of reovirus isolates and ability to discriminate between normal and Ras-transformed cells

Penny Rudd and Guy Lemay

Département de Microbiologie et Immunologie, Université de Montréal, PO Box 6128, Station centre-ville, Montréal, Québec, Canada, H3C 3J7

Correspondence
Guy Lemay
guy.lemay{at}umontreal.ca


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mammalian reoviruses exhibit a propensity to replicate in transformed cells. It is currently believed that the interferon-inducible RNA-dependent protein kinase (PKR), an intracellular host-cell resistance factor that is inhibited by an activated Ras-dependent pathway in transformed cells, is responsible for this discrimination. In the present study, reovirus isolates differing in their sensitivity to interferon were obtained by chemical mutagenesis, and examined for their replicative properties in parental and Ras-transformed mouse NIH-3T3 cells. It was observed that most isolates can bypass resistance mechanisms of parental cells at high m.o.i., and that there is a correlation between the ability to discriminate between transformed and parental cells, and interferon sensitivity. Most interestingly, an interferon-hypersensitive mutant virus was more dependent on Ras activation than any other viral isolate. Altogether, this suggests that optimal reovirus isolates could be selected to attack tumour cells depending on the nature of the alterations in interferon-inducible pathways found in these cells.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the last few years, it has been recognized that viruses are not only foes to fight but can become useful tools in our fight against diseases. One field where viruses can be used is the treatment of cancer, as ‘so-called’ oncolytic viruses (Bell et al., 2002; Chiocca, 2002; Mullen & Tanabe, 2002; Wildner, 2001).

Classical non-fusogenic mammalian virus of the genus Orthoreovirus (herein referred to as ‘reoviruses’) are among the best candidates for such an application in oncolytic therapy against various cancers (Alain et al., 2002; Coffey et al., 1998; Etoh et al., 2003; Hirasawa et al., 2002, 2003; Kilani et al., 2003; Norman et al., 2002; Norman & Lee, 2000; Thirukkumaran et al., 2003; Wilcox et al., 2001; Yang et al., 2003). These viruses present the advantage of being naturally oncolytic, in contrast to viruses that need to be attenuated or somehow manipulated before being used. Also, mammalian reoviruses exhibit only low, or lack whatsoever, pathogenic potential in human adults, an advantage in any future therapeutic use (Virgin et al., 1996).

An oncolytic virus must be able to efficiently destroy cancer cells without causing pathology, thus sparing normal untransformed cell counterparts. The ability to destroy only transformed cells defines the ‘therapeutic index’ of the virus. The discrimination exhibited by a given virus or viral isolate could depend on both the nature of the alterations found in cancer cells, and the nature of the virus itself determining its sensitivity to resistance mechanisms present in untransformed cells.

The ability of reoviruses to preferentially replicate in, and destroy, transformed cells has been known for quite a long time (Duncan et al., 1978; Hashiro et al., 1977). However, only recent advances in cellular biology has allowed a better understanding of the nature of the alterations found in Ras-transformed cells that are responsible for preferential reovirus replication (Strong et al., 1998; Strong & Lee, 1996).

Multiplication of reoviruses can be controlled at the translational level by interferon-inducible pathways involving the cellular interferon-inducible dsRNA-dependent protein kinase, PKR (Gupta et al., 1982; Miyamoto & Samuel, 1980; Nilsen et al., 1982; Samuel, 1998; Wiebe & Joklik, 1975). Biologically active PKR is found in normal untransformed cells, such as ‘flat’ mouse NIH-3T3 fibroblasts exhibiting highly developed contact inhibition of the cellular monolayer in cultured cells. PKR inhibits reovirus multiplication and this is generally believed to occur mainly via an increased level of phosphorylation of the eIF-2{alpha} translation initiation factor (Clemens & Elia, 1997; Proud, 1992; Samuel et al., 1997). Many different viruses have evolved different mechanisms to counteract PKR, stressing the importance of this resistance factor in viral multiplication (Gale & Katze, 1998; Katze, 1993, 1995). In reoviruses, PKR inhibition was shown to rely mostly on the {sigma}3 viral protein that competes with PKR for the dsRNA activator (Bergeron et al., 1998; Imani & Jacobs, 1988; Jacobs & Langland, 1998; Lloyd & Shatkin, 1992; Samuel, 1998; Schiff, 1998; Schmechel et al., 1997; Yue & Shatkin, 1997). However, the resistance conferred by the {sigma}3 protein is certainly incomplete since reoviruses are not protected against high levels of PKR activity, as found in most untransformed cells and in interferon-treated cells.

The resistance to reovirus multiplication observed in NIH-3T3 mouse fibroblasts is apparently due to such a high level of active PKR. Ras transformation of these cells interferes with PKR activity (Mundschau & Faller, 1992, 1994) and allows reovirus multiplication, thus giving an ‘oncolytic’ potential to these viruses (Strong et al., 1998). This oncolytic potential can allow the virus to selectively destroy tumour cells in vivo and has generated much interest for eventual clinical applications in humans (http://www.oncolyticsbiotech.com/).

In the present study, mammalian reovirus serotype 3 isolates (MRV-3; strain Dearing, genus Orthoreovirus, family Reoviridae) differing in interferon sensitivity were obtained by chemical mutagenesis and examined for their ability to replicate in parental or Ras-transformed NIH-3T3 cells. It was observed that most isolates can bypass resistance mechanisms of parental cells at high m.o.i. and that there is at least a partial correlation between the ability to discriminate transformed cells from parental cells and interferon sensitivity. Most interestingly, an interferon-hypersensitive mutant virus was more dependent on Ras activation than any other viral isolate. It is thus likely that specific viral isolates could be chosen to attack different tumour cells and that due caution should be exerted in the choice of viral strains that will be used in clinical settings.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
Mouse fibroblasts L929 cells were originally obtained from the ATCC (Manassas, VA, USA) and were propagated in MEM medium supplemented with 5 % fetal bovine serum (FBS). SC1 feral mouse embryo fibroblasts cells were also obtained from the ATCC and propagated in MEM medium supplemented with 1 % non-essential amino acids and 5 % FBS. Parental NIH-3T3 murine fibroblast cells and their Harvey-RasG12V-transformed counterparts (herein referred to as Ras-transformed) were a generous gift from Dr Robert Nabi (Département de Pathologie et Biologie Cellulaire, Université de Montréal). These cells were originally obtained from Dr Michael Lisanti (Whitehead Institute for Biomedical Research) (Koleske et al., 1995) and were routinely propagated in DMEM medium supplemented with 1 % non-essential amino acids, vitamins and 10 % bovine calf serum. They were passaged on a regular basis at a cell density of 300 000 cells per 10 cm-diameter culture dish to avoid spontaneous transformation. Original stock of reovirus serotype 3 (Dearing) was obtained from ATCC and propagated at an m.o.i. of 1 p.f.u. per cell in mouse L929 cells.

Chemical mutagenesis.
The Dearing strain of MRV-3 was subjected to nitrosoguanidine (NTG) mutagenesis by using a modification of a procedure described by Patrick et al. (2001) for avian reovirus mutagenesis. Briefly, NTG (Sigma-Aldrich) was dissolved at 50 mg ml–1 in DMSO, filter-sterilized and diluted at 5, 20, 50 or 200 µg ml–1 in complete tissue culture medium. Mouse L929 fibroblasts were seeded the day before at a density of 106 cells per 60 mm-diameter Petri dish. Infection was done with 107 infectious units (as determined by TCID50 titration) of virus diluted in 0·5 ml of serum-free culture medium. Adsorption was done for 1 h at 4 °C before addition of each dilution of NTG in separate Petri dishes. Infected cells were then incubated 48 h at 37 °C, at which time cell lysis was essentially complete. Following three cycles of freeze–thaw (–80 °C to room temperature), viral suspension was then titrated over a 9-day period on L929 cells to determine the remaining virus titre following mutagenic treatment. All virus titrations were done using the TCID50 method on L929 cells in 96-well microplates as previously described (Danis & Lemay, 1993). Viruses treated with 200 µg ml–1 were then amplified by four consecutive passages on either L929 or SC1 cells. These pools were then used to isolate viral clones.

Isolation of viral cloned isolates.
All viral isolates were cloned using two consecutive cloning steps. First, the original virus stock or the mutagenized virus passaged in L929 cells or SC1 cells were diluted and plated in 96-well microplates seeded with L929 cells such as to obtain less than 50 % of visibly infected wells after 7 days. Viruses were then propagated on L929 cell monolayers and recloned by plaque formation on L929 cell monolayers overlaid with agar. Isolated plaques were picked and virus propagated again in L929 cells. Final viral stocks were titrated using the TCID50 method. Many isolates were obtained and 16 were examined briefly. From these, four virus isolates were used and better characterized in the rest of the study: the H8-2 isolate from the original unmutagenized stock, P4L-12 from mutagenized virus stock propagated on L929 cells, and the P4SC1-31 and P4SC1-42 from the mutagenized stock propagated in SC1 cells.

Effect of interferon: synthesis of viral and cellular proteins.
To compare interferon sensitivity between different viral clones, L929 cells (5x105 cells seeded in a 100 mm-diameter Petri dish) were pretreated with concentrations of 0, 25, 50 or 250 IU mouse {beta}-interferon (Calbiochem) ml–1 for 24 h before being infected at an m.o.i. of 10 p.f.u. per cell. In experiments involving parental or Ras-transformed NIH-3T3 cells, cells were plated at 5x105 per 100 mm-diameter Petri dish and treated at a near-saturating concentration of interferon (250 IU ml–1). Infected cells were incubated in MEM containing 2 % heat-decomplemented FBS and interferon concentration was maintained at the same level. Metabolic radiolabelling of infected and uninfected controls was performed 24 (L929 cells) or 48 h (parental and Ras-transformed NIH-3T3 cells) post-infection at 37 °C. Briefly, medium was replaced with methionine- and serum-free medium with 25 µCi Tran35S-Label ml–1 (7 Ci mmol–1 or 259 MBq mmol–1; Perkin Elmer) and incubated for 2 h. Cells were then washed with PBS, scraped from the Petri dish and recovered by centrifugation in an Eppendorf microcentrifuge (1000 r.p.m. for 2 min). Cells were then lysed in TBS buffer (10 mM Tris/HCl pH 7·5, 150 mM sodium chloride containing 1 % NP-40). Nuclei were pelleted by centrifugation in a microcentrifuge (10 000 r.p.m. for 20 min) and supernatant recovered for analysis by SDS-PAGE (10 % acrylamide/bis-acrylamide gel) and autoradiography on a Kodak X-Omat AR film (Amersham).

Immunoblotting analysis.
The synthesis and accumulation of viral proteins in parental or Ras-transformed NIH-3T3 cells was determined by immunoblotting. Cells were seeded at a concentration of 5x105 cells per 10 cm-diameter Petri dish and infected with various isolates at different m.o.i. for 48 h at 37 °C. Cells were then recovered by scraping, and lysed in 1 ml RIPA buffer [25 mM Tris/HCl, pH 7·2; 5 mM sodium-EDTA, 1 % NP-40, 0·5 % sodium deoxycholate, 0·05 % SDS, 50 mM sodium fluoride, 10 mM sodium orthovanadate and a cocktail of protease inhibitors, as recommended by the manufacturer (Roche)]. Analysis was achieved by immunoblotting using a rabbit anti-reovirus antiserum (Lee Biomolecular) followed by a phosphatase-conjugated anti-rabbit immunoglobulin antibody at a 1 : 3000 dilution (Cedarlane). Incubations with antibodies were performed in TBS buffer containing 0·1 % Tween 20 and 1 % powdered skimmed milk for 1 h at room temperature followed by three washes in TBS containing 1 % Tween 20. Antigen–antibody complexes were detected using NBT-BCIP substrate (Invitrogen Life Technologies).

Virus production.
Parental and Ras-transformed NIH-3T3 cells were infected at an m.o.i. of 250 p.f.u. per cell in 60 mm-diameter Petri dish. At 4 days post-infection, a 100 µl aliquot of medium was collected and analysed by immunoblotting using the anti-reovirus antiserum. The Petri dish were then submitted to three cycles of freeze–thaw before being titrated in 96-well microplates.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Rationale
Chemical mutagenesis was used to obtain a panel of viral isolates that differ in their sensitivity to interferon. It was reasoned that these viruses should also differ in their sensitivity to PKR, and therefore in their ability to discriminate between normal and transformed (or cancerous) cells. It was previously reported that serotype 1 reovirus is resistant to interferon while serotype 3 is sensitive (Jacobs & Ferguson, 1991). However, in preliminary studies, isolates of both serotypes were found to vary in their sensitivity depending of their laboratory of origin (data not shown). Thus, serotype was not considered as a factor in the study and the classical reovirus serotype 3 Dearing was chosen for mutagenic treatment. However, the difference in interferon sensitivity previously observed between serotypes, as well as our own observation of a thermosensitive mutant exhibiting increased interferon resistance (Bergeron et al., 1998), both supported the idea that reovirus isolates could differ in their sensitivity to interferon.

Generation of viral mutant isolates
Chemical (NTG) mutagenesis on a serotype 3 Dearing reovirus stock was done using modifications of classical procedures (Fields & Joklik, 1969; Ikegami & Gomatos, 1968; Patrick et al., 2001), as described in Methods. Recovery of infectious virus following treatment at different concentrations of the mutagen was quantified, as summarized in Table 1. Concentration of 200 µg ml–1, resulting in less than 0·002 % of virus survival, was used thereafter to maximize our chances of obtaining mutants while ensuring recovery of infectious viruses. The resulting viruses were passaged in L929 or SC1 cells; and viral isolates were biologically cloned from the mixed viral populations using consecutive limiting dilution and plaque purification, as described in Methods.


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Table 1. Effect of mutagenic NTG treatment on reovirus infectivity

Virus stock (reovirus serotype 3 Dearing) was treated with NTG as a mutagenic agent as described in Methods. Decreased in infectious titre was measured by titration.

 
Determination of interferon sensitivity in viral isolates
Biological clones of viruses, herein referred to as ‘viral isolates’, were used to infect L929 cells in the presence or absence of interferon treatment. It was previously determined that maximal effect of interferon on different viruses, as well as maximal induction of PKR, is achieved at a concentration of 500 IU ml–1 in L929 cells (Bergeron et al., 1998; Danis et al., 1997; Jacobs & Ferguson, 1991). This so-called ‘saturating’ concentration was thus used for initial characterization of the viral isolates in the present study.

Mouse L929 cells were thus exposed to interferon 24 h before and during viral infection. Synthesis of viral proteins was then detected by metabolic radiolabelling as described in Methods. All 16 different cloned viral isolates were initially examined and generated a panel of viruses exhibiting varying degrees of sensitivity to interferon in L929 cells, as judged by a reduction in synthesis of viral proteins (data not shown).

The four isolates used hereafter represent the overall variation in sensitivity that was observed. Isolate H8-2 is a cloned viral isolate from the original unmutagenized virus stock of wild-type reovirus serotype 3 Dearing; the three mutant viruses were obtained after four passages of the mutagenized virus stock in L929 cells (P4L-12 isolate) or SC1 cells (P4SC1-31 and P4SC1-42). The four viruses were then examined more extensively by varying the concentration of interferon, as shown in Fig. 1. Among these four strains, two appeared only moderately sensitive to interferon: H8-2, the cloned isolate of wild-type virus and P4SC1-42. With either of these two viruses, there was still a significant level of viral proteins accumulated in infected cells even at 250 IU interferon ml–1. In contrast, the other two viral isolates (P4L-12 and P4SC1-31) were notably sensitive to interferon with an important decrease of viral proteins even at low (25 IU ml–1) concentrations of interferon.



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Fig. 1. Effect of interferon on reovirus isolates: synthesis of viral proteins. The four representative reovirus isolates that were later retained for further characterization are presented. Each virus stock was used at an m.o.i. of 10 to infect L929 cells that were either left untreatred or treated with an increasing concentration of mouse {beta}-interferon from 24 h before infection, as described in Methods. Synthesis of viral proteins was detected by metabolic radiolabelling 24 h post-infection; radiolabelled proteins were analysed by SDS-PAGE and autoradiography. The portion of the gel that encompasses the major µ1C viral capsid protein is indicated by arrows.

 
Sensitivity to interferon was examined further by quantification of the decrease in virus titre in interferon-treated L929 cells (Fig. 2). As expected, at most a 10-fold decrease was observed with wild-type virus (H8-2); isolate P4SC1-42 had a similar sensitivity, as was observed when protein synthesis was examined. The P4SC1-31 virus exhibited an increased sensitivity of up to 40-fold decrease in virus titre. The P4L-12 isolate is clearly the most interesting virus with a more than 1000-fold decrease in virus titre upon interferon treatment, making it the most interferon-sensitive reovirus isolate in this cell system, one that could likely be referred to as a ‘hypersensitive’ virus.



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Fig. 2. Effect of interferon on reovirus isolates: virus titre. Viral stocks prepared from infected L929 cells were titrated by TCID50 on L929 cells as described in Methods. Treatment with interferon at 500 IU ml–1 was performed starting at 24 h before titration in 96-well microplate. Results are presented as the mean of three independent experiments with error bars representing standard deviation of the mean.

 
Infection of untransformed and Ras-transformed cells
The different viral isolates were then used to infect Ras-transformed NIH-3T3 cells or their normal counterparts. An m.o.i. of 10 was previously shown to allow a good discrimination between parental and transformed cells (data not shown), conditions similar to the one originally reported (Strong et al., 1998). This m.o.i. was thus initially used in our study.

As expected, all 16 viral isolates tested were able to efficiently infect Ras-transformed NIH-3T3 cells although they varied somewhat in their ability to synthesize viral proteins in untransformed cells (data not shown). To examine further if interferon sensitivity somehow correlates with the virus' ability to preferentially replicate in Ras-transformed cells, the four viral isolates exhibiting the whole range of sensitivity to interferon from wild-type low sensitivity (H8-2) to the interferon-hypersensitive virus (P4L-12) were studied further.

With increasing m.o.i., the different viral isolates were gradually able to bypass the restriction to synthesis of viral proteins observed in parental NIH-3T3 cells; at an m.o.i. of 1000 p.f.u. per cell, all isolates were able to replicate almost as efficiently in parental and Ras-transformed cells except for P4L-12 (Fig. 3). The P4SC1-31 isolate, exhibiting an intermediate sensitivity to interferon, also showed a good discrimination between parental and transformed cells up to an m.o.i. of 100 p.f.u. per cell, replicating better than the H8-2 virus in Ras-transformed cells but less efficiently in parental NIH-3T3 cells. However, with this virus, the discrimination was lost at the highest m.o.i., the virus replicating somewhat better than the wild-type virus in both cell lines. The two interferon-sensitive isolates (P4L-12 and P4SC1-31) also appeared to synthesize viral proteins at an increased level in Ras-transformed cells compared with the other viral isolates. Altogether, the P4L-12 isolate was clearly the best to discriminate Ras-transformed cells from parental NIH-3T3 cells (Fig. 3).



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Fig. 3. Multiplication of reovirus in Ras-transformed and parental NIH-3T3 cells. The four viral isolates that were chosen for further analysis were propagated, titrated on L929 cells and used to infect parental (–) or Ras-transformed (+) NIH-3T3 cells at increasing m.o.i. Proteins were recovered 48 h post-infection and analysed by immunoblotting using anti-reovirus antiserum as described in Methods. The position of the major µ1C capsid protein is indicated by arrows.

 
Virus production in parental and Ras-transformed NIH-3T3 cells
The mutant viral isolate, P4L-12, and the control wild-type isolate, H8-2, were retained to perform additional experiments. To verify that the synthesis of viral proteins also reflects production of infectious virus, parental and Ras-transformed cells were infected with the two viral isolates at high (250 p.f.u. per cell) m.o.i. Total production of infectious virus was measured by viral titration.

The production of infectious virus was higher in Ras-transformed cells with both viruses but, again, the restriction observed in parental NIH-3T3 cells was more than 200-fold more stringent for the P4L-12 virus (Fig. 4a), resulting in an approximately 40-fold better discrimination index, defined as the ratio of replication in Ras-transformed compared with parental cells.



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Fig. 4. Virus production in parental and Ras-transformed NIH-3T3 cells. Parental and Ras-transformed cells were infected at an m.o.i. of 250 p.f.u. per cell of either the wild-type isolate H8-2 or the P4L-12 isolate. Following freeze–thaw of cells and medium, infectious virus produced after 4 days was measured by determination of virus titre as described in Methods. In parallel, a small aliquot of the tissue culture supernatant of cells infected at either an m.o.i. of 10 or 250 was collected before freezing and analysed by immunoblotting using anti-reovirus antiserum.

 
Virus release was also examined in infected cells by immunoblotting of cell supernatant (Fig. 4b). At 4 days post-infection, cell lysis appeared minimal for parental NIH-3T3 cells, as judged by phase-contrast microscopy and trypan blue staining. In Ras-transformed cells cytopathic effect was only apparent at high m.o.i., trypan blue staining indicated between 30 and 50 % of cell death in these conditions. Virus release was seen in Ras-transformed cells for both viruses at high m.o.i. while, at low m.o.i. it was only observed for the P4L-12 virus. In parental cells, only the H8-2 virus was detected in the medium and only at high m.o.i.

Effect of interferon in parental and Ras-transformed NIH-3T3 cells
The effect of interferon in parental versus Ras-transformed cells was finally examined. In this experiment, radiolabelling was performed to look at the synthesis of cellular as well as viral proteins (Fig. 5). As expected, in the absence of interferon, synthesis of viral proteins was detected in both cell types with the H8-2 virus at high m.o.i. but only in Ras-transformed cells at low m.o.i.; discrimination between the two cell types was thus limited with this virus at high m.o.i. and only observed at lower m.o.i. [compare lane 3 with 4 in (a)]. As also expected, the P4L-12 virus exhibited a better discrimination between parental and Ras-transformed cells. Synthesis of viral proteins in parental cells was barely detectable even at high m.o.i. [lane 7, (a)]; furthermore, synthesis of cellular proteins was strongly inhibited, even at low m.o.i., in Ras-transformed but not in parental cells [compare lane 6 with 5 in (a)]. At high m.o.i., the P4L-12 virus essentially killed the cells under the conditions used, thus explaining reduced detection of even the viral proteins [lane 8, (a)]. Treatment with a high concentration (250 IU ml–1) of interferon was protective at low m.o.i. with either H8-2 [compare lane 2 in (b) and (a)] or P4L-12 [compare lane 6 in (b) and (a)]; at high m.o.i., there was a significant protection in parental cells for the H8-2 virus [compare lane 3 in (b) and (a)], thus favouring the discrimination between parental and Ras-transformed cells [compare lane 4 to lane 3 in (b)]. Even though it is highly sensitive to interferon, the P4L-12 virus was still the most efficient in Ras-transformed cells even under interferon treatment [lane 8, (b)].



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Fig. 5. Effect of interferon in parental and Ras-transformed cells. Parental (–) or Ras-transformed (+) NIH-3T3 cells were infected with either the H8-2 or P4L-12 virus isolate at the indicated m.o.i. (b) Cells were pretreated and treated with 250 interferon IU ml–1 as described in Methods. Proteins were analysed by radiolabelling at 48 h post-infection followed by SDS-PAGE and autoradiography. Positions of major viral proteins ({lambda} class, µ1C and {sigma}3) are indicated by arrows.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Different viruses are presently considered as putative anti-cancer agents via their so-called ‘oncolytic’ potential, or ability to discriminate between transformed and normal cells, or between cancerous cells and normal tissues in vivo. Mammalian reoviruses are almost unique in their natural ability to discriminate between normal and transformed cells without prior genetic engineering of the virus, while most other viruses require the selective inactivation of viral genes to acquire an adequate discriminative or therapeutic index, i.e. relative ability to replicate and destroy cancerous cells compared with their normal counterparts.

Prior studies have strongly suggested that the interferon-inducible cellular protein kinase PKR is the main factor restricting reovirus replication in untransformed cells while direct or indirect activation of the Ras oncogene counteracts this effect (Strong et al., 1998; Strong & Lee, 1996); an inhibition of PKR by Ras was noted earlier outside the context of viral infection (Mundschau & Faller, 1992, 1994). PKR is believed to be the main factor restricting reovirus infection following interferon treatment (Jacobs & Langland, 1998; Samuel, 1998) and it is thus likely that viruses exhibiting increased sensitivity to interferon, a property that can be easily assayed, will also exhibit increased sensitivity to the PKR-dependent intracellular resistance mechanisms observed in untransformed cells.

Classical reovirus isolates exhibit only limited sensitivity to interferon that was reported to vary between strains (Jacobs & Ferguson, 1991; Samuel, 1998; Sherry et al., 1998). This is most likely due to the virus' ability to counteract PKR via attachment of the dsRNA activator by the viral {sigma}3 protein (Jacobs & Langland, 1998; Schiff, 1998). The {sigma}3 protein is a major structural protein of the outer capsid that also clearly has a role in translation regulation and PKR inhibition via the presence of two dsRNA-binding motifs (Denzler & Jacobs, 1994; Imani & Jacobs, 1988; Mabrouk et al., 1995; Miller & Samuel, 1992; Wang et al., 1996). It is generally accepted that the binding to dsRNA and binding to the µ1C structural protein for capsid assembly are two mutually exclusive events (Bergeron et al., 1998; Huismans & Joklik, 1976; Liemann et al., 2002; Miller & Samuel, 1992). This suggests that viruses harbouring a {sigma}3 protein with reduced ability to interfere with PKR could be more efficient in their capsid assembly. Differences in the ability to interact with µ1C between virus strains was also shown to correlate with inhibition of protein synthesis in infected cells, a phenomenon that is probably dependent upon PKR activity (Schmechel et al., 1997). Finally, in accordance with the idea that interferon/PKR resistance is inversely correlated with efficient viral assembly, a thermosensitive virus mutant, with a {sigma}3 protein unable to assemble with µ1, also exhibits an increased resistance to interferon (Bergeron et al., 1998). Altogether, these results prompted us to examine the possibility of selecting mutant viruses with increased sensitivity to interferon, these viruses should be able to replicate in Ras-transformed cells while being strongly inhibited in untransformed cells.

Since the approaches for manipulation of the reovirus by ‘reverse genetics' are still under development, classical genetic approaches are the only tools available to modify the viral genome. Classical chemical mutagenesis did allow us to isolate a panel of viruses among which novel viral isolates derived from classical reovirus serotype 3 were shown to differ in their sensitivity to interferon. Additional work will be needed to determine the nature of the genetic alterations present in the different viral isolates, especially in the hypersensitive P4L-12 virus. It will be especially interesting to determine if the {sigma}3-encoding S4 gene is altered and to study further the biological properties of the protein. The crystallographic structure of the {sigma}3–µ1 hexameric complex should also contribute to our interpretation of the data (Liemann et al., 2002; Olland et al., 2001), if either {sigma}3 or µ1 is indeed altered in the mutants.

Interestingly, mutant viruses that are more sensitive to interferon (P4L-12 and P4SC1-31) are not only restricted in the synthesis of their proteins in parental NIH-3T3 cells, but also appear to be more efficient in Ras-transformed cells than a wild-type virus (Fig. 3). It is possible that {sigma}3–µ1 interaction is stronger in these mutants, thus favouring viral assembly to the detriment of the PKR-interfering ability of free unassembled {sigma}3. This will be the exact opposite situation of the ts453 mutant of {sigma}3 where virus assembly is precluded at the non-permissive temperature while affinity to dsRNA and ability to inhibit PKR is increased (Bergeron et al., 1998).

Relative synthesis of viral proteins also correlates with infectious virus production. Discrimination between parental and Ras-transformed cells was at least 40-fold better for the P4L-12 virus (Fig. 4a). Slightly higher titre observed for the H8-2 virus, despite lower synthesis of viral proteins, could be explained by earlier virus production for the P4L-12 virus, as suggested by important viral release in the cell supernatant (Fig. 4b).

The mutants that we obtained, together with other reovirus strains that differ in their response to PKR and/or interferon (Sherry et al., 1998; Schmechel et al., 1997), could certainly help us in any future study toward a better understanding of the viral and cellular factors that affect the interferon-induced antiviral effect in different cell types. Further studies are also needed to establish definitely the mechanism responsible for the viral discrimination between normal and Ras-transformed cells. The correlation observed between sensitivity to interferon of our four viral isolates and the discrimination between normal and Ras-transformed cells, pleads in favour of the importance of an interferon-regulated mechanism in determining the relative ability of a cell to support reovirus multiplication. Preliminary data suggest that the level of phosphorylation of eIF2-{alpha}, the best known substrate of interferon-induced PKR, may not be solely responsible for this correlation. It is possible that localized or transient effects are most important and this certainly deserves to be studied further using our different viral isolates. Interestingly, similar observations were recently reported in the translational control of the oncolytic activity of vesicular stomatitis virus. The authors raised the possibility that downstream regulation could be responsible for the defective antiviral effect of PKR in transformed cells, rather than a direct effect on PKR activity or eIF-2{alpha} phosphorylation (Balachandran & Barber, 2004). This point certainly deserves to be examined further. Recent data also indicate that the RalGEF/p38 pathway is involved in permissiveness to reovirus replication that is conferred by Ras (Norman et al., 2004). The link with PKR is not yet determined and comparisons between virus isolates could certainly contribute to these studies.

Finally, the different viral isolates described herein, especially the interferon hypersensitive P4L-12 virus strain, should be examined further for their replicative potential in different transformed cells, especially of human origins. Although P4L-12 appears as the most interesting virus for future clinical applications, it cannot be excluded that viruses with intermediate sensitivity could be appropriate in given cell types. Interestingly, the interferon-hypersensitive virus P4L-12 was still able to replicate and kill Ras-transformed cells in the presence of high concentration of interferon (Fig. 5). This suggests that interferon treatment could be used to restrict replication of the virus in normal tissue without interfering with its oncolytic activity. Future work should also investigate the potency of these novel viral isolates in the context of murine tumours produced by inoculation of human tumour cells in nude mice, as used in initial studies of wild-type reovirus oncolytic activity (Coffey et al., 1998).


   ACKNOWLEDGEMENTS
 
We thank Carole Danis for expert technical support leading to smooth operation of the laboratory and for her involvement in some parts of this study. We thank Dionissios Baltzis and Dr Antonis Koromilas (Lady Davis Research Institute) for their help with the preliminary immunoblotting experiments for detection of phospho-eIF2{alpha}. We thank Nicholas Svitek and Sapha Barkati for numerous helpful discussions. This work was supported by a grant from the Cancer Research Society Inc. (to G. L.). G. L. was the recipient of a senior scholarship from the Fonds de la Recherche en Santé du Québec. We also thank the ‘Faculté des études supérieures' of ‘Université de Montréal’ for financial support to P. R.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 21 September 2004; accepted 23 December 2004.



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