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
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ABSTRACT |
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INTRODUCTION |
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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
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
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
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.
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METHODS |
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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 ml1 in DMSO, filter-sterilized and diluted at 5, 20, 50 or 200 µg ml1 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 freezethaw (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 ml1 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 -interferon (Calbiochem) ml1 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 ml1). 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 ml1 (7 Ci mmol1 or 259 MBq mmol1; 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. Antigenantibody 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 freezethaw before being titrated in 96-well microplates.
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RESULTS |
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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 ml1, 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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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 ml1. 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 ml1) concentrations of interferon.
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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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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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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 ml1) 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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DISCUSSION |
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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
3 protein (Jacobs & Langland, 1998
; Schiff, 1998
). The
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
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
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 3-encoding S4 gene is altered and to study further the biological properties of the protein. The crystallographic structure of the
3µ1 hexameric complex should also contribute to our interpretation of the data (Liemann et al., 2002
; Olland et al., 2001
), if either
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
3µ1 interaction is stronger in these mutants, thus favouring viral assembly to the detriment of the PKR-interfering ability of free unassembled
3. This will be the exact opposite situation of the ts453 mutant of
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-
, 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
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
).
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ACKNOWLEDGEMENTS |
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Received 21 September 2004;
accepted 23 December 2004.
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