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Showing papers on "NSP1 published in 1999"


Journal ArticleDOI
TL;DR: Antiserum prepared against an amino-terminal fragment of rubella virus (RUB) nonstructural polyprotein was used to study RUB-infected Vero cells, indicating that these structures may be the sites of viral RNA synthesis.
Abstract: Antiserum prepared against an amino-terminal fragment of rubella virus (RUB) nonstructural polyprotein was used to study RUB-infected Vero cells. Replicase protein P150 was associated with vesicles and vacuoles of endolysosomal origin and later with large, convoluted, tubular membrane structures. Newly incorporated bromouridine was associated with the same structures and specifically with small membrane invaginations, spherules, indicating that these structures may be the sites of viral RNA synthesis.

70 citations


Patent
01 Nov 1999
TL;DR: In this paper, the authors proposed a recombinant rotavirus fusion protein vaccine composition, which consists of an adjuvant and a fusion partner protein in genetic association with the RPs.
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.

46 citations


Journal ArticleDOI
TL;DR: The results demonstrate that the substantial diversity of NSP1 observed among group A rotaviruses (GAR) also exists within GBRs and that a high degree of diversity also exists among NSP5 of G BRs, in contrast to GAR NSP 5.
Abstract: An ovine group B rotavirus (GBR) isolate, KB63, was isolated from faeces of a young goat with diarrhoea in Xinjiang, People’s Republic of China. Sequence determination and comparison of genes 6 and 11 with the corresponding sequences of GBR strains ADRV and IDIR showed that they were the cognate genes encoding NSP1 and NSP5, respectively. While the overall identities of nucleotide sequences between these two genes and the corresponding genes of strains ADRV and IDIR were in the range 52·6–57·2%, the identities of deduced amino acid sequences were only 34·9–46·3%. These results demonstrate that the substantial diversity of NSP1 observed among group A rotaviruses (GAR) also exists within GBRs and that a high degree of diversity also exists among NSP5 of GBRs, in contrast to GAR NSP5. The NSP1 gene of KB63 contains three ORFs, whereas the NSP1 genes of other GBR strains contain only two. ORFs 2 and 3 of the KB63 gene may be derived from a single ORF corresponding to ORF2 of other GBR strains by the usage of a stop codon created by an upstream single base deletion and single point mutations. In vitro expression studies showed that ORFs 1 and 2, but not 3, of gene 6 can be translated, suggesting that ORF2 may encode a C-terminally truncated, potentially functional product. It may play a role, together with the product of ORF1, in virus replication, as the virus can be passaged further in kids. Similarly, gene 11 can be translated in vitro. Like its counterpart in GARs, the protein encoded by gene 11 was shown to be phosphorylated in vitro.

26 citations


Journal ArticleDOI
TL;DR: Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals and supports the reclassification of Vee subtype-variety IF and subtype II viruses.

20 citations


Journal ArticleDOI
TL;DR: Phylogenetic analysis of all the non-structural genes of group A, B and C rotaviruses suggests that these viruses have diverged at a constant rate from a common ancestor.
Abstract: Genes 6, 7 and 9 of human group C rotavirus 'Bristol' strain, encoding non-structural proteins (NSP) 3, 1 and 2, respectively, were cloned and sequenced. Human group C rotavirus genome segment 6 is 1350 bp and contains a single ORF of 1231 nucleotides (encoding 402 amino acids). Genome segment 7 is 1270 bp and encodes a protein of 394 amino acids and genome segment 9 is 1037 bp and encodes a 312 amino acid protein. The human group C rotavirus genes 6, 7 and 9 showed 78, 67 and 88% sequence identity, respectively, to the corresponding porcine group C rotavirus genes. The derived protein sequences were compared with those of the porcine 'Cowden' group C and mammalian group A rotavirus strains. The human group C rotavirus NSP1 protein sequence is one amino acid longer than the porcine group C equivalent. In common with group A and porcine group C rotaviruses, the human group C rotavirus NSP1 protein has a zinc finger motif. Human group C rotavirus NSP2 has two hydrophobic heptad repeat regions, a basic, RNA-binding domain and a basic, proline-rich region. Human group C rotavirus NSP3 has both single- and double-stranded RNA-binding domains and several hydrophobic heptad repeat regions, one of which forms a leucine zipper. This work completes the molecular characterization of the non-structural proteins of a human group C rotavirus. Phylogenetic analysis of all the non-structural genes of group A, B and C rotaviruses suggests that these viruses have diverged at a constant rate from a common ancestor.

20 citations


Journal ArticleDOI
TL;DR: Reassortment of reassortants with the mutated NSP1 gene and RNA segments from heterologous strains normally replicated in cultured cells showed almost identical growth curve to that of KU, while KU showed a better replication than Aff5–16.
Abstract: Rotavirus clones Aff5–10 and Aff5–16 isolated from a bovine rotavirus strain Aff5 possess NSP1 gene which has a point mutation generating a nonsense codon and a 500 base-deletion, respectively. As a result, the two Aff5 clones encode truncated NSP1 product which lacks cysteine-rich region forming zinc finger motif. In order to analyze reassortment of these mutated NSP1 gene with RNA segments from heterologous strains, we investigated a number of reassortant clones derived from coinfection with either Aff5–10, Aff5–16 or a reference strain Aff5–13 (possessing intact NSP1 gene) and either simian rotavirus SAff11 or human rotavirus KU. In coinfection with SAff11 and Aff5–13, selection rates of Aff5–13 segments in reassortants ranged approximately from 20 to 70% (46% for NSP1 gene). In contrast, in the reassortment between SAff11 and Aff5–10 or between SAff11 and Aff5–16, selection rates of NSP1 gene from Aff5–10 and Aff5–16 were only 1% (one clone) and 0%, respectively. In reassortants from crosses KU × Aff5-clones, selection rate of Aff5–13 NSP1 gene decreased to 15%, while 11 reassortants with Aff5–10 NSP1 gene (31%) and one reassortant with Aff5–16 NSP1 gene (2%) were isolated. Reassortants with Aff5–10 NSP1 possessed a single gene (segment 9 or 11) from KU in the genetic background of Aff5–10. One reassortant clone (cl-55) with Aff5–16 NSP1 gene possessed KU gene segments 3, 4, and 8–11. When single-step growth curves were compared, the reassortant cl-55 showed almost identical growth curve to that of KU, while KU showed a better replication than Aff5–16. These results indicated that although Aff5–10 or Aff5–16 NSP1 gene encoding the truncated NSP1 is selected into reassortants much less efficiently than normal NSP1 gene, the reassortants with the mutated NSP1 gene and RNA segments from heterologous strains normally replicated in cultured cells. Thus, cysteine-rich region of NSP1 was not considered essential for genome segment reassortment with heterologous virus.

14 citations


01 Dec 1999
TL;DR: YN87448 virus strain is a new sindbis-like virus strain and identified as a member of Alphavirus by the serological method, and can not produce a fatal disease in adult mice.
Abstract: OBJECTIVE To determine the complete nucleotide sequence of the nonstructural gene of YN87448 virus stain which was firstly isolated from a female patient with fever in Yunnan Province in 1986, and identified as a member of Alphavirus by the serological method. METHODS The complete nucleotide sequence of the nonstructural region gene of YN87448 virus strain was determined with nine clones, which were obtained by using reverse transcription and polymerase chain reaction (RT-PCR), and by linking nine overlapping fragments into pGEM-T vector respectively. RESULTS The complete nucleotide sequence of nonstructural gene of YN87448 virus strain was 7,613 nucleotides long exclusive of the 5' cap, encoding four nonstructural proteins, nsP1, nsP2, nsP3, nsP4, and contained one initiator(ATG) and two stop codons (TGA). In comparison with the consensus sequence of S.A.AR86, the homogeneity of the nucleotide sequence between YN87448 virus strain and sindbis-like virus isolate S.A.AR86 was 98.8%. YN87448 virus strain can not produce a fatal disease in adult mice. In comparison with the consensus sequence of S.A.AR86, there is a 54 nucleotide insertion from 5,256 bp to 5,309 bp, 3 nucleotide (AGT) deletion at 5,603 bp in nsP3 region, and an opal termination codon between the nsP3 and the nsP4 genes in YN87448. virus strain. This sequence has been put into Gene Bank and No. is AF103734. CONCLUSION YN87448 is a new sindbis-like virus strain.

14 citations


Journal ArticleDOI
TL;DR: The results indicated that viral growth and genome segment reassortment with other viruses may not be influenced by the presence of heterologous NSP1 and its expression level, while genomic diversity of N SP1 genes might have been associated with the relative adaptability to the genetic background of SAff11.
Abstract: Function of rotavirus NSP1 was analyzed by using single-NSP1 gene-substitution reassortants, SKF, SDF, and SNF which have the NSP1 gene derived from human rotaviruses KU, DS-1, and canine rotavirus K9, respectively, in the genetic background of simian rotavirus SAff11. The NSP1 genes from KU, DS-1, K9, and SAff11 exhibited 58–76% nucleotide sequence identity to one another. No substantial difference in viral growth was observed among the reassortants and SAff11. However, production of NSP1 was not detected in SNF when viral proteins were labelled with 35S-methionine during replication in MAff104 cells, in contrast to SAff11, SKF and SDF which exhibited evident expression of NSP1. Difference in reassortant formation was examined among the reassortant clones generated between human rotavirus strain 69M and either of SAff11, SKF or SNF. Although reassortant formation rate was significantly lower in the cross 69M × SNF than the other crosses, selection rates of RNA segments from parent strain 69M in the resultant reassortants was similar among the crosses. Selectivity of homolog- ous and heterologous NSP1 genes in SAff11 background was also analyzed by mixed infection and multiple passages among the single-NSP1 gene-reassortants and/or SAff11. KU NSP1 gene was selected most frequently, whereas homologous (SAff11) NSP1 gene was least efficiently segregated. These results indicated that viral growth and genome segment reassortment with other viruses may not be influenced by the presence of heterologous NSP1 and its expression level, while genomic diversity of NSP1 genes might have been associated with the relative adaptability to the genetic background of SAff11.

9 citations


Dissertation
01 Sep 1999
TL;DR: The function of NSP1 in the replication cycle and the importance of its presence in early replication complexes has not been determined and it was proposed that N SP1 formed a previously unrecognised complex with these proteins.
Abstract: Rotavirus encodes six structural and six non-structural proteins. In contrast to the structural proteins, the functional roles of the non-structural proteins are not well defined beyond a realisation that they must have a role in the viral replication cycle. A fuller understanding of the replication cycle must therefore rest on determining the specific roles played by the non-structural proteins. Non-structural protein NSP1 shows high levels of sequence divergence. A generally well conserved cysteine-rich region at the amino-terminus may form a zinc finger structure. It has been shown to possess non-specific RNA-binding activity, and has been found associated with the smallest of three replication intermediates (RIs) found in infected cells, together with the viral proteins VP1, VP3 and NSP3. VP2 and VP6 are added sequentially to the pre-core RI to form the core RI and single-shelled RI respectively. The function of NSP1 in the replication cycle and the importance of its presence in early replication complexes has not been determined. The intermolecular interactions that occur between the components of the RIs have not been defined. Protein-protein interactions between NSP1 and VP1, VP2, VP3, and NSP3, from the UKtc strain of bovine rotavirus, were investigated using a variety of approaches, the first of which was the yeast two-hybrid system. In this assay a self-interaction of NSP1 was not detected. Protein-protein interactions between NSPl and VPl, VP2, VP3, and NSP3, were also not detected. Both the full-length protein and a truncated NSPl, consisting of only the amino terminal third of the protein, were tested. A direct self-interaction of NSP3 was shown and quantified. Radio-immunoprecipitation analysis of in vitro translated viral proteins using specific anti-NSP1 serum was also employed. However, it failed to detect direct protein-protein interactions between NSP1 and VPI, VP2, and VP3. Immunoprecipitation of UKtc rotavirus-infected celllysates with anti-NSP1 serum showed the co-precipitation of viral proteins VPl, VP2, VP3NP4, VP6 and NSP3, with NSP1. It was proposed that NSP1 formed a previously unrecognised complex with these proteins. Immunoprecipitation of nuclease-treated infected cell lysates showed a reduction in the co-precipitation of VP2, VP3NP4 and NSP3 with NSP1. No reduction in the co-precipitation of VP6 was seen. The association of the complex proteins may be mediated by RNA binding. Immunoprecipitation with an anti-VP6 monoclonal antibody reciprocally precipitated small amounts of NSP1, VP2, VP3/VP4, and NSP3, with VP6.

1 citations