NSP1

About: NSP1 is a research topic. Over the lifetime, 248 publications have been published within this topic receiving 12044 citations.

Novel Functions of the Alphavirus Nonstructural Protein nsP3 C-Terminal Region

Abstract: The functions of the alphavirus-encoded nonstructural protein nsP3 during infection are poorly understood. In contrast, nsP1, nsP2, and nsP4 have known enzymatic activities and functions. A functional analysis of the C-terminal region of nsP3 of Semliki Forest virus revealed the presence of a degradation signal that overlaps with a sequence element located between nsP3 and nsP4 that is required for proteolytic processing. This element was responsible for the short half-life (1 h) of individually expressed nsP3, and it also was functionally transferable to other proteins. Inducible cell lines were used to express native nsP3 or truncated mutants. The removal of 10 C-terminal amino acid (aa) residues from nsP3 increased the half-life of the protein approximately 8-fold. While the deletion of 30 C-terminal aa residues resulted in a similar stabilization, this deletion also changed the cellular localization of nsP3. This truncated mutant no longer exhibited a punctate localization in the cytoplasm, but instead filamentous stretches could be formed around the nuclei of induced cells, suggesting the existence of an additional functional element upstream of the degradation signal. C-terminally truncated uncleavable polyprotein P12(CA)3del30 was localized diffusely, which is in contrast to P12(CA)3, which is known to be associated with vesicle membranes. The induction of nsP3 or its truncated forms reduced the efficiency of virus multiplication in corresponding cells by affecting different steps of the infection cycle. The expression of nsP3 or a mutant lacking the 10 C-terminal aa residues repressed the establishment of infection, while the expression of nsP3 lacking 30 C-terminal aa residues led to the reduced synthesis of subgenomic RNA.

Whole genomic characterization of a human rotavirus strain B219 belonging to a novel group of the genus Rotavirus.

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

Rotavirus subunit vaccine

Abstract: The present invention is directed to the generation and use of recombinant rotavirus fusion proteins as immunogens to produce a protective immune response from immunized individuals. In one embodiment, the present invention contemplates a recombinant rotavirus fusion protein vaccine composition comprising a rotavirus subunit protein or immunogenic fragment thereof, and an adjuvant in combination with the recombinant rotavirus subunit fusion protein. In one aspect of this embodiment, the recombinant rotavirus fusion protein comprises a rotavirus subunit protein and a fusion partner protein in genetic association with the rotavirus subunit protein, wherein the fusion partner protein does not interfere with expression and immunogenicity of the rotavirus subunit protein, the fusion partner protein prevents complex formation by the rotavirus subunit protein, and the fusion partner protein facilitates purification of the recombinant rotavirus fusion protein. In another aspect of this embodiment, the rotavirus subunit protein is selected from the group consisting of VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 or NSP5. In yet another aspect of this embodiment, the rotavirus subunit protein is VP6.

A human rotavirus with rearranged genes 7 and 11 encodes a modified NSP3 protein and suggests an additional mechanism for gene rearrangement.

Abstract: Group A rotaviruses are the main cause of viral gastroenteritis in infants and in the young of many animal species. Their genome consists of 11 segments of double-stranded RNA (dsRNA) which can be separated by polyacrylamide gel electrophoresis (PAGE). Electropherotype profiles of rotavirus dsRNA typically show four size classes of segments according to their molecular weight (10). Variations in the mobility of individual RNA segments allow a genetic characterization of rotavirus strains. However, group A rotaviruses showing unusual electropherotypes in which segments of standard size are replaced by rearranged forms of larger size have been described. Such viruses with a rearranged genome (for a review, see reference 9) were first isolated from chronically infected immunodeficient children (30) and later recovered either from asymptomatically infected immunocompetent children (5) or from animals (4, 33, 41). Rotaviruses with genome rearrangements were also generated in vitro by serial passage at a high multiplicity of infection of animal (16, 38), or human (19, 27) strains. Rotaviruses carrying rearranged genes are generally not defective, and the rearranged segments can reassort in vitro and replace their normal counterparts structurally and functionally (1, 6, 14). Gene rearrangements in human rotaviruses recovered from stool samples mostly involve segment 11 and less frequently involve segments 6, 8, 9, and 10. It is not known whether the rearrangements in segment 11 occur more frequently or if viruses with a rearrangement in segment 11 have some selective advantage so that they are detected more easily (10). Gene rearrangements generated in vitro have also been reported for segment 5 of bovine (16, 42) and segment 7 of human (19, 27) rotaviruses. Nucleotide sequences of rearranged genes from several group A rotavirus strains have been described (3, 12, 13, 15, 25, 27, 28, 36, 38, 42). In most cases, the rearrangement resulted from a partial head-to-tail duplication of the gene: the sequence included a normal 5′ untranslated region (UTR) followed by a normal open reading frame (ORF). The duplication started from various positions after the stop codon and extended up to the 3′ end, leading to a long 3′ UTR (9). Thus, the rearranged gene expressed a normal protein product (3, 27, 38). However, Tian et al. described two bovine rotavirus variants with rearrangements in the gene 5 that modified the ORF (42). The resulting viruses retained their capacity to grow in cell culture, although they expressed modified NSP1 proteins (15, 42). So far, no mosaic structures due to an intermolecular recombination have been described in rearranged genes. Thus, genome rearrangements have been proposed to play a part in the evolution of rotaviruses (beside point mutations and gene reassortments) and to contribute to their diversity (9, 39). Moreover, it has been suggested that rearranged segments containing a partial duplication of the ORF might be more efficient templates for dsRNA synthesis than are their homologous wild-type counterparts and thus may be preferentially selected during viral replication (29). The mechanism by which genome rearrangements occur in rotavirus genes has yet not been defined, and different models have been proposed (see reference 9 for a review). Current hypotheses suggest that the RNA-dependant RNA polymerase of the virus may jump back on its template during either the transcription (plus-strand synthesis) (20) or the replication (minus-strand synthesis) (9) step. Direct repeats that might favor the polymerase switch have been found close to the rearrangement site in some cases (3, 13, 20, 38) but not in others (25, 36). In this paper we report the analysis of two rearranged genes (gene 7 and gene 11) in a group A human rotavirus isolated from an immunodeficient child. The rearrangement in gene 7 was very unusual because it contained two complete ORFs. The rearranged gene 7 underwent further evolution in vitro, with a change in the ORF leading to the expression of a modified NSP3 protein. Furthermore, the comparison of the two rearranged genes to their normal homologues and the computer modeling of their mRNAs led us to propose a mechanism for rearrangements in rotavirus genes based on the existence of secondary structures between the 3′ and 5′ ends of the plus-strand RNAs. Similarly to the model of picornaviruses in which regions of high local secondary structure such as hairpins or stem-loops have been proposed as hot spots for RNA recombination (22, 35, 43, 46), secondary structures in rotavirus mRNAs might correspond to hot spots for genome rearrangements.

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.