Abstract
The orf virus (ORFV) is among the parapoxvirus genus of the poxviridae family, but little is known about the proteolytic pathways of ORFV encoding proteins. By contrast, the proteolysis mechanism of the vaccinia virus (VV) has been extensively explored. Vaccinia virus core protein P4a undergoes a proteolytic process that takes place at a conserved cleavage site Ala-Gly-X (where X is any amino acid) and participates in virus assembly. Bioinformatics analysis revealed that an ORFV encoding protein, ORFV086, has a similar structure to the vaccinia virus P4a core protein. In this study, we focus on the kinetic analysis and proteolysis mechanism of ORFV086. We found, via kinetic analysis, that ORFV086 is a late gene that starts to express at 8 h post infection at mRNA level and 12–24 h post infection at the protein level. The ORFV086 precursor and a 21 kDa fragment can be observed in mature ORFV virions. The same bands were detected at only 3 h post infection, suggesting that both the ORFV086 precursor and the 21 kDa fragment are viral structural proteins. ORFV086 was cleaved from 12 to 24 h post infection. The cleavage took place at different sites, resulting in seven bands with differing molecular weights. Sequence alignment revealed that five putative cleavage sites were predicted at C-terminal and internal regions of ORFV086. To investigate whether those cleavage sites are involved in proteolytic processing, full length and several deletion mutant ORFV086 recombinant proteins were expressed and probed. The GGS site that produced a 21 kDa cleavage fragment was confirmed by identification of N/C-terminal FLAG epitope recombinant proteins, site-directed mutagenesis and pulse-chase analysis. Interestingly, chase results demonstrated that, at late times, ORFV086 is partially cleaved. Taken together, we concluded that GGS is a cleavage site in ORFV086 and produces a 21 kDa fragment post infection. Both ORFV086 precursor and the 21 kDa fragment are structural proteins of mature ORFV virions. ORFV086 and its cleaved products are indispensable for correct assembly of mature viral particles and this proteolytic processing of ORFV086 may play an essential role in viral morphogenic transition.
Highlights
The orf virus (ORFV), the prototype member of the Poxviridae Parapoxvirus genus (Diel et al, 2011), is a double-stranded DNA virus
Reverse transcription-polymerase chain reaction (RT-PCR) kinetic analysis revealed the synthesis of ORFV086 could be detected 8 h after infection with NA1/11, and could be detected only in those cells cultured in the absence of AraC, an inhibitor of late poxvirus transcription
In experiments of transient expression of recombinant proteins fused with a C-terminal FLAG epitope and genetic mutations of the ORFV086 precursor, the only certainty is that the 23 kDa product is 4A-GGS-C derived from the 4A-086 precursor cleaved at the GGS site which was further confirmed in the pulse-chase assay
Summary
The orf virus (ORFV), the prototype member of the Poxviridae Parapoxvirus genus (Diel et al, 2011), is a double-stranded DNA virus. The genome of the orf virus is 138kb and is rich in G+C content (64%) (Delhon et al, 2004) Both ends of the genome encode immunomodulatory proteins and are highly variable, while genes in the central region of the genome (ORF009ORFV111) are highly conserved and play key roles in replication, assembly and viral release (Mercer et al, 1987, 2006; Cottone et al, 1998; Delhon et al, 2004). Research regarding the replication, assembly, morphogenesis and immune mechanisms of the ORFV is scarce
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
