RESUMO
The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.
Assuntos
Vírus da Dengue/enzimologia , Vírus da Dengue/genética , Serina Endopeptidases/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Clonagem Molecular , Vírus da Dengue/classificação , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/genética , RNA Helicases , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
We report that expression of a nearly full-length cDNA clone of the L-A double-stranded RNA virus causes virus loss in a wild-type strain of Saccharomyces cerevisiae. We show that in this system exclusion of the L-A virus is independent of the presence of the packaging site or of cis sites for replication and transcription and completely dependent on expression of functional recombinant gag and gag-pol fusion protein. Thus, this exclusion is not explained in terms of overexpression of packaging signals. Mutation of the chromosomal SKI2 gene, known to repress the copy number of double-stranded RNA cytoplasmic replicons of S. cerevisiae, nearly eliminates the exclusion. We suggest that exclusion is due to competition by proteins expressed from the plasmid for a possibly limiting cellular factor. Our hypotheses on exclusion of L-A proteins may also apply to resistance to plant viruses produced by expression of viral replicases in transgenic plants.
Assuntos
Proteínas de Fusão gag-pol/genética , Produtos do Gene gag/genética , Vírus de RNA/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Interferência Viral/genética , Acetiltransferases , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Fusão gag-pol/biossíntese , Produtos do Gene gag/biossíntese , Genes Virais/genética , Vetores Genéticos/genética , Dados de Sequência Molecular , Acetiltransferase N-Terminal B , Vírus de RNA/crescimento & desenvolvimento , RNA de Cadeia Dupla/genética , RNA Viral/genética , Proteínas Virais/genética , Replicação ViralRESUMO
To assess the role of codon context on the efficiency of eukaryotic suppression of termination codons, we have compared, in a rabbit cell-free translation system, the readthrough efficiency related to two synthetic transcripts differing by the codon context around an amber codon. The codon contexts are derived from tobacco mosaic virus (TMV) and Moloney murine leukemia virus (Mo-MuLV) RNAs. The Mo-MuLV-like codon context does not promote suppression. Substituting TMV-derived triplets in the Mo-MuLV-like codon context shows that the two codons downstream from the TMV UAG signal are important determinants of suppression, as recently demonstrated in vivo.
Assuntos
Códon , Vírus da Leucemia Murina/genética , Biossíntese de Proteínas , Regiões Terminadoras Genéticas/genética , Vírus do Mosaico do Tabaco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Livre de Células , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA Viral , Eletroforese em Gel de Poliacrilamida , Genes Sintéticos , Dados de Sequência Molecular , RNA Viral , CoelhosRESUMO
An increasing number of examples of translational regulation at the level of termination has been recently reported in eukaryotes. This paper reviews our present knowledge on this topic and proposes an understanding of these regulations by relating the study of viral gene expression to a comprehensive view of the mechanisms and components of the translational process.
Assuntos
Células , Células Eucarióticas , Regulação da Expressão Gênica , Terminação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Animais , Códon , Genes Virais , RNA de Transferência/genética , Retroviridae/genética , Supressão GenéticaRESUMO
Cross-protection in plants is the phenomenon whereby a plant preinoculated with a mild virus strain becomes resistant to subsequent inoculation by a related severe strain. It has been used on a large scale in cases where no resistant plants are available. Although several hypotheses have been proposed to explain the molecular mechanism underlying cross-protection, no single hypothesis can account for all the data obtained. Recently, a phenomenon akin to cross-protection has been achieved in transformed plants harboring the cDNA of a part of a viral RNA genome. These results obtained by genetic engineering raise new hopes for obtaining plants resistant to virus infection.
Assuntos
Vírus de Plantas , Plantas/microbiologia , Engenharia Genética , Vírus de Plantas/genética , Vírus de Plantas/patogenicidade , Plantas/genéticaRESUMO
Two amber suppressor tRNAs have been isolated from calf liver. They are different from previously identified naturally occurring amber suppressors of eukaryotes in so far as they are neither tRNATyr nor tRNAGln. They are leucine iso-acceptors and their nucleotide sequence indicates that they harbour a CAA and a CAG anticodon respectively. Both species are functional as amber suppressors as demonstrated by readthrough of the amber codon which terminates the 126 kd protein gene of tobacco mosaic virus RNA. The results bring new information in the discussion of codon-anticodon recognition and regulation of termination in eukaryotic protein synthesis.
Assuntos
RNA de Transferência/genética , Supressão Genética , Animais , Sequência de Bases , Bovinos , Fígado/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA de Transferência/isolamento & purificação , RNA de Transferência/metabolismo , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência de Tirosina/genética , Tirosina/metabolismoRESUMO
Naturally occurring antibodies to left-handed Z-DNA have been shown to be present in the sera of human patients with systemic lupus erythematosus (SLE). These antibodies are of two types. One type reacts with both denatured DNA and Z-DNA. The other type is specific for Z-DNA and remained in the serum after removal of the cross-reactive antibody by extensive absorption on a denatured DNA affinity column. The antibodies appear to be specific for SLE and do not appear frequently in other rheumatic diseases. The presence of the antibody in SLE is correlated with the clinical manifestations of the disease, in parallel with antibodies to native and denatured DNA.
