Showing papers on "NSP1 published in 2003"

Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4.

Abstract: Long electropherotype with Subgroup I specificity is a common feature of animal rotaviruses. In an epidemic of infantile gastroenteritis in Manipur, India, long but SG I strains predominated in the outbreak in the year 1987–88. One such strain isolated from that region, following the outbreak had G9P [19] specificity. As this is a rare combination, the gene sequences encoding VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 of this strain were analyzed. All these genes except VP7 were closely related to porcine rotaviruses (95–99% identity at amino acid level) and clustered with the porcine strains in phylogenetic analysis. In addition, it had subgroup I nature and belonged to NSP4 genotype B which is characteristic of animal rotaviruses. This is the first report of a rotavirus with VP6 and NSP4, two crucial proteins thought to be involved in host range restriction and pathogenicity, were of porcine origin and caused diarrhoea in a human host. Among the genes of this strain sequenced so far, only VP7 had highest identity to human strains at amino acid level. This study suggests reassortment may be occurring between human and other animal strains and some of the reassortant viruses may be virulent to humans.

An Attenuating Mutation in nsP1 of the Sindbis-Group Virus S.A.AR86 Accelerates Nonstructural Protein Processing and Up-Regulates Viral 26S RNA Synthesis

Abstract: The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.

Equine arteritis virus non-structural protein 1, an essential factor for viral subgenomic mRNA synthesis, interacts with the cellular transcription co-factor p100.

Abstract: Non-structural protein 1 (nsp1), the N-terminal subunit of the replicase polyprotein of the arterivirus Equine arteritis virus (EAV), is essential for viral subgenomic mRNA synthesis, but fully dispensable for genome replication. However, at the molecular level, the role of nsp1 in EAV subgenomic mRNA synthesis is poorly understood. A yeast two-hybrid screen did not reveal interactions between EAV nsp1 and other viral non-structural proteins or the nucleocapsid protein, although both nsp1 and the nucleocapsid protein were found to form homomers. Subsequently, a yeast two-hybrid screen of a HeLa cell cDNA library was performed using nsp1 as bait. Remarkably, this analysis revealed (potential) interactions between EAV nsp1 and factors that are involved in host cell transcriptional regulation. The interaction of nsp1 with one of these proteins, p100, a transcription co-activator that also interacts with regulatory proteins of other viruses, was confirmed by mutual co-immunoprecipitation from lysates of EAV-susceptible mammalian cells.

Serologic and Genomic Characterization of a G12 Human Rotavirus in Thailand

Abstract: The G and P type specificity of the human rotavirus strain T-152 (G12P[9]) isolated in Thailand was serologically confirmed with G12-specific monoclonal antibodies prepared in this study by using a reference G12 strain, L26, as an immunizing antigen and a P[9]-specific monoclonal antibody, respectively. The genomic relationship of strain T-152 with representative human rotavirus strains was examined by means of Northern blot analysis. The results showed that T152 is closely related to strain AU-1 (G3P[9]). Gene 5 (NSP1 gene) of T152, which did not hybridize with those of any other strains examined, was characterized by sequence determination. The T152 NSP1 gene is 1,652 nucleotides in length, encodes 493 amino acids, and exhibits low identity to those of representative human and animal rotaviruses.

Genomic Diversity Through Gene Reassortment and Antigenic Drift and Molecular Epidemiology of Rotaviruses in India

Abstract: A review. Long term epidemiol. studies on both symptomatic and asymptomatic rotavirus infections during the period 1988 to 1999 in Bangalore revealed a consistently high rate (.apprx.60%) of asymptomatic infection of neonates born in hospitals/clinics by unusual P[11]G10 type strains. The prototype neonatal strain I321 was shown to be a reassortant between a P[11]G10 bovine rotavirus and a human rotavirus with all the genes, except for two genes encoding the nonstructural proteins NSP1 and NSP3, derived from the bovine parent. Neonatal infections were totally dominated by I321-like strains. Neonatal infections appear to confer protection against subsequent rotavirus illness as evident from the two-year follow-up study of I321-infected newborn children as well as by the significant reduction in rotavirus illness in Bangalore for the last 8 years. Another reassortant neonatal strain 116E (P[11]G9) was also isolated in New Delhi. G3 type strains were more prevalent in symptomatic infections in Bangalore throughout the study period. The order of prevalence was G3 (36.6) > G2 (22.0%) > G1 (17.1%) > G4 (6.0%). There was no change in the epidemiology situation during this period. In contrast, serotype G1 was found to be more prevalent in other regions of the country. Majority of the G2-like strains were nontypeable in serotyping ELISA due to broad cross-reactivity to serotyping mAbs. This appears to be due to amino acid substitutions in the antigenic regions of VP7 as well as in VP4 and these strains probably represent a G2 subtype. An unusual G2 strain assocd. with diarrhoea, having 'long' e-type and SG1 specificity has been isolated in Manipur and appears to have been evolved by gene reassortment between a P[4]G2 human strain and a porcine strain. Direct transmission of bovine G8 strains from cattle was observed to cause severe diarrhoea in children in Mysore. Serotype G10 was found to dominate infections in cattle in India and represented 55.0% - 85.0% of the isolates in different farms across the country. Serotype G6 was negligible in Indian cattle and G8 strains were detected in all the regions (5.8%). Another surprising observation was the presence of G3 strains in significant nos. (10.7%) in all the regions. The prototype bovine G3 strain G3 was shown to be a reassortant between a bovine G8 strain and an animal G3 strain (simian, canine or equine). A novel rotavirus was isolated from a single farm in Bangalore and was shown to represent the new serotype P[21]G15. The findings from our lab. are of great significance since they provided strong epidemiol. basis for the origin of P[11]G10 and P[11]G9 reassortant asymptomatic neonatal strains in different regions of the country. Age-old Indian traditions, close proximity of majority of the Indian population with cattle, and environmental conditions appear to facilitate inter-species transmission of rotaviruses between humans and cattle and cattle and animals and evolution of novel strains in India.

Translational regulation of rotavirus gene expression.

Abstract: Rotavirus mRNAs are transcribed from 11 genomic dsRNA segments within a subviral particle. The mRNAs are extruded into the cytoplasm where they serve as mRNA for protein synthesis and as templates for packaging and replication into dsRNA. The molecular steps in the replication pathway that regulate the levels of viral gene expression are not well defined. We have investigated potential mechanisms of regulation of rotavirus gene expression by functional evaluation of two differentially expressed viral mRNAs. NSP1 (gene 5) and VP6 (gene 6) are expressed early in infection, and VP6 is expressed in excess over NSP1. We formulated the hypothesis that the amounts of NSP1 and VP6 were regulated by the translational efficiencies of the respective mRNAs. We measured the levels of gene 5 and gene 6 mRNA and showed that they were not significantly different, and protein analysis indicated no difference in stability of NSP1 compared with VP6. Polyribosome analysis showed that the majority of gene 6 mRNA was present on large polysomes. In contrast, sedimentation of more than half of the gene 5 mRNA was subpolysomal. The change in distribution of gene 5 mRNA in polyribosome gradients in response to treatment with low concentrations of cycloheximide suggested that gene 5 is a poor translation initiation template compared with gene 6 mRNA. These data define a regulatory mechanism for the difference in amounts of VP6 and NSP1 and provide evidence for post-transcriptional control of rotavirus gene expression mediated by the translational efficiency of individual viral mRNAs.

Virus-based vectors for gene expression in mammalian cells: Sindbis virus

Abstract: Publisher Summary Alphaviruses are relatively simple RNA-enveloped viruses that have been valuable models for learning about virus replication and transmission. Their wide host range, small genome length, simple gene organization, and high level of gene expression provided the original motivation to adapt them as gene-expression vectors. This chapter focuses mainly on Sindbis virus (SIN), the type species of the Alphavirus genus in the Togaviridae family. In nature, it is transmitted by a number of species of mosquitoes to vertebrates (mammals, birds, reptiles and amphibia), principally birds. Two other members of this genus, Semliki Forest virus (SFV) and Venezuelan equine encephalitis virus (VEEV) are also being developed as vectors. VEEV is a known human pathogen but a vaccine strain exists that is the basis for the VEEV vectors. The ways in which SIN vectors are being used cover a wide spectrum. In addition to the illustrations given in this chapter, there are many examples in which proteins were expressed to be able to study their cellular localization, modifications, and function. Alphaviruses enter cells through the endosomal pathway. SIN attaches to the cell surface using one of several potential receptors. It is then taken up into an endosome, that, when acidified, triggers the viral surface glycoproteins to fuse the virus membrane with the endosomal membrane, thus depositing the internal nucleocapsid into the cytoplasm. The genomic RNA is capped and polyadenylated, and is the mRNA for the viral nonstructural polyprotein. The polyprotein is proteolytically processed to produce the individual subunits, nsP1 through nsP4 by the nsP2 protease. The nonstructural proteins serve as the replicase-transcriptase that uses the genomic RNA as template to produce a full-length, negative-sense complement, which in turn is template for synthesis of additional genomic RNAs . The negative sense complement additionally contains a promoter that is used to produce a subgenomic mRNA for translation of the structural polyprotein . The capsid protein located at the amino-terminus is an autoprotease that cleaves itself from the nascent polypeptide, which is then inserted into cellular membranes and is cleaved by host cell proteases to produce the glycoproteins E1 and PE2 (precursor of the virus glycoprotein E2) and a small hydrophobic 6K protein. The capsid protein binds to genomic RNA, facilitated by a packaging signal in the nsP1-encoding sequences, to form nucleocapsids. (Packaging signals of other alphaviruses map to different parts of the nonstructural proteins coding region). The nucleocapsids interact with the cytoplasmic tail of the E2 glycoprotein, resulting in the budding and release of progeny virions through the host plasma membrane.