Showing papers on "NSP1 published in 2001"

A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus.

Abstract: The genome expression of positive-stranded RNA viruses starts with translation rather than transcription. For some viruses, the genome is the only viral mRNA and expression is regulated primarily at the translational level and by limited proteolysis of polyproteins. Other virus groups also generate subgenomic mRNAs later in the reproductive cycle. For nidoviruses, subgenomic mRNA synthesis (transcription) is discontinuous and yields a 5′ and 3′ coterminal nested set of mRNAs. Nidovirus transcription is not essential for genome replication, which relies on the autoprocessing products of two replicase polyproteins that are translated from the genome. We now show that the N-terminal replicase subunit, nonstructural protein 1 (nsp1), of the nidovirus equine arteritis virus is in fact dispensable for replication but crucial for transcription, thereby coupling replicase expression and subgenomic mRNA synthesis in an unprecedented manner. Nsp1 is composed of two papain-like protease domains and a predicted N-terminal zinc finger, which was implicated in transcription by site-directed mutagenesis. The structural integrity of nsp1 is essential, suggesting that the protease domains form a platform for the zinc finger to operate in transcription.

Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication

Abstract: The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.

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.

Sequence analysis of genes encoding structural and nonstructural proteins of a human group B rotavirus detected in Calcutta, India.

Abstract: Nucleotide sequences of RNA segments encoding structural proteins(VP4, VP6, and VP7) and nonstructural proteins(NSP1 and NSP3) of a human group B rotavirus CAL-1, which was detected in Calcutta, India, were determined and their relatedness with cognate genes of other group B rotaviruses was analyzed. The CAL-1 genes showed generally high sequence identities (more than 90%) to those of human group B rotavirus, adult diarrheal rotavirus (ADRV) in China, while identities with bovine, murine, and ovine viruses were considerably lower (58–73%). Among RNA segments analyzed, sequence identity of the VP6 gene was relatively high compared with other gene segments. In the CAL-1 VP7 sequence, many characteristics were shared by ADRV, but not by other animal group B rotaviruses. In contrast, VP4 and NSP3 of CAL-1 were single animo acid and 23 amino acids longer than those of ADRV strain, respectively, due to differences of a few nucleotides. These findings suggested that human group B rotaviruses CAL-1 and ADRV might have originated from a common ancestral virus distinct from animal group B rotaviruses reported so far, while some notable sequence differences indicated the distinct nature of these viruses. J. Med. Virol. 64:583–588, 2001. © 2001 Wiley-Liss, Inc.

Antigenic and Molecular Analyses Reveal that the Equine Rotavirus Strain H-1 is Closely Related to Porcine, but not Equine, Rotaviruses: Interspecies Transmission from Pigs to Horses?

Abstract: We have sequenced the genes encoding the inner capsid protein VP6 and the outer capsid glycoprotein VP7 of the subgroup (SG) I equine rotavirus strain H-1 (P9[7], G5). The VP6 and VP7 proteins of the equine rotavirus strain H-1 shared a high degree of sequence and deduced amino acid identity with SG I porcine strains and serotype G5 porcine strains, respectively. Previous sequence analyses of the genes encoding the outer capsid spike protein VP4 and the nonstructural proteins NSP1 and NSP4 of equine H-1 strain also revealed a high degree of sequence and deduced amino acid homology with the prototype porcine rotavirus strain OSU (P9[7], G5). We have also confirmed and extended the VP4 and VP7 antigenic relatedness of equine rotavirus strain H-1 to porcine strains of P9[7] and G5 serotype specificities isolated in the United States, Venezuela, Argentina, and Australia based on cross-neutralization studies. In addition, the pathogenicity of tissue culture-adapted equine H-1, H-2, FI-14, FI-23, and L338, and porcine OSU rotavirus strains was compared in the neonatal mouse model. The 50% diarrhea dose (DD50) of equine H-1 was similar to that of porcine OSU and equine H-2 and L338 strains, while the DD50 of equine H-2 was > or = 50 or 315-fold lower than those of equine FI-14 or FI-23, respectively. Our sequence comparison of NSP4 of the rotavirus strains tested potentially identified amino acid residue 136, within the variable region spanning amino acids 130 to 141, as playing a role in virulence. Taken together, there is strong support to suggest that the equine rotavirus strain H-1 may represent an example of interspecies transmission from pigs to horses.