Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses.

A cDNA containing the complete coding sequence of the Bunyamwera virus (family Bunyaviridae) L genome segment has been constructed and cloned into two recombinant vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus P7.5 promoter; in the second, the L gene is under control of the bacteriophage T7 phi 10 promoter, and expression of the L gene requires coinfection with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. Both systems express a protein which is the same size as the Bunyamwera virus L protein and is recognized by a monospecific L antiserum. The expressed L protein was shown to be functional in synthesizing Bunyamwera virus RNA in a nucleocapsid transfection assay: recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently, total cellular RNA was analyzed by Northern (RNA) blotting. No Bunyamwera virus RNA was detected in control transfections, but in cells which had previously been infected with recombinant vaccinia viruses expressing the L protein, both positive- and negative-sense Bunyamwera virus S segment RNA was detected. The suitability of this system to delineate functional domains within the Bunyamwera virus L protein is discussed.     

Three monoclonal antibodies against measles virus F protein cross-react with cellular stress proteins.

In a group of 11 monoclonal antibodies specifically reacting with the measles virus fusion protein, three antibodies also immunoprecipitated other proteins, in particular a 79,000-molecular-weight protein from virus-infected cells. The cross-reacting 79,000-molecular-weight protein was shown to be a virus-induced host stress protein. This protein could be induced by (i) different paramyxoviruses, (ii) heat shock of uninfected HeLa cells, and (iii) 2-deoxyglucose, tunicamycin, or L-canavanine treatment of different mammalian cell lines. Immunofluorescence of stressed HeLa cells localized the cross-reacting host protein(s) mainly in the cytoplasm. The significance of these results in relation to autoimmunity is discussed.     

痘苗病毒/T7RNA聚合酶辅助的原核基因在真核细胞中的表达

痘苗病毒/T7RNA聚合酶这一瞬时表达系统由于具有很多优于其他表达系统的特点而被广泛地应用于表达外源蛋白.TB-Chen株轮状病毒的VP6 DNA编码片段插入到原核质粒pETL的噬菌体T7启动子和终止子之间,获得重组表达质粒pET-VP6.构建好的重组表达质粒pET-VP6通过脂质体转染到真核细胞MA104中,用携带噬...     

The ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles

Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two-open reading frame 3a (ORF3a) and ORF7a-have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a -1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.     

In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release.

We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.     

Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs.

We have characterized the proteins and mRNAs of bovine respiratory syncytial (BRS) virus strain 391-2 and constructed cDNA clones corresponding to 9 of the 10 BRS virus mRNAs. The proteins of BRS virus-infected cells were compared with the proteins from human respiratory syncytial (HRS) virus-infected cells. Nine proteins specific to BRS virus-infected cells, corresponding to nine HRS virus proteins, were identified. Only a BRS virus polymerase protein remains to be identified. The BRS virus G glycoprotein showed major antigenic differences from the HRS virus G glycoprotein by immunoprecipitation and Western (immuno-) blot analysis, whereas the BRS virus F, N, M, and P proteins showed antigenic cross-reactivity with their HRS virus counterparts. Analysis of RNAs from BRS virus-infected cells showed virus-specific RNAs which had electrophoretic mobilities similar to those of mRNAs of HRS virus but which hybridized poorly or not at all with HRS virus-specific probes in Northern (RNA) blot analysis. To analyze the BRS virus RNAs further, cDNA clones to the BRS virus mRNAs were generated. Nine separate groups of clones were identified and shown to correspond to nine BRS virus mRNAs by Northern blot analysis. A 10th BRS virus large mRNA was identified by analogy with the HRS virus polymerase mRNA. These data show that like HRS virus, BRS virus has 10 genes coding for 10 mRNAs.     

Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide.

Previous nucleotide sequence analysis of RNA segment 7 of influenza B virus indicated that, in addition to the reading frame encoding the 248 amino acid M1 protein, there is a second overlapping reading frame (BM2ORF) of 585 nucleotides that has the coding capacity for 195 amino acids. To search for a polypeptide product derived from BM2ORF, a genetically engineered beta-galactosidase-BM2ORF fusion protein was expressed in Escherichia coli and a polyclonal rabbit antiserum was raised to the purified fusion protein. This antiserum was used to identify a polypeptide, designated BM2 protein (Mr approximately equal to 12,000), that is synthesized in influenza B virus-infected cells. To understand the mechanism by which the BM2 protein is generated from influenza B virus RNA segment 7, a mutational analysis of the cloned DNA was performed and the altered DNAs were expressed in eukaryotic cells. The expression patterns of the M1 and BM2 proteins from the altered DNAs indicate that the BM2 protein initiation codon overlaps with the termination codon of the M1 protein in an overlapping translational stop-start pentanucleotide, TAATG, and that the expression of the BM2 protein requires 5'-adjacent termination of M1 synthesis. Our data suggest that a termination-reinitiation scheme is used in translation of a bicistronic mRNA derived from influenza B virus RNA segment 7, and this strategy has some analogy to prokaryotic coupled stop-start translation of tandem cistrons.     

Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.     

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.     

mRNA sequence and deduced amino acid sequence of the mumps virus small hydrophobic protein gene.

The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.     

Cloning of influenza cDNA ino M13: the sequence of the RNA segment encoding the A/PR/8/34 matrix protein

A strategy has been developed for sequencing the single-stranded RNA genes of influenza virus. Restriction fragments derived from double-stranded cDNA copies of total influenza RNA were cloned into the bacteriophage M13mp2 and sequenced by the dideoxy technique. Sequences were extended and overlapped by using the virion RNA as template and priming with small restriction fragments. In the course of this strategy, the nucleotide sequence of segment 7 (1027 nucleotides) was completed and provides the primary structure of the matrix protein (27, 861 daltons). In addition, there is a second long reading frame which partly overlaps the reading frame of the matrix protein.     

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.