Showing papers on "NSP1 published in 2012"

Severe Acute Respiratory Syndrome Coronavirus nsp1 Facilitates Efficient Propagation in Cells through a Specific Translational Shutoff of Host mRNA

Abstract: Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is an enveloped virus containing a single-stranded, positive-sense RNA genome. Nine mRNAs carrying a set of common 5' and 3' untranslated regions (UTR) are synthesized from the incoming viral genomic RNA in cells infected with SCoV. A nonstructural SCoV nsp1 protein causes a severe translational shutoff by binding to the 40S ribosomal subunits. The nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5'UTR of host mRNA. However, the mechanism by which SCoV viral proteins are efficiently produced in infected cells in which host protein synthesis is impaired by nsp1 is unknown. In this study, we investigated the role of the viral UTRs in evasion of the nsp1-mediated shutoff. Luciferase activities were significantly suppressed in cells expressing nsp1 together with the mRNA carrying a luciferase gene, while nsp1 failed to suppress luciferase activities of the mRNA flanked by the 5'UTR of SCoV. An RNA-protein binding assay and RNA decay assay revealed that nsp1 bound to stem-loop 1 (SL1) in the 5'UTR of SCoV RNA and that the specific interaction with nsp1 stabilized the mRNA carrying SL1. Furthermore, experiments using an SCoV replicon system showed that the specific interaction enhanced the SCoV replication. The specific interaction of nsp1 with SL1 is an important strategy to facilitate efficient viral gene expression in infected cells, in which nsp1 suppresses host gene expression. Our data indicate a novel mechanism of viral gene expression control by nsp1 and give new insight into understanding the pathogenesis of SARS.

Genetic Evidence of a Long-Range RNA-RNA Interaction between the Genomic 5′ Untranslated Region and the Nonstructural Protein 1 Coding Region in Murine and Bovine Coronaviruses

Abstract: Higher-order RNA structures in the 5' untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ∼36% nucleotide sequence divergence. In a previous study, each of three 5'-end-proximal cis-acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stem-loops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ∼200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5' UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5' UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5' UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture.

Suppression of immune responses in pigs by nonstructural protein 1 of Porcine reproductive and respiratory syndrome virus

Abstract: Porcine reproductive and respiratory syndrome (PRRS) is characterized by a delayed and defective adaptive immune response. The viral nonstructural protein 1 (NSP1) of the PRRS virus (PRRSV) is able to suppress the type I interferon (IFN) response in vitro. In this study, recombinant adenoviruses (rAds) expressing NSP1 (rAd-NSP1), glycoprotein 5 (GP5) (rAd-GP5), and the NSP1-GP5 fusion protein (rAd-NSP1-GP5) were constructed, and the effect of NSP1 on immune responses was investigated in pigs. Pigs inoculated with rAd-NSP1 or rAd-NSP1-GP5 had significantly lower levels of IFN-γ and higher levels of the immunosuppressive cytokine IL-10 than pigs inoculated with rAd-GP5, wild-type adenovirus, or cell culture medium alone. The antibody response to vaccination against classic swine fever virus (CSFV) was significantly decreased by inoculation of NSP1 7 d after CSFV vaccination in pigs. Thus, NSP1-mediated immune suppression may play an important role in PRRSV pathogenesis.

Purification, crystallization and preliminary X-ray analysis of nonstructural protein 2 (nsp2) from avian infectious bronchitis virus

Abstract: Avian infectious bronchitis virus (IBV) is a member of the group III coronaviruses, which differ from the other groups of coronaviruses in that they do not encode the essential pathogenic factor nonstructural protein 1 (nsp1) and instead start with nsp2. IBV nsp2 is one of the first replicase proteins to be translated and processed in the viral life cycle; however, it has an entirely unknown function. In order to better understand the structural details and functional mechanism of IBV nsp2, the recombinant protein was cloned, overexpressed in Escherichia coli, purified and crystallized. The crystals diffracted to 2.8 A resolution and belonged to space group P21, with unit-cell parameters a = 57.0, b = 192.3, c = 105.7 A, β = 90.8°. Two molecules were found in the asymmetric unit; the Matthews coefficient was 3.9 A3 Da−1, corresponding to a solvent content of 68.2%.

Rearranged bovine rotavirus production through cultivation of virus by high multiplicity of infection (moi) in cell culture and amplification of non-structural genes using rt-pcr

Abstract: Objective: Group A rotaviruses (GARV) are responsible for the vast majority of severe diarrhea worldwide that kills an estimated 600,000-870,000 children annually. Since infantile gastroenteritis is a main health problem, therefore diagnosis and treatment of this disease is crucial. Gene rearrangements have been detected in vitro during serial passages of the virus at a high multiplicity of infection (MOI) in cell culture, as well as in chronically infected immunodeficient individuals. In this study, we developed an RT-PCR method to detect and diagnose the standard and gene rearranged bovine rotavirus. Methods: Rotavirus RNA was extracted from confluent monolayers of infected MA-104 cells, stained with silver nitrate, and then electrophoresed in a 10% polyacrylamide gel. The full-length gene products that encoded the NSP1, 2, and 3 genes of the standard and rearranged rotavirus were amplified by RT-PCR using specific primers. Results: We observed rearranged NSP1 and NSP3 genes that had different migration patterns seen with polyacrylamide gel electrophoresis. NSP1, 2, and 3 gene segments from standard and rearranged rotaviruses were amplified by RT-PCR, then the complete nucleotide sequence of each gene was subjected to sequencing. The results showed the generation of gene rearrangement through serial passages of the bovine rotavirus RF strain. Conclusion: Serial passage of rotavirus in cell culture at a high MOI and chronic infection in immunodeficient target groups might alter rotavirus evolution. The methods utilized for detection and characterization of rotaviruses are continually evolving and being refined. Data collection is necessary to understand the molecular and antigenic features of the rotavirus in order to have a successful implementation of rotavirus studies and the development of a rotavirus vaccine. This study shows the importance of genetic variation and can provide valuable information about the amplification, diversity, biology, and evolution of rotaviruses.

The role of NSP1 in rotavirus pathogenesis

Abstract: NSP1, a non-structural protein encoded by rotavirus gene segment 5, has been suggested as a virulence determinant for rotavirus and to function as an antagonist of the interferon signalling pathway. Although non-essential for rotavirus replication in cell culture, and is the least conserved in all rotavirus proteins, NSP1 from different rotavirus strains of different species has been demonstrated to interact with several cellular proteins involved in the IFNβ induction pathway. NSP1 from a bovine rotavirus strain (UKtc) has been shown to interact with and to degrade IRF3 in a proteasome dependent manner whereas NSP1 from a porcine rotavirus strain (OSU) fails to target IRF3 but is able to interfere with IFNβ production via similar targeting of β-TrCP. The research presented in this thesis sought to gain a better understanding of the molecular determinants of NSP1 specificity for targeting the IFNβ pathway by mapping the regions in NSP1 sequences responsible for targeting specific cellular proteins. NSP1 hybrid constructs with sequences from both UKtcNSP1 and OSUNSP1 were generated and their interactions with both IRF3 and β-TrCP were tested in a series of assays. The initial attempts to map interaction sites using the mammalian two-hybrid assay were not successful. No reporter plasmid signal was generated indicating the expected interaction. The failure of this assay might be due to the insufficient expression of the NSP1 proteins as subsequent modification of the expression vector was shown to improve the expression level of NSP1 proteins in subsequent reporter assay analysis. Using IFNβ promoter reporter assays to demonstrate the functional consequence of NSP1 action in IRF3, it was found that the constructs containing the entire Cterminal part of UKtcNSP1 were able to reduce IRF3-induced IFNβ promoter activity. Such constructs also caused IRF3 degradation in a proteasome dependent manner in agreement with previous studies. However, the sequence containing the last 135 amino acids from UKtcNSP1 was not sufficient for these activities. Collectively, these data suggested that the sequence between amino acid position 165 and 135 from the C-terminus are required for this interaction and subsequent degradation of IRF3. Similar experiments focused on determining the interaction site for β-TrCP on NSP1 were more difficult to interpret according the data presented. Unexpectedly in the light of published data, not only OSUNSP1 was able to degrade β-TrCP but UKtcNSP1 appeared to have the similar effect, as well as two reciprocal pairs of NSP1 hybrid constructs. In summary, it appears that sequences from the C-terminal part of UKtcNSP1 can function in a heterogeneous NSP1 context to target IRF3 from human cells. Further analysis is clearly required to fulfil the understanding of the role of NSP1 in rotavirus pathogenesis, including its interaction with β-TrCP.