RESUMO
UNLABELLED: The untranslated regions (UTR) present at the ends of bunyavirus genome segments are required for essential steps in the virus life cycle and provide signals for encapsidation by nucleocapsid protein and the promoters for RNA transcription and replication as well as for mRNA transcription termination. For the prototype bunyavirus, Bunyamwera virus (BUNV), only the terminal 11 nucleotides (nt) of the segments are identical. Thereafter, the UTRs are highly variable both in length and in sequence. Furthermore, apart from the conserved termini, the UTRs of different viruses are highly variable. We previously generated recombinant BUNV carrying the minimal UTRs on all three segments that were attenuated for growth in cell culture. Following serial passage of these viruses, the viruses acquired increased fitness, and amino acid changes were observed to accumulate in the viral polymerase (L protein) of most mutant viruses, with the vast majority of the amino acid changes occurring in the C-terminal region. The function of this domain within L remains unknown, but by using a minigenome assay we showed that it might be involved in UTR recognition. Moreover, we identified an amino acid mutation within the polymerase that, when introduced into an otherwise wild-type BUNV, resulted in a virus with a temperature-sensitive phenotype. Viruses carrying temperature-sensitive mutations are good candidates for the design of live attenuated vaccines. We suggest that a combination of stable deletions of the UTRs together with the introduction of temperature-sensitive mutations in both the nucleocapsid and the polymerase could be used to design live attenuated vaccines against serious pathogens within the family Bunyaviridae. IMPORTANCE: Virus growth in tissue culture can be attenuated by introduction of mutations in both coding and noncoding sequences. We generated attenuated Bunyamwera viruses by deleting sequences within both the 3' and 5' untranslated regions (UTR) on each genome segment and showed that the viruses regained fitness following serial passage in cell culture. The fitter viruses had acquired amino acid changes predominantly in the C-terminal domain of the viral polymerase (L protein), and by using minigenome assays we showed that the mutant polymerases were better adapted to recognizing the mutant UTRs. We suggest that deletions within the UTRs should be incorporated along with other specific mutations, including deletion of the major virulence gene encoding the NSs protein and introduction of temperature-sensitive mutations, in the design of attenuated bunyaviruses that could have potential as vaccines.
Assuntos
Adaptação Biológica , Vírus Bunyamwera/enzimologia , Evolução Molecular , RNA Polimerase Dependente de RNA/metabolismo , Deleção de Sequência , Regiões não Traduzidas , Proteínas Virais/metabolismo , Vírus Bunyamwera/genética , Vírus Bunyamwera/crescimento & desenvolvimento , RNA Polimerase Dependente de RNA/genética , Inoculações Seriadas , Proteínas Virais/genéticaRESUMO
The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.
Assuntos
Vírus Bunyamwera/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Animais , Vírus Bunyamwera/enzimologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Epitopos/genética , Regulação Viral da Expressão Gênica , Rim , RNA Polimerase Dependente de RNA/genética , Recombinação Genética , Replicon/genética , Células VeroRESUMO
The small (S) genomic segment of Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. In order to elucidate the function of NSs, we established a plasmid-based minireplicon system using mammalian cells that express large amounts of T7 RNA polymerase. Expression of N, the viral polymerase protein (L), and a minireplicon containing a reporter gene was sufficient to reconstitute functional virus nucleocapsids. Coexpression of NSs, however, led to a dose-dependent decrease in reporter activity without affecting expression of controls. The inhibition could not be reversed by overexpression of N, L or the minireplicon, indicating that the NSs effect was not caused by a reduction in virus gene expression. The NSs proteins of two other members of the Bunyavirus genus, Guaroa virus and Lumbo virus, were also inhibitory in our system. The intracellular localisation of Bunyamwera virus NSs was investigated and found to be predominantly cytoplasmic, but intranuclear inclusion was also detected. Taken together, these data suggest that, in mammalian cells, the bunyavirus NSs protein controls the activity of the viral polymerase by a highly conserved mechanism.
Assuntos
Vírus Bunyamwera/genética , Vírus Bunyamwera/fisiologia , RNA Viral/biossíntese , Replicon/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Animais , Western Blotting , Vírus Bunyamwera/efeitos dos fármacos , Vírus Bunyamwera/enzimologia , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cricetinae , Citoplasma/metabolismo , Citoplasma/virologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Imunofluorescência , Genes Reporter/genética , Dados de Sequência Molecular , Nucleocapsídeo/biossíntese , Nucleocapsídeo/genética , Transporte Proteico , RNA Viral/genética , Replicon/efeitos dos fármacos , Alinhamento de Sequência , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/farmacologia , Proteínas Virais , Replicação Viral/efeitos dos fármacosRESUMO
RNA dependent RNA polymerase has been demonstrated in purified Lumbo virus (Bunyavirus) which contains a single stranded segmented RNA. Divalent cations (Mn++ and Mg++) are required for optimal in vitro activity. Reaction products can be specifically annealed with the viral genome.
