RESUMO
Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.
Assuntos
Corpos de Inclusão Viral/metabolismo , Reoviridae/metabolismo , Spodoptera/virologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Corpos de Inclusão Viral/química , Corpos de Inclusão Viral/genética , Fases de Leitura Aberta , Reoviridae/química , Reoviridae/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Here is described the isolation of a naturally occurring A-type inclusion body (ATI)-negative vaccinia-like virus, Belo Horizonte virus (VBH), obtained from a mousepox-like outbreak in Brazil. The isolated virus was identified and characterized as an orthopoxvirus by conventional methods. Molecular characterization of the virus was done by DNA cross-hybridization using Vaccinia virus (VACV) DNA. In addition, conserved orthopoxvirus genes such as vaccinia growth factor, thymidine kinase and haemagglutinin were amplified by PCR and sequenced. All sequences presented high similarity to VACV genes. Based on the sequences, phenograms were constructed for comparison with other poxviruses; VBH clustered consistently with VACV strains. Attempts to amplify the ATI gene (ati) by PCR, currently used to identify orthopoxviruses, were unsuccessful. Results presented here suggest that most of the ati gene is deleted in the VBH genome.
Assuntos
Corpos de Inclusão Viral/genética , Vaccinia virus/genética , Animais , Sequência de Bases , Southern Blotting , DNA Viral/genética , DNA Viral/isolamento & purificação , Deleção de Genes , Camundongos , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Vaccinia virus/classificação , Vaccinia virus/isolamento & purificaçãoRESUMO
The lack of knowledge about the natural host of Vaccinia virus (VV) along with the description of human infections caused by poxviruses after smallpox eradication has increased the need to characterize poxviruses isolated from the wild. Moreover, in the past years poxviruses have been widely studied as potential vaccination tools, with the discovery of several genes implicated in the evasion of the host immune response involved in virus pathogenesis. Among them, an Interferon (IFN)-binding protein was identified in the supernatant of VV strain WR infected cells coded by the B18R gene. It was shown that many other Orthopoxviruses also encode and express this soluble receptor although some VV strains such as Lister and modified Ankara, which were less reactogenic vaccines, do not. The BeAn 58058 virus (BAV) has been recently characterized and proposed to be an Orthopoxvirus. BAV was also shown to be less virulent in animal models than VV Lister. Here we report the identification of an IFN-alpha/betaR gene in the BAV genome with 99% of sequence identity with the VVWR B18R gene. The identified gene encodes a B18R-like IFN binding protein as demonstrated by its capacity to inhibit the IFN-mediated protection of VERO cells against EMC virus. In order to better characterize the virus we have searched for the A type inclusion body (ATI) gene currently used in the classification of Orthopoxviruses but did not detect it in the BAV genome. We have also sequenced the BAV thymidine kinase (TK) gene, a poxvirus-conserved gene, which, as expected, showed high homology with the TK gene of other poxviruses. Phylogenetic trees were constructed based on sequences of the IFN-alpha/betaR and TK genes from several poxviruses and in both cases BAV was placed in the same cluster as other VV strains. These observations strengthened the hypothesis that this virus is a variant of the VV vaccine used in Brazil. However the explanation for the BAV lack of virulence remains to be discovered.