Showing papers on "NSP1 published in 2005"

Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3.

Abstract: IFN regulatory factor 3 (IRF3), a constitutively expressed protein localizing largely to the cytoplasm, is a primary effector of the innate immune response. Infection can trigger the phosphorylation, dimerization, and nuclear translocation of IRF3, where the factor stimulates the expression and release of IFN. In this study, we determined that the rotavirus gene 5 product, nonstructural protein 1 (NSP1), interacts with IRF3 in the infected cell. To understand the importance of the interaction, we compared IRF3 activation by rotaviruses expressing wild-type and C-truncated forms of NSP1. The analysis showed that IRF3 underwent dimerization and nuclear translocation and stimulated IFN promoter activity in infected cells expressing truncated NSP1. In contrast, infected cells expressing wild-type NSP1 were characterized by the rapid degradation of IRF3 during the replication cycle, severe decreases in IRF3 dimerization and nuclear translocation, and lack of IFN promoter activity. The implication of these results, that wild-type NSP1 is an antagonist of the IFN-signaling pathway, was confirmed in transient expression assays, which showed that wild-type NSP1, but not the C-truncated protein, induced the degradation of IRF3 fusion proteins. Related experiments indicated that NSP1 mediates IRF3 degradation through a proteasome-dependent pathway. The critical role of NSP1 in promoting cell-to-cell spread of rotavirus was demonstrated by using gene 5-specific short interfering RNAs in plaque assays. Although several viruses have been described that subvert the innate immune response by preventing IRF3 activation, rotavirus is identified as one that accomplishes this task by inducing the degradation of IRF3.

The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication.

Abstract: The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.

Identification of Adult Mouse Neurovirulence Determinants of the Sindbis Virus Strain AR86

Abstract: Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.

Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA.

Abstract: Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by a novel coronavirus (SARS-CoV), which spread to over 30 countries in early 2003. Until recently, no specific vaccines and effective drugs have been available to protect patients from infection by this virus. To exploit a new strategy to fight this disease, we investigated the effect of interference RNA (RNAi) on the virus infection and replication with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), plaque-forming, Western-blot and real-time PCR assays. Results showed that the plasmid-derived siRNAs targeting the non-structural protein 1 (NSP1) sequence of the SARS-CoV genome could specifically inhibit the expression of the NSP1 sequence and effectively suppress the replication and propagation of SARS-CoV in cultured Vero E6 cell lines. The expression of the Spike and Nucleoprotein genes of SARS-CoV at mRNA and protein levels in small interfering (si)RNA-expressing cells was significantly less than that in controls when analysed with PCR and Western-blot assays, 3 days post SARS-CoV infection. Our study provides strong evidence that the NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the effect of the siRNAs probably mainly resulted from effects on global reduction of subgenome synthesis and subsequent protein expression of SARS-CoV.