The non‐enveloped fusogenic avian and Nelson Bay reoviruses encode homologous 10 kDa non‐structural transmembrane proteins. The p10 proteins localize to the cell surface of transfected cells in a type I orientation and induce efficient cell–cell fusion. Mutagenic studies revealed the importance of conserved sequence‐predicted structural motifs in the membrane association and fusogenic properties of p10. These motifs included a centrally located transmembrane domain, a conserved cytoplasmic basic region, a small hydrophobic motif in the N‐terminal domain and four conserved cysteine residues. Functional analysis indicated that the extreme C‐terminus of p10 functions in a sequence‐independent manner to effect p10 membrane localization, while the N‐terminal domain displays a sequence‐dependent effect on the fusogenic property of p10. The small size, unusual arrangement of structural motifs and lack of any homologues in previously described membrane fusion proteins suggest that the fusion‐associated small transmembrane (FAST) proteins of reovirus represent a new class of membrane fusion proteins.

Reovirus, a potential cancer therapy, replicates more efficiently in Ras-transformed cells than in non-transformed cells. It was presumed that increased translation was the mechanistic basis of reovirus oncolysis. Analyses of each step of the reovirus life cycle now show that cellular processes deregulated by Ras transformation promote not one but three viral replication steps. First, in Ras-transformed cells, proteolytic disassembly (uncoating) of the incoming virions, required for onset of infection, occurs more efficiently. Consequently, threefold more Ras-transformed cells become productively infected with reovirus than non-transformed cells, which accounts for the observed increase of reovirus proteins in Ras-transformed cells. Second, Ras transformation increases the infectious-to-noninfectious virus particle ratio, as virions purified from Ras-transformed cells are fourfold more infectious than those purified from non-transformed cells. Progeny assembled in non- and Ras-transformed cells appear similar by electron microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, suggesting that Ras transformation introduces a subtle change necessary for virus infectivity. Finally, reovirus release, mediated by caspase-induced apoptosis, is ninefold more efficient in Ras-transformed cells. The combined effects of enhanced virus uncoating, infectivity, and release result in >100-fold differences in virus titers within one round of replication. Our analysis reveals previously unrecognized mechanisms by which Ras transformation mediates selective viral oncolysis.

Human mast cells are found in skin and mucosal surfaces and next to blood vessels. They play a sentinel cell role in immunity, recognizing invading pathogens and producing proinflammatory mediators. Mast cells can recruit granulocytes, and monocytes in allergic disease and bacterial infection, but their ability to recruit antiviral effector cells such as natural killer (NK) cells and T cells has not been fully elucidated. To investigate the role of human mast cells in response to virus-associated stimuli, human cord blood-derived mast cells (CBMCs) were stimulated with polyinosinic⅐polycytidylic acid, a double-stranded RNA analog, or infected with the double-stranded RNA virus, reovirus serotype 3 Dearing for 24 hours. CBMCs responded to stimulation with polyinosinic⅐polycytidylic acid by producing a distinct chemokine profile, including CCL4, CXCL8, and CXCL10. CBMCs produced significant amounts of CXCL8 in response to low levels of reovirus infection, while both skin-and lungderived fibroblasts were unresponsive unless higher doses of reovirus were used. Supernatants from CBMCs infected with reovirus induced substantial NK cell chemotaxis that was highly dependent on CXCL8 and CXCR1. These results sug- IntroductionMast cells are long-lived resident tissue cells found close to blood vessels, and are numerous at sites in close proximity to the external environment such as the skin and airways (reviewed in Galli et al 1 and Metz and Maurer 2 ). From these strategic locations they can quickly recognize and respond to invading pathogens. They are also relatively resistant to ultraviolet (UV) and gamma irradiation. [3][4][5] Upon activation, mast cells produce a wide array of mediators, including granule-associated products, such as histamine, and preformed and de novo synthesized cytokines, chemokines, and lipid mediators. They can activate and recruit effector cells, including eosinophils, 6 neutrophils, 7 and monocytes. 8 Their role in innate immune responses to bacterial infections has been clearly delineated, however their involvement in viral infections is not well understood. Mast cells express Toll-like receptor 3 (TLR3), which recognizes viral double-stranded RNA (dsRNA), 9 and they can produce type I interferons when activated through this receptor. 10 Studies examining the permissiveness of mast cells to viruses show that they can be infected by, and respond to, dengue virus, 11,12 HIV, 13,14 and respiratory syncytial virus. 15 Human mast cells produce the chemokines CCL3, CCL4, and CCL5 when infected with dengue virus, 16 and mouse mast cells produce CCL4 and CCL5 when infected with Newcastle disease virus, all of which are known natural killer (NK) cell and T-cell chemoattractants. 17 NK cells are large granular lymphocytes that can kill virally infected cells, and are crucial for the clearance of viruses during infections (reviewed in Lodoen and Lanier 18 ). The chemokines and chemokine receptors necessary for the infiltration of NK cells into virally infected tissues have recently begun to be uncover...

Reovirus is the first naturally occurring human virus reported to exploit activated Ras signaling in the host cell for infection, and is currently undergoing clinical trials as a cancer therapeutic. Recent evidence suggests that Ras transformation promotes three reoviral replication steps during the first round of infection: uncoating of the incoming virion, generation of progeny viruses with enhanced infectivity, and virus release through enhanced apoptosis. Whether oncogenic Ras also enhances reovirus spread in subsequent rounds of infection through other mechanisms has not been examined. Here, we show that compared with nontransformed cells, Ras-transformed cells are severely compromised not only in their response to IFN-β, but also in the induction of IFN-β mRNA following reovirus infection. Defects in both IFN-β production and response allow for efficient virus spread in Ras-transformed cells. We show that the MEK/ERK pathway downstream of Ras is responsible for inhibiting IFN-β expression by blocking signaling from the retinoic acid-inducible gene I (RIG-I) which recognizes viral RNAs. Overexpression of wild-type RIG-I restores INF-β expression in reovirus-infected Rastransformed cells. In vitro-synthesized viral mRNAs also invoke robust RIG-I-mediated IFN-β production in transfected nontransformed cells, but not in Ras-transformed cells. Collectively, our data suggest that oncogenic Ras promotes virus spread by suppressing viral RNA-induced IFN-β production through negative regulation of RIG-I signaling. Cancer Res; 70(12); 4912-21. ©2010 AACR.

Immunosuppression associated with ovarian cancer (OC) and resultant peritoneal carcinomatosis (PC) hampers the efficacy of many promising treatment options, including immunotherapies. It is hypothesized that oncolytic virus-based therapies can simultaneously kill OC and mitigate immunosuppression. Currently, reovirus-based anticancer therapy is undergoing phase I/II clinical trials for the treatment of OC. Hence, this study was focused on characterizing the effects of reovirus therapy on OC and associated immune microenvironment. Our data shows that reovirus efficiently killed OC cells and induced higher expression of the molecules involved in antigen presentation including major histocompatibility complex (MHC) class I, β2-microglobulin (β2M), TAP-1, and TAP-2. In addition, in the presence of reovirus, dendritic cells (DCs) overcame the OC-mediated phenotypic suppression and successfully stimulated tumor-specific CD8+ T cells. In animal studies, reovirus targeted local and distal OC, alleviated the severity of PC and significantly prolonged survival. These therapeutic effects were accompanied by decreased frequency of suppressive cells, e.g., Gr1.1+, CD11b+ myeloid derived suppressor cells (MDSCs), and CD4+, CD25+, FOXP3+ Tregs, tumor-infiltration of CD3+ cells and higher expression of Th1 cytokines. Finally, reovirus therapy during early stages of OC also resulted in the postponement of PC development. This report elucidates timely information on a therapeutic approach that can target OC through clinically desired multifaceted mechanisms to better the outcomes.