Showing papers on "NSP1 published in 2008"
TL;DR: The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed a common origin between human Wa-like rotavirus strains and porcine rotav virus strains.
Abstract: Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.
778 citations
TL;DR: It is demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that Sars- CoV nSp1 most probably plays a critical role in SARS -CoV virulence.
Abstract: The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.
396 citations
TL;DR: In this article, substitutions and deletions of the presumed catalytic cysteine and histidine residues of PCPα and PCPβ were introduced into a porcine reproductive and respiratory syndrome virus (PRRSV) infectious cDNA clone.
Abstract: The two N-terminal cleavage products, nsp1α and nsp1β, of the replicase polyproteins of porcine reproductive and respiratory syndrome virus (PRRSV) each contain a papain-like autoproteinase domain, which have been named PCPα and PCPβ, respectively. To assess their role in the PRRSV life cycle, substitutions and deletions of the presumed catalytic cysteine and histidine residues of PCPα and PCPβ were introduced into a PRRSV infectious cDNA clone. Mutations that inactivated PCPα activity completely blocked subgenomic mRNA synthesis, but did not affect genome replication. In contrast, mutants in which PCPβ activity was blocked proved to be non-viable and no sign of viral RNA synthesis could be detected, indicating that the correct processing of the nsp1β/nsp2 cleavage site is essential for PRRSV genome replication. In conclusion, the data presented here show that a productive PRRSV life cycle depends on the correct processing of both the nsp1α/nsp1β and nsp1β/nsp2 junctions.
109 citations
TL;DR: The basic structures of all B219 viral proteins are considered to be similar to those of the known rotavirus groups.
Abstract: Novel rotavirus strains B219 and ADRV-N derived from adult diarrheal cases in Bangladesh and China, respectively, are considered to belong to a novel rotavirus group (species) distinct from groups A, B, and C, by genetic analysis of five viral genes encoding VP6, VP7, NSP1, NSP2, and NSP3 In this study, the nucleotide sequences of the remaining six B219 gene segments encoding VP1, VP2, VP3, VP4, NSP4, and NSP5 were determined The nucleotide sequences of the group B human rotavirus VP1 and VP3 genes were also determined in order to compare the whole genome of B219 with those of group A, B, and C rotavirus genomes The nucleotide and deduced amino acid sequences of all B219 gene segments showed considerable identity to the ADRV-N (strain J19) sequences (877-943% and 887-987%, respectively) In contrast, sequence identity to groups A-C rotavirus genes was less than 61% However, functionally important domains and structural characteristics in VP1-VP4, NSP4, and NSP5, which are conserved in group A, B, or C rotaviruses, were also found in the deduced amino acid sequences of the B219 proteins Hence, the basic structures of all B219 viral proteins are considered to be similar to those of the known rotavirus groups
47 citations
TL;DR: Results implicate nsP2 in regulation of minus-strand synthesis and suggest that different regions of the nsP 2 MTase-like domain differentially modulate host defense mechanisms, independent of its role as the viral protease.
Abstract: The plus-strand RNA genome of Sindbis virus (SINV) encodes four nonstructural proteins (nsP1 to nsP4) that are involved in the replication of the viral RNA. The ∼800-amino-acid nsP2 consists of an N-terminal domain with nucleoside triphosphatase and helicase activities and a C-terminal protease domain. Recently, the structure determined for Venezuelan equine encephalitis virus nsP2 indicated the presence of a previously unrecognized methyltransferase (MTase)-like domain within the C-terminal ∼200 residues and raised a question about its functional importance. To assess the role of this MTase-like region in viral replication, highly conserved arginine and lysine residues were mutated to alanine. The plaque phenotypes of these mutants ranged from large/wild-type to small plaques with selected mutations demonstrating temperature sensitive lethality. The proteolytic polyprotein processing activity of nsP2 was unaffected in most of the mutants. Some of the temperature-sensitive mutants showed reduction in the minus-strand RNA synthesis, a function that has not yet been ascribed to nsP2. Mutation of SINV residue R615 rendered the virus noncytopathic and incapable of inhibiting the host cell translation but with no effects on the transcriptional inhibition. This property differentiated the mutation at R615 from previously described noncytopathic mutations. These results implicate nsP2 in regulation of minus-strand synthesis and suggest that different regions of the nsP2 MTase-like domain differentially modulate host defense mechanisms, independent of its role as the viral protease.
40 citations
TL;DR: A safe non-infectious replicon was constructed by replacing the S gene with the enhanced green fluorescent protein (eGFP) gene, which confirmed previous observation that nsp16, but not nsp1 or nsp2, was essential for efficient viral replication or transcription.
22 citations
TL;DR: It is found that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing.
Abstract: Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.
21 citations
Dissertation•
01 Jan 2008
TL;DR: It is determined that rotavirus NSP1 functions as an interferon antagonist by directing the proteasome-mediated degradation of IFNβ induction signaling components, most likely by acting as an E3 ubiquitin ligase.
Abstract: Rotaviruses cause severe gastroenteritis in mammals, including humans and livestock. Most rotavirus proteins have known functions, either as a mediator of virus replication or as a component of the infectious virus particle. The function of nonstructural protein 1 (NSP1) was unknown. However, it has been suggested that the function of NSP1 involved interactions with cellular proteins. Using the NSP1 encoded by a bovine rotavirus as the bait protein of a yeast-two hybrid interaction trap, interferon regulatory factor 3 (IRF3) was identified as an NSP1-interacting protein. Due to the importance of IRF3 in initiating an interferon response, we hypothesized that NSP1 acts to antagonize the interferon system. A comprehensive set of experiments yielded the following observations. Interferon-β (IFNβ) induction was blocked in wild-type, but not NSP1 null, infected cells. Expression of NSP1 in the absence of infection resulted in proteasome-mediated degradation of IRF3. A cysteine-rich zincbinding region near the amino-terminus of all known NSP1 sequences resembles a domain found in hundreds of E3 ubiquitin ligases. Mutational analysis of the zinc-binding domain was consistent with NSP1 acting as an E3 enzyme. The NSP1 of a murine strain of rotavirus also interacted with IRF3. The NSP1 proteins encoded by the bovine and murine rotavirus strains showed low sequence homology (37% identity), which indicated that directing degradation of IRF3 may be a common property of NSP1 proteins from many different rotavirus strains. Screening of simian, bovine, and porcine rotavirus strains indicated that the porcine strain OSU was the only wild-type strain that was unable to direct IRF3 degradation. Characterization of OSU showed that this strain was able to block IFNβ induction by antagonizing NFκB signaling, rather than IRF3 signaling. While analyzing NFκB signaling in rotavirus infected cells, we found that the F box protein, βTrCP, of the Skp1/Cul1/F box multi-subunit E3 enzyme was targeted for degradation by OSU NSP1 and the NSP1 of a bovine rotavirus strain. Together, the results presented in this dissertation have determined that rotavirus NSP1 functions as an interferon antagonist by directing the proteasome-mediated degradation of IFNβ induction signaling components, most likely by acting as an E3 ubiquitin ligase.