Investigation of orf virus structure and morphogenesis using recombinants expressing FLAG-tagged envelope structural proteins: evidence for wrapped virus particles and egress from infected cells.

Orf virus (ORFV) is the type species of the genus Parapoxvirus, but little is known about the structure or morphogenesis of the virus. In contrast, the structure and morphogenesis of vaccinia virus (VACV) has been extensively studied. VACV has two main infectious forms, mature virion (MV) and extracellular virion (EV). The MV is wrapped by two additional membranes derived from the trans-Golgi to produce a wrapped virion (WV), the outermost of which is lost by cellular membrane fusion during viral egress to form the EV. Genome sequencing of ORFV has revealed that it has homologues of almost all of the VACV structural genes. Notable exceptions are A36R, K2L, A56R and B5R, which are associated with WV and EV envelopes. This study investigated the morphogenesis and structure of ORFV by fusing FLAG peptide to the structural proteins 10 kDa, F1L and ORF-110 to form recombinant viruses. 10 kDa and F1L are homologues of VACV A27L and H3L MV membrane proteins, whilst ORF-110 is homologous to VACV A34R, an EV membrane protein. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG-F1L and FLAG-10 kDa were displayed on the surface of infectious particles, whereas ORF-110-FLAG could not be detected. Western blot analysis of solubilized recombinant ORF-110-FLAG particles revealed that ORF-110-FLAG was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking. Fluorescent microscopy confirmed the prediction that ORF-110-FLAG localized to the Golgi in virus-infected cells. Finally, immunogold labelling of EVs showed that ORF-110-FLAG became exposed on the surface of EV-like particles as a result of egress from the cell.