Characterization of Spring viremia of carp virus mRNA species and the 3′ sequence of the viral RNA

The base sequence at the 3' end of Spring viremia of carp virus RNA was determined by using a terminal label and chemical RNA sequencing procedures. By comparison with the 3' termini of VSV-Indiana and VSV-New Jersey viruses there is complete homology for the first 20 nucleotides but less homology thereafter. The 5' termini of SVCV mRNA species isolated from infected BHK-21 cells have a basic 5' terminal capped sequence of 7mGppp(m)A(m)p(m)A(m)pNp… i.e. similar to that of SVCV in vitro synthesized mRNA species as well as VSV in vitro and in vivo mRNA species. The coding capabilities of the individual SVCV mRNA species have also been analyzed; the NS mRNA species yielded two translation products, NS1 and NS2.     

Coding strategy of rice stripe virus: major nonstructural protein is encoded in viral RNA segment 4 and coat protein in RNA complementary to segment 3

The two major proteins found in plants infected with rice stripe virus (RSV), coat protein and a major nonstructural protein (major NS), were purified and their partial amino acid sequences were determined. Oligonucleotides were synthesized according to the amino acid sequence information, and used as probes for Northern blot analyses of four single-stranded RNA species (segments 1-4) and four double-stranded RNA species of RSV. The results indicated that the coding strategy of RSV was similar to those of ambisense viruses: Segment 3 coded for the coat protein in the (-) sense while segment 4 coded for the major NS protein in the (+) sense.     

The influenza C virus NS gene: evidence for a spliced mRNA and a second NS gene product (NS2 protein)

It has previously been shown that the shortest RNA (RNA 7) of influenza C viruses codes for a nonstructural (NSI) protein (Nakada et al. (1985) J. Virol. 56, 221-226). Experiments reported here indicate that RNA 7 also directs the synthesis of a second nonstructural protein via a spliced mRNA. The amino terminal 62 codons of this NS2 protein appear to be shared with the NS1 protein and the carboxyl terminal 59 amino acids are unique (derived from a +1 open reading frame in the mRNA). Although the size of the C virus NS2 protein is comparable to that of the A and B virus NS2 proteins, the overall arrangement of the C virus NS gene is quite different from that of the A and B virus NS genes. The second (+1) open reading frame of the C virus NS gene is completely overlapped by that of the NS1 protein, whereas the second (+1) open reading frame of the A and B virus NS genes extends to the 3' end of the RNA and only partially (or in some strains not at all) overlaps the NS1 open reading frame.     

Identification of nonstructural proteins encoded by viruses of the Bunyamwera serogroup (family Bunyaviridae)

The proteins synthesized in BHK cells infected with nine members of the Bunyamwera serogroup (family Bunyaviridae, Bunyavirus genus) were analyzed by polyacrylamide gel electrophoresis. In addition to the virus structural proteins, a number of virus-coded nonstructural proteins were detected. One protein, designated NS1, was shown to be related to the nucleocapsid protein by one-dimensional peptide mapping. A second protein, NS2, was mapped to the M RNA segment by gel electrophoretic analysis of the proteins synthesized in cells infected with reassortants of Batai, Bunyamwera, and Maguari viruses of known genotype. A third protein, NS3, was mapped to the S RNA segment by its pattern of labeling with [35S]cysteine in cells infected with reassortant viruses: the NS3 protein was only labeled when the S RNA segment of Bunyamwera virus was present. The mapping of NS3 was confirmed by in vitro translation of mRNAs which hybridized to recombinant plasmids containing S gene-specific sequences.     

Deglycosylation of the NS1 protein of dengue 2 virus,strain 16681: Construction and characterization of mutant viruses

Summary. The dengue 2 virus (DENV-2) NS1 glycoprotein contains two potential sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 produced in infected cells is glycosylated at both of these sites. We used site-directed mutagenesis of a DENV-2, strain 16681, full length infectious clone to create mutant viruses lacking the Asn-130, Asn-207 or both of these NS1 glycosylation sites in order to investigate the effects of deglycosylation. Ablation of both NS1 glycosylation sites resulted in unstable viruses that acquired numerous additional mutations; these viruses were not further characterized. Viruses altered at the Asn-130 site exhibited growth characteristics similar to the wild-type (WT) 16681 virus in LLC-MK₂ cells and reduced growth in C6/36 cells. Viruses mutated at the Asn-207 site achieved similar titers in LLC-MK₂ cells compared to WT, however, the appearance of cytopathic effect was delayed and growth of these viruses in C6/36 cells was also reduced compared to WT virus. The plaque size of mutant viruses altered at the Asn-130 site did not differ from that of the WT virus, while mutants altered at the Asn-207 site exhibited a reduced and mixed plaque size. Temperature sensitivity studies comparing the growth of the viruses at 37°C and 39°C showed no significant differences compared to the WT virus. Immunofluorescent antibody staining of infected cells showed that for WT 16681 virus or the Asn-130 site mutant viruses NS1 was located throughout the cytoplasm, however, Asn-207 site mutant virus NS1 protein appeared to be localized to the perinuclear region. Viruses deglycosylated at either site exhibited a significant reduction in mouse neurovirulence compared to the WT virus. The results of our studies indicate that glycosylation of the DENV-2 virus NS1 protein may influence NS1 protein processing/transport as well as the pathogenicity of the virus.     

Antigenic variation in the influenza A virus nonstructural protein, NS1

The antigenic structure of the nonstructural (NS1) protein encoded by influenza type A virus was examined using monoclonal antibodies prepared against purified NS1 inclusions isolated from the cytoplasm of infected cells. Topographical analysis by competitive radioimmunoassay indicated that three different overlapping antigenic regions were present on the NS1 of A/WSN/33 (H1N1). Immunoprecipitation studies using infected cell lysates showed that antigenic determinants on A/WSN/33 NS1 are common to NS1 proteins encoded by a wide range of viruses of human, swine, equine, and avian origin. Several avian strains, however, were found to encode antigenically variant NS1 proteins which had either extensive changes in one or more antigenic regions or small changes in epitopes within a region suggestive of antigenic drift. There was no correlation between surface antigen subtype and the antigenic profile of the NS1 protein. The antigenic relationships of NS1 proteins shown in this study are in agreement with the available sequence data.     

Sequence characterization of the membrane protein gene of paramyxovirus simian virus 5

The complete nucleotide sequence of the membrane (M) protein gene of the paramyxovirus simian virus 5 (SV5) was determined from cDNA clones of viral mRNAs. The M gene boundaries were determined by (i) primer extension sequencing on M mRNA; (ii) nuclease S1 analysis; and (iii) primer extension sequencing on viral genomic RNA. The M gene mRNA consisted of 1371 templated nucleotides. It contains a single large open reading frame that can encode a protein of 377 amino acids with a predicted Mr = 42,253. The authenticity of the predicted M protein coding sequence was confirmed by synthesis of the M protein from mRNA synthesized from cDNA. The predicted M amino acid sequence indicated it is an overall hydrophobic protein carrying a net positive charge. Alignment of the SV5 protein amino acid sequence with the M protein sequences of other paramyxoviruses indicated that these viruses fall into the following two groups: (1) SV5, mumps virus, and Newcastle disease virus; or (2) Sendai, parainfluenza virus type 3, measles virus, and canine distemper virus, with mumps virus M sequence being the most closely related to SV5.     

Cell culture-adaptive mutations in the NS5B gene of hepatitis C virus with delayed replication and reduced cytotoxicity

Remarkable advances have been made through recent demonstrations of hepatitis C virus (HCV) replication in cultured cells transfected with in vitro synthesized viral genome RNA. From the HCV JFH1 (genotype 2a) subcultured successively in Huh-7 cells we have identified several missense mutations near the junction of NS5A and NS5B genes. Reverse genetic analysis indicated that two mutations in the N-terminal region of NS5B replicase caused delayed viral RNA replication and protein expression in the early stage of infection. However, the mutant viruses showed significantly alleviated effects on cell growth inhibition, proteolysis of viral proteins, apoptotic DNA cleavage, and induction of antiviral responses, giving rise to a 100-fold higher titer compared to the parental JFH1 virus in a more extended time period. These results suggested that delayed replication and reduced cytotoxicity can be characteristic features of cell culture-adaptive mutants with enhanced infectivity.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

Characterization of a unique protein produced by influenza A virus recovered from a long-term persistent infection

Virus isolated from a persistent infection initiated in BHK cells with influenza A/WSN/33 (H1N1) produced an unusual pattern of protein synthesis in productive infections of BHK cells: The levels of NP and M1 proteins were slightly reduced compared to an infection with wild-type WSN, while the other proteins (Pb1, Pb2, Pa, HA, NS1, and NS2) were synthesized at very low or undetectable levels. In addition, a new viral protein with an approximate molecular weight of 11 kDa (Pi protein) is made (Frielle et al., Virology 138, 103-117, 1984). When viral RNA was analyzed by the Northern blot technique, a deletion was found in the NS gene segment and in NS1 mRNA; all other RNAs were full-sized. Immunoprecipitation of in vitro translation products demonstrated that the Pi protein reacts specifically with anti-NS1 serum. In addition, the Pi protein, like the NS1 of the parental wild-type virus, accumulated in the nucleus of infected cells. These results indicate that the Pi protein is a mutated form of the NS1 protein encoded by a deleted NS segment and suggest that this mutation may be involved in the expression of the persistent virus phenotype.     

Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection

Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.     

Differences in the electrophoretic migration rates of polypeptides and RNAs of recent isolates of influenza B viruses

Summary The electrophoretic migration rates of structural and non-structural poly-peptides of 38 influenza B viruses isolated in epidemics in 1978–1980 and antigenically closely related to B/Singapore/222/79 virus were compared using high resolution SDS polyacrylamide gels. Thirty of the viruses could be distinguished from the prototype B/Singapore/222/79 virus by electrophoretic migration rate differences in HA, 17 by differences in NP and 27 by differences in mobility of the NS1 polypeptide. Mobility differences of NP, NS1 and HA polypeptides was noted in influenza B viruses isolated in the UK in the same year. In addition, electrophoretic mobility of³²P labelled virus RNAs varied for certain UK isolates and indicated heterogeneity in genes 2, 3, 4 and 8 coding for polymerase proteins 2 and 1, nucleoprotein (NP) and non-structural protein (NS1) respectively.With 3 Figures     

Genetic variability of hepatitis C virus non-structural protein 3 and virus-specific CD8+ response in patients with chronic hepatitis C

Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus.     

Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization

In a previous paper (G. Winkler, V. B. Randolph, G. R. Cleaves, T. E. Ryan, and V. Stollar, 1988, Virology 162, 187-196) we showed that the newly synthesized dengue-2 virus nonstructural protein, NS1, exists briefly as a monomer and then undergoes dimerization. We demonstrate here that the dimerization of NS1 is associated with a change in hydrophobicity and sedimentability of this protein. Newly synthesized monomeric NS1 is a hydrophilic and water-soluble protein which cannot be pelleted at 75,000 g. After dimerization, however, NS1 showed increased hydrophobicity in a Triton X-114 phase partitioning system and was completely pelletable at 75,000 g; these findings are consistent with NS1 becoming membrane-associated. In experiments in which infected cells were treated with tunicamycin it was shown that the glycosylation of NS1 was not required for either the dimerization or the membrane association.     

Progressive truncation of the Non-Structural 1 gene of H7N1 avian influenza viruses following extensive circulation in poultry

In order to support eradication efforts of avian influenza (AI) infections in poultry, the implementation of "DIVA" vaccination strategies, enabling the Differentiation of Infected from Vaccinated Animals have been recommended by international organisations. A system, based on the detection of antibodies to the Non-Structural 1 (NS1) protein of AI has been proposed but the success of such a system lies in the conservation of the NS1 protein among different AI isolates. With this in mind, the ns1 gene of 40 influenza A viruses isolated from a spectrum of avian species was sequenced and compared phylogenetically. The isolates included both low pathogenicity (LPAI) (n=22) and highly pathogenic (HPAI) (n=18) viruses of the H7 subtype and were representative of the avian influenza viruses that circulated in Northern Italy from 1999 to 2003. Size variation in the predicted amino acid sequence of each NS1 was revealed with two different levels of carboxy-terminal truncation being observed. Of the 40 isolates analysed, 16 had a full-length NS1 protein of 230 aa, 6 had a truncated protein of 220 aa and 18 had an intermediate truncation resulting in a protein of 224 aa. All of the H7N1 HPAI isolates possessed the intermediate carboxy-terminal truncation. In addition, all of the H7N1 LPAI viruses circulating at the beginning of the epidemic had a full length NS1 while those circulating towards the end of the period had a truncated protein. To determine whether modifications to NS1 could be a result of laboratory manipulation, two strains (A/ty/Italy/977/99 and A/ck/Italy/1082/99) with a full length NS1 were inoculated into 10-day-old embryonated chicken and 12-day-old embryonated turkey eggs via the allantoic route for 20 blind passages and sequenced at passages 3, 10, and 20. No truncation was observed following these serial passages. To determine whether the truncation involved an immunogenic region of the NS1 protein a peptide spanning residues 219 aa to 230 aa was synthesized and tested in an indirect ELISA against sera obtained from turkeys experimentally infected with a virus strain known to have a full length NS1 protein. The peptide proved to be immunogenic highlighting the fact that the variations of the NS1 protein presented in this work must to be taken into consideration when developing a diagnostic test based on the identification of antibodies to the NS1 protein.