Localization of the varicella-zoster virus encapsidation proteins in transfected and infected cells

Varicella-zoster virus (VZV) DNA encapsidation likely involves at least seven proteins encoded by open reading frames (ORFs) 54, 45/42, 43, 34, 30, 26, and 25. In contrast to proteins encoded by the herpes simplex virus encapsidation gene homologs ULs 6, 15, 17, 25, 28, 32, and 33, none of the putative VZV encapsidation proteins has been characterized. All seven of the VZV encapsidation proteins were detected in transfected cells via indirect immunofluorescence using murine anti-V5 monoclonal primary antibodies and FITC-tagged secondary antibodies. To study the localization of individual encapsidation proteins in the context of infection, ORF-specific antisera were developed in rabbits or guinea pigs. pORF30-V5 showed varied localization patterns when observed by indirect immunofluorescence. To further investigate localization of the proposed terminase subunit, subcellular fractionation studies were performed. The 87 kDa ORF30 gene product was detected in nuclear extracts via immunoblot analysis using the pORF30-specific antiserum. This finding is consistent with studies showing that the terminase subunits of HSV-1 localize to the nucleus. pORF45/42-V5 was shown to localize primarily to the cytoplasm of transfected MeWo cells. Localization of the ORF45/42 gene product in the context of VZV infection was examined in multiple cell lines using an affinity-purified rabbit pORF45/42-specific antiserum. In contrast to the cytoplasmic localization observed in transfected cells, pORF45/42 showed distinct nuclear staining in all cell lines. The results strongly suggested that the ORF45/42 gene product was expressed in the nucleus of VZV-infected cells, which is consistent with the function and localization of a viral terminase. Furthermore, the results suggested that an as yet undetermined viral gene product was involved in the translocation of ORF45/42 protein into the nucleus of infected cells. pORF34 expression in transfected MeWo cells was primarily cytoplasmic with some nuclear localization. Anti-pORF34 antiserum developed in guinea pigs was used to examine localization in the context of VZV infection. pORF34 was shown to localize to both the cytoplasm and nucleus of infected MeWo cells. Nuclear staining accumulated into globular structures consistent with replication compartments, nuclear sites which contain essential DNA replication components. This is consistent with the proposed function of pORF34, a viral capsid protein. Indirect immunofluorescence analysis provided a means to study subcellular localization and perform initial characterization of the encapsidation gene products of VZV