ABSTRACT
Programmed death of infected cells is used by multicellular organisms to counter viral infections. Sheeppox virus encodes for SPPV14, a potent inhibitor of Bcl-2-mediated apoptosis. We reveal the structural basis of apoptosis inhibition by determining crystal structures of SPPV14 bound to BH3 motifs of proapoptotic Bax and Hrk. The structures show that SPPV14 engages BH3 peptides using the canonical ligand-binding groove. Unexpectedly, Arg84 from SPPV14 forms an ionic interaction with the conserved Asp in the BH3 motif in a manner that replaces the canonical ionic interaction seen in almost all host Bcl-2:BH3 motif complexes. These results reveal the flexibility of virus-encoded Bcl-2 proteins to mimic key interactions from endogenous host signalling pathways to retain BH3 binding and prosurvival functionality.
Subject(s)
Apoptosis Regulatory Proteins/chemistry , Capripoxvirus/chemistry , Viral Proteins/chemistry , bcl-2-Associated X Protein/chemistry , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Host-Pathogen Interactions , Models, Molecular , Protein Conformation , Protein Domains , Viral Proteins/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
The fusion gene (ORF 117) sequences of twelve (n = 12) capripox virus isolates namely sheeppox (SPPV) and goatpox (GTPV) viruses from India were demonstrated for their genetic and phylogenetic relationship among them. All the isolates were confirmed for their identity by routine PCR before targeting ORF 117 gene for sequence analysis. The designed primers specifically amplified ORF 117 gene as 447 bp fragment from total genomic DNA extracted from all the isolates. Sequence analysis revealed a significant percentage of identity among GTPV, SPPV and between them at both nucleotide and amino acid levels. The topology of the phylogenetic tree revealed that three distinct clusters corresponding to SPPV, GTPV and lumpy skin disease virus was formed. However, SPPV Pune/08 and SPPV Roumanian Fanar isolates were clustered into GTPV group as these two isolates showed a 100 and 99.3 % identity with GTPV isolates of India at nt and aa levels, respectively. Protein secondary structure and 3D view was predicted and found that it has high antigenic index and surface probability with low hydrophobicity, and it can be targeted for expression and its evaluation to explore its diagnostic potential in epidemiological investigation in future.
Subject(s)
Capripoxvirus/genetics , Cattle Diseases/virology , Goat Diseases/virology , Poxviridae Infections/veterinary , Sheep Diseases/virology , Vaccinia virus/genetics , Viral Fusion Proteins/genetics , Animals , Capripoxvirus/chemistry , Capripoxvirus/classification , Cattle , Genetic Variation , Goats , India , Molecular Sequence Data , Phylogeny , Poxviridae Infections/virology , Sequence Homology, Amino Acid , Sheep , Vaccinia virus/chemistry , Viral Fusion Proteins/chemistryABSTRACT
BACKGROUND: Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA. RESULTS: A single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/µl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device. CONCLUSIONS: This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.
