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Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

ABSTRACT: Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV) and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005) molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.

SUBMITTER: Assis FL

PROVIDER: S-EPMC3506599 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

Assis Felipe L FL Almeida Gabriel M F GM Oliveira Danilo B DB Franco-Luiz Ana P M AP Campos Rafael K RK Guedes Maria I M MI Fonseca Flávio G FG Trindade Giliane S GS Drumond Betânia P BP Kroon Erna G EG Abrahão Jônatas S JS

PloS one 20121126 11

Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have ...[more]

PMID: 23189200

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Project description:For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the ΔA56 virus, the G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter's G9 subcomponent. Although the G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

Dataset Information

Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

Publications

Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

Similar Datasets

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