Uncoating Reo: Uncovering the Steps Critical for Oncolysis

See pages 1512 and 1522 In the last few years, it has become clear that viruses not only are foes but may also be possible therapeutic allies in the fight against cancer. Expressions such as “oncolytic virotherapy” now appear regularly in the literature. Mammalian reovirus(es), herein referred to as “reovirus,” is a nonenveloped double-stranded RNA virus that is distributed widely in nature. It has a large host range and shows only low pathogenicity in humans. The idea that reovirus could act as an oncolytic agent was first suggested in the 1970s, when it was observed that it preferentially replicated in transformed cells in vitro.1Hashiro G Loh PC Yau JT The preferential cytotoxicity of reovirus for certain transformed cell lines.Arch Virol. 1977; 54: 307-315Crossref PubMed Scopus (118) Google Scholar However, this aspect of reovirus' biology was neglected for almost 30 years until Patrick Lee's group reported that Erb-B or Ras transformation allows reovirus replication in mouse NIH-3T3 cells that are poorly permissive under usual infection conditions.2Strong JE Coffey MC Tang D Sabinin P Lee PW The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus.EMBO J. 1998; 17: 3351-3362Crossref PubMed Scopus (461) Google Scholar Later work showed that this effect was achieved via the RalGEF/p38 effector of Ras.3Norman KL Hirasawa K Yang AD Shields MA Lee PW Reovirus oncolysis: the Ras/RalGEF/p38 pathway dictates host cell permissiveness to reovirus infection.Proc Natl Acad Sci USA. 2004; 101: 11099-11104Crossref PubMed Scopus (147) Google Scholar Initially, this increased viral replication was assumed to be caused by the inhibition of the interferon-inducible RNA-activated cellular protein kinase (PKR) in cells expressing an activated Ras pathway. This inhibition of PKR should increase synthesis of viral proteins, whereas normally PKR activation subsequent to reovirus infection should prevent viral replication via an inhibition of protein synthesis.4Shmulevitz M Marcato P Lee PW Unshackling the links between reovirus oncolysis, Ras signaling, translational control and cancer.Oncogene. 2005; 24: 7720-7728Crossref PubMed Scopus (73) Google Scholar In agreement with this model, it was also reported that a reovirus mutant with increased interferon sensitivity—and thus postulated to be more sensitive to the effect of PKR—showed limited growth in parental NIH-3T3 cells but replicated efficiently in Ras-transformed cells.5Rudd P Lemay G Correlation between interferon sensitivity of reovirus isolates and ability to discriminate between normal and Ras-transformed cells.J Gen Virol. 2005; 86: 1489-1497Crossref PubMed Scopus (31) Google Scholar However, PKR inhibition may not always directly correlate with the ability of reovirus to replicate in a given cell type. In fact, PKR can increase replication of some reovirus strains in mouse embryo fibroblasts.6Smith JA Schmechel SC Raghavan A Abelson M Reilly C Katze MG et al.Reovirus induces and benefits from an integrated cellular stress response.J Virol. 2006; 80: 2019-2033Crossref PubMed Scopus (117) Google Scholar Two articles published in this issue of Molecular Therapy by the groups of Lee (Marcato et al.7Marcato P Shmulevitz M Pan D Stoltz D Lee PWK Ras transformation mediates reovirus oncolysis by enhancing virus uncoating, particle infectivity, and apoptosis-dependent release.Mol Ther. 2007; 15: 1522-1530Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and Forsyth (Alain et al.8Alain T Kim TSY Lun XQ Liacini A Schiff LA Senger DL et al.Proteolytic disassembly is a critical determinant for reovirus oncolysis.Mol Ther. 2007; 15: 1512-1521Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) shed new light on the mechanisms responsible for the preferential replication of reovirus in transformed cells, leading to viral oncolysis. The data presented suggest that multiple events in the viral replication cycle, rather than simply translational regulation via PKR inhibition, are regulated by the transformed status of the host cell. Uncoating of virions at early times after infection was identified in both articles as a critical step that determines the ability of reovirus to preferentially infect Ras-transformed rather than parental NIH-3T3 cells. Removal of outer capsid proteins of the reovirus virion during uncoating normally relies on the activity of cellular lysosomal proteases such as cathepsins.9Chandran K Nibert ML Animal cell invasion by a large nonenveloped virus: reovirus delivers the goods.Trends Microbiol. 2003; 11: 374-382Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Proteolytic activity is thus potentially limiting for reovirus multiplication. It was previously demonstrated that pretreatment of virions with extracellular proteases allows efficient reovirus infection of cells that are otherwise poorly infected.10Golden JW Linke J Schmechel S Thoemke K Schiff LA Addition of exogenous protease facilitates reovirus infection in many restrictive cells.J Virol. 2002; 76: 7430-7443Crossref PubMed Scopus (55) Google Scholar,11Wetzel JD Chappell JD Fogo AB Dermody TS Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.J Virol. 1997; 71: 299-306Crossref PubMed Google Scholar Similarly, during establishment of reovirus persistence, host cells acquire resistance to the virus by decreasing the level of active cathepsins, whereas coevolution results in increased ability of the virus to uncoat even at reduced protease levels.12Dermody TS Molecular mechanisms of persistent infection by reovirus.Curr Top Microbiol Immunol. 1998; 233: 1-22PubMed Google Scholar In the new articles, cleavage of reovirus outer capsid proteins is shown to be more rapid and complete in Ras-transformed as compared with parental NIH-3T3 cells. In addition, Alain et al. demonstrate that protease treatment of reovirus virions significantly increases their ability to infect parental cells. A similar observation was previously reported13Lemay G Barkati S Rudd P Danis C Uncoating and interferon sensitivity of reovirus isolates: effect on replication in transformed cells.Eighth International Symposium on Double-Stranded RNA Viruses. Il Ciocco, Castelvecchio Pascoli, Italy2003Google Scholar and is consistent with the increase in cathepsin levels in human tumor cells and Ras-transformed NIH-3T3 cells.14Chambers AF Colella R Denhardt DT Wilson SM Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells.Mol Carcinog. 1992; 5: 238-245Crossref PubMed Scopus (87) Google Scholar Alain et al. used the same experimental approach to demonstrate that the resistance of certain human glioma cell lines15Wilcox ME Yang W Senger D Rewcastle NB Morris DG Brasher PM et al.Reovirus as an oncolytic agent against experimental human malignant gliomas.J Natl Cancer Inst. 2001; 93: 903-912Crossref PubMed Scopus (194) Google Scholar to reovirus infection is due to lack of efficient uncoating in vitro, whereas tumors established from these cells are sensitive to reovirus because of a “protease cloud” in the tumor microenvironment, which includes increased cathepsin activity. Interestingly, the possibility that viral persistence could lead to reovirus-resistant tumor cells was recently investigated.16Alain T Kim M Johnston RN Urbanski S Kossakowska AE Forsyth PA et al.The oncolytic effect in vivo of reovirus on tumour cells that have survived reovirus cell killing in vitro.Br J Cancer. 2006; 95: 1020-1027Crossref PubMed Scopus (29) Google Scholar,17Kim M Egan C Alain T Urbanski SJ Lee PW Forsyth PA et al.Acquired resistance to reoviral oncolysis in Ras-transformed fibrosarcoma cells.Oncogene. 2007; (published online 8 January 2007 (doi:10.1038/sj.onc.1210189))Google Scholar Again, tumors produced by cells that are resistant in vitro are sensitive to reovirus oncolysis in vivo. In addition to early uncoating events, Marcato et al. report that late events, leading to assembly of infectious virions, are also affected by the Ras status of the host cells. Virions released from transformed cells exhibit a higher specific infectivity, an original and unexpected observation. Gross alteration of virion morphology was ruled out, suggesting a subtle effect on virion structure or stability. One possibility, not discussed by the authors, is increased synthesis of the σ1 viral host cell-binding protein in transformed cells, due to PKR inhibition. Synthesis of σ1 is believed to be more sensitive to PKR than that of other reovirus proteins.18Samuel CE Brody MS Biosynthesis of reovirus-specified polypeptides. 2-aminopurine increases the efficiency of translation of reovirus s1 mRNA but not s4 mRNA in transfected cells.Virology. 1990; 176: 106-113Crossref PubMed Scopus (19) Google Scholar It is also possible that the cellular environment of Ras-transformed cells relieves the pressure to maintain virus resistance to PKR, or sensitivity to uncoating proteases, and thus could select viruses with better replicative properties. Further work will be needed to distinguish between these possibilities and to determine if the specific infectivity of virions released from different types of human cells are also affected by cellular transformation. In evaluating oncolytic potential, virus replication, as well as the ability of the virus to preferentially destroy transformed/cancer cells, must be taken into account and may not necessarily be related. Marcato et al. indicate that apoptosis preferentially occurs in Ras-transformed NIH-3T3 cells as compared with parental cells. Using inhibitors of apoptosis, they demonstrate that apoptosis contributes to viral release and thus to viral propagation. This result is consistent with previous reports indicating that an increase in apoptosis, rather than viral replication per se, may be responsible for the sensitivity of human and mouse cancer cell lines to reovirus oncolysis.19Smakman N van den Wollenberg DJ Elias SG Sasazuki T Shirasawa S Hoeben RC et al.KRAS(D13) promotes apoptosis of human colorectal tumor cells by ReovirusT3D and oxaliplatin but not by tumor necrosis factor-related apoptosis-inducing ligand.Cancer Res. 2006; 66: 5403-5408Crossref PubMed Scopus (40) Google Scholar,20Smakman N van den Wollenberg DJ Borel Rinkes IH Hoeben RC Kranenburg O Sensitization to apoptosis underlies KrasD12-dependent oncolysis of murine C26 colorectal carcinoma cells by reovirus T3D.J Virol. 2005; 79: 14981-14985Crossref PubMed Scopus (46) Google Scholar Finally, accumulating evidence suggests that the importance of Ras activation for reovirus oncolysis and its mode of action should be reconsidered. Analysis of a panel of human colorectal tumor cell lines has shown that viral replication and cell lysis do not correlate with Ras activation status19Smakman N van den Wollenberg DJ Elias SG Sasazuki T Shirasawa S Hoeben RC et al.KRAS(D13) promotes apoptosis of human colorectal tumor cells by ReovirusT3D and oxaliplatin but not by tumor necrosis factor-related apoptosis-inducing ligand.Cancer Res. 2006; 66: 5403-5408Crossref PubMed Scopus (40) Google Scholar, even though Ras inactivation in certain murine and human cell lines prevents viral-induced apoptosis.19Smakman N van den Wollenberg DJ Elias SG Sasazuki T Shirasawa S Hoeben RC et al.KRAS(D13) promotes apoptosis of human colorectal tumor cells by ReovirusT3D and oxaliplatin but not by tumor necrosis factor-related apoptosis-inducing ligand.Cancer Res. 2006; 66: 5403-5408Crossref PubMed Scopus (40) Google Scholar,20Smakman N van den Wollenberg DJ Borel Rinkes IH Hoeben RC Kranenburg O Sensitization to apoptosis underlies KrasD12-dependent oncolysis of murine C26 colorectal carcinoma cells by reovirus T3D.J Virol. 2005; 79: 14981-14985Crossref PubMed Scopus (46) Google Scholar Alain et al.did not detecta change in the Ras activation status in reovirus-sensitive tumors derived from in vitro–resistant glioma cells. Furthermore, mouse L929 cells—classically used for reovirus replication—have a negligible level of Ras activation. Thus, despite its importance in some cellular models, such as NIH-3T3 cells, Ras activation may not be sufficient or necessary for reovirus oncolysis. Future research efforts directed toward a better understanding of reovirus interactions with various host cells—as exemplified by the two articles herein—should facilitate the selection of viruses optimized for replication in transformed/cancer cells while sparing parental cells. Of interest for the pursuit of this objective is the recent description of a long-awaited plasmid-based reverse genetics system for double-stranded RNA viruses.21Kobayashi T Antar AAR Boehme KW Danthi P Eby EA Guglielmi KM et al.A plasmid-based reverse genetics system for animal double-stranded RNA viruses.Cell Host Microbe. 2007; 1: 147-157Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar Each reovirus gene could be subjected to site-directed mutagenesis and introduced into an infectious virus using a relatively simple transfection procedure. Together with the interest generated by the potential of reovirus as an oncolytic agent, this novel reverse genetics approach is likely to mark the beginning of an exciting era in reovirus research.

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