Showing papers on "NSP1 published in 1996"

Nondefective rotavirus mutants with an NSP1 gene which has a deletion of 500 nucleotides, including a cysteine-rich zinc finger motif-encoding region (nucleotides 156 to 248), or which has a nonsense codon at nucleotides 153-155.

Abstract: We isolated two nondefective bovine rotavirus mutants (A5-10 and A5-16 clones) which have nonsense mutations in the early portion of the open reading frame of the NSP1 gene In the NSP1 gene (1,587 bases long) of A5-10, a nonsense codon is present at nucleotides 153 to 155 just upstream of the coding region (nucleotides 156 to 230) of a cysteine-rich Zn finger motif A5-16 gene 5 (1,087 bases long) was found to have a large deletion of 500 bases corresponding to nucleotides 142 to 641 of a parent A5-10 NSP1 gene and to have a nonsense codon at nucleotides 183 to 185, which resulted from the deletion Expression of gene 5-specific NSP1 could not be detected in MA-104 cells infected with the A5-10 or A5-16 clone or in an in vitro translation system using the plasmids with gene 5 cDNA from A5-10 or A5-16 Nevertheless, both A5-10 and A5-16 replicated well in cultured cells, although the plaque size of A5-16 was extremely small

Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein.

Abstract: We identified mutations in the gene for nsP2, a nonstructural protein of the alphavirus Sindbis virus, that appear to block the conversion of the initial, short-lived minus-strand replicase complex (RCinitial) into mature, stable forms that are replicase and transcriptase complexes (RCstable), producing 49S genome or 26S mRNA. Base changes at nucleotide (nt) 2166 (G-->A, predicting a change of Glu-163-->Lys), at nt 2502 (G-->A, predicting a change of Val-275-->Ile), and at nt 2926 (C-->U, predicting a change of Leu-416-->Ser) in the nsP2 N domain were responsible for the phenotypes of ts14, ts16, and ts19 members of subgroup 11 (D.L. Sawicki and S.G. Sawicki, Virology 44:20-34, 1985) of the A complementation group of Sindbis virus RNA-negative mutants. Unlike subgroup I mutants, the RCstable formed at 30 degrees C transcribed 26S mRNA normally and did not synthesize minus strands in the absence of protein synthesis after temperature shift. The N-domain substitutions did not inactivate the thiol protease in the C domain of nsP2 and did not stop the proteolytic processing of the polyprotein containing the nonstructural proteins. The distinct phenotypes of subgroup I and 11 A complementation group mutants are evidence that the two domains of nsP2 are essential and functionally distinct. A detailed analysis of ts14 found that its nsPs were synthesized, processed, transported, and assembled at 40 degrees C into complexes with the properties of RCinitial and synthesized minus strands for a short time after shift to 40 degrees C. The block in the pathway to the formation of RCstable occurred after cleavage of the minus-strand replicase P123 or P23 polyprotein into mature nsP1, nsP2, nsP3, and nsP4, indicating that structures resembling RCstable, were formed at 40 degrees C. However, these RCstable or pre-RCstable structures were not capable of recovering activity at 30 degrees C. Therefore, failure to increase the rate of plus-strand synthesis after shift to 40 degrees C appears to result from failure to convert RCinitial to RCstable. We conclude that RCstable is derived from RCinitial by a conversion process and that ts14 is a conversion mutant. From their similar phenotypes, we predict that other nsP2 N-domain mutants are blocked also in the conversion of RCinitial to RCstable. Thus, the N domain of nsP2 plays an essential role in a folding pathway of the nsPs responsible for formation of the initial minus-strand replicase and for its conversion into stable plus-strand RNA-synthesizing enzymes.

Recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against NSP2.

Abstract: Replication of the rotavirus genome involves two steps: (i) transcription and extrusion of transcripts and (ii) minus-strand RNA synthesis in viral complexes containing plus-strand RNA. In this study, we showed evidence for the importance of the viral nonstructural protein of rotavirus, NSP2, in the replication of viral RNAs. RNA-binding properties of NSP2 were tested by UV cross-linking in vivo (in rotavirus-infected MA104 cells and recombinant baculovirus-expressing NSP2-infected Sf9 cells). In rotavirus-infected cells, NSP2 is bound to the 11 double-stranded RNA genomic segments of rotavirus. Quantitative analysis (using hydrolysis by RNase A) is consistent with NSP2 being directly bound to partially replicated viral RNA. Using various monoclonal antibodies and specific antisera against the structural (VP1, VP2, and VP6) and nonstructural (NSP1, NSP2, NSP3, and NSP5) proteins, we developed a solid-phase assay for the viral replicase. In this test, we recovered a viral RNA-protein complex with replicase activity only with a monoclonal antibody directed against NSP2. Our results indicated that these viral complexes contain the structural proteins VP1, VP2, and VP6 and the nonstructural protein NSP2. Our results show that NSP2 is closely associated in vivo with the viral replicase.

Structure and function of rotavirus NSP1.

Abstract: Studies on the structure and function of the nonstructural proteins (NSP1-NSP5) of rotaviruses are important for dissection of the morphogenesis and replication processes of rotavirus. Above all, NSP1, the product of gene 5, has several interesting features, such as extreme sequence diversity, a highly conserved cysteine-rich region, RNA-binding activity, accumulation on the cytoskeleton, and non-random segregation in reassortment. Recently, comparable NSP1 sequence analysis has been performed on a number of rotavirus strains from various species. Furthermore, characterization of mutants with rearranged NSP1 genes has helped to elucidate the structure-function interaction of NSP1. We isolated and characterized two interesting mutants which have a large deletion including the cysteine-rich region or a nonsense codon at the early portion in the open reading frame (ORF) of the NSP1 gene. In this report, we summarize the structure and function of NSP1.

G (VP7) serotype-dependent preferential VP7 gene selection detected in the genetic background of simian rotavirus SA11.

Abstract: We previously found the preferential selection of VP7 gene from a parent rotavirus strain SA11 with G serotype 3 (G3) in the sequential passages after mixed infection of simian rotavirus SA11 and SA11-human rotavirus single-VP7 gene-substitution reassortants with G1, G2, or G4 specificity. However, it has not been known whether or not VP7 genes derived from other strains with G3 specificity (G3-VP7 gene) are preferentially selected in the genetic background of SA11. To address this question, mixed infections followed by multiple passages were performed with a reassortant SA11-L2/KU-R1 (SKR1) (which possesses VP7 gene derived from G1 human rotavirus KU and other 10 genes of SA11 origin) and one of the five G3-rotaviruses, RRV, K9, YO, AK35, and S3. After the 10th passage, selection rates of SA11-L2/KU-R1 gene 9 (G1-VP7 gene) and gene 5 (NSP1 gene) reduced considerably (0 to 20.4%) in the clones obtained from all the coinfection experiments, while all or some of other segments were preferentially selected from SKR1 depending on the pairs of coinfection. When viral growth kinetics was examined, SKR1 exhibited better growth and reached a higher titer than any G3 viruses. Although the generated reassortants with VP7 gene and NSP1 gene derived from G3 viruses showed almost similar growth kinetics to that of SKR1 during the first 20 h of replication, the titers of these reassortants were higher than that of SKR1 after 36h postinfection. The results obtained in this study suggested that G3-VP7 gene is functionally more adapted to the genetic background of SA11.

Synthesis of Semliki Forest virus RNA polymerase components nsP1 through nsP4 in Saccharomyces cerevisiae by expression of cDNA encoding the nonstructural polyprotein.

Abstract: A Semliki Forest virus nonstructural polyprotein, P1234, expressed in the yeast Saccharomyces cerevisiae in the absence of a replicative RNA template appeared to be properly cleaved into nsP1 to nsP4. All nsPs were membrane associated, and nsP2 was also transported to the nucleus. The membrane fraction containing nsPs showed guanine-7-methyltransferase and guanylyltransferase-like activities, typical for Semliki Forest virus nsP1.