Roles of Nonstructural Protein nsP2 and Alpha/Beta Interferons in Determining the Outcome of Sindbis Virus Infection

Abstract

Alphaviruses are a group of significant human and animal pathogens. The nearly 30 members of this genus are transmitted by mosquitoes to higher vertebrates that serve as amplifying hosts (54). Alphaviruses cause different diseases but have similar replication strategies and life cycles. In insect vectors, they cause persistent lifelong infections and viral replication does not critically affect the viability of the hosts (51). These viruses also often establish persistent infection in cultured mosquito cells. In contrast, vertebrate hosts usually develop acute infection that often results in disease prior to virus clearance by the immune system (27, 31). The infection of susceptible vertebrate cells typically leads to rapid cytopathic effect (CPE) (54) and cell death.Sindbis virus (SIN) is the prototype member of the Alphavirus genus. It can replicate productively in a wide variety of cell lines of insect and vertebrate origin and causes age-dependent encephalomyelitis in mice (28). As do all other alphaviruses, SIN enters the cells via receptor-mediated endocytosis. pHdependent fusion of viral and endosomal membranes leads to release of nucleocapsids into the cytoplasm (12) followed by nucleocapsid disassembly and genome replication (64).The SIN genome is a single RNA molecule of positive polarity, 11.7 kb in length. It contains a 5Ј methylguanylate cap and a 3Ј polyadenylate tract and is translated by host-cell machinery similar to cellular mRNAs (52). The 5Ј two-thirds of the genome encodes the nonstructural proteins (nsPs), which comprise the viral components of the RNA replicase/transcriptase required for replication of the viral genome and transcription of subgenomic RNA. The 4.1-kb subgenomic RNA corresponds to the 3Ј third of the genome and is translated into structural proteins that form virus particles. Replication of SIN is very rapid and leads to high-level accumulation of virusspecific RNAs and structural proteins. Finally, a large number of viral particles released by budding from the cell surface disseminate the infection. Viral replication profoundly affects cell metabolism, with downregulation of host cell protein synthesis playing the central role (31). Cells lose integrity and die within 24 to 48 h postinfection (p.i.). In many cell types, death is accompanied by apoptotic changes (37). Replication of viral RNA and/or accumulation of SIN nsPs is sufficient to cause translational shutoff and cell death. However, expression of viral structural proteins significantly accelerates the development of CPE (18).In recent studies, we selected SIN self-replicating RNAs (replicons) that were capable of persisting in some vertebrate cell lines for an unlimited number of passages without causing CPE (1, 16). These noncytopathic replicons replicated less efficiently than the parent and contained point mutations in the gene encoding one of the nsPs, nsP2. One adaptive mutation, P 726 3L, was at the same position as that found in the SIN-1 variant that also exhibits reduced cytopathogenicity (11,63).

Prenylome profiling reveals S-farnesylation is crucial for membrane targeting and antiviral activity of ZAP long-isoform

“…HEK293T cells were transfected using XtremeGENE 9 DNA Transfection Reagent (Roche) whereas HeLa cells and MEFs were transfected using Lipofectamine 2000 (Invitrogen). Sindbis virus encoding the enhanced green fluorescent protein (EGFP) from a duplicated subgenomic promoter (TE/5′2J/GFP) has been previously reported (46). Stocks were prepared and titers determined on BHK-J cells with 10-fold serial dilutions of sample, and then plaques were visually enumerated after crystal violet staining, as previously described (25); multiplicities of infection (MOI) were calculated based on BHK-J-derived titers.…”

Identification of a Soluble NADPH Oxidoreductase (BmNOX) with Antiviral Activites in the Gut Juice ofBombyx mori

“…13,14) In Sindbis virus-host cell interactions, a viral nonstructural protein, nsP2, which is a component of the replicative enzyme complex required for replication and transcription of viral RNAs, plays a role in suppressing the antiviral response in Sindbis virus-infected cells. 15) In this study, we report the identification, purification, and characterization of a 26.5-kDa gut-juice protein showing antiviral activities. The expression of this protein in larvae from seven multivoltine and six bivoltine strains of B. mori was also evaluated to draw y To whom correspondence should be addressed.…”

A Semliki Forest virus vector engineered to express IFNα induces efficient elimination of established tumors

“…It has been reported that this shut-off affects many proteins involved in the IFN response, a mechanism developed by alphaviruses to escape antiviral innate responses. 38 This virus-induced type I IFN response can synergize with other cytokines or tumor antigens expressed from the SFV vector, resulting in good antitumor responses. 27,28,41 However, we have shown that overexpression of IFNa from SFV in the absence of other therapeutic transgenes is non-redundant and, moreover, essential to achieve a therapeutic antitumor activity.…”

“…37 In addition, BHK cells can support the replication of noncytopathic alphavirus RNA mutant replicons, in contrast to cell lines with an intact type I IFN response where those replicons are rapidly lost. [38][39][40] Not only is the production of SFV-IFN feasible but IFNa expression could also be achieved in infected cells independently of endogenous type I responses. We have observed that on infection with SFV-IFN, similar levels of IFNa are expressed in TC-1 and L929 cells, in spite of the fact that TC-1, the main tumor target cells used in our study did not seem to develop a type I IFN response after infection with SFV-LacZ control virus (Figure 1f).…”