In cells coexpressing ORF49p and ORF44F129Sp, ORF44F129Sp did not accumulate on the TGN and was not coimmunoprecipitated with ORF49p (data not shown)

In cells coexpressing ORF49p and ORF44F129Sp, ORF44F129Sp did not accumulate on the TGN and was not coimmunoprecipitated with ORF49p (data not shown). To examine whether 129F functions specifically in the conserved interaction in VZV, alanine scanning was performed around 129F (Fig. is required for the efficient production of infectious progeny virus mediated by Pax1 the conserved interaction between the two proteins. INTRODUCTION Varicella-zoster virus (VZV) is a member of the human alphaherpesvirus subfamily and the etiologic agent of two diseases: varicella is the result of primary infection with VZV, and herpes-zoster is caused by reactivation of the virus from the latent state (1). VZV shares many features, especially a tropism for epithelial and neural tissues, with other human alphaherpesvirus members, including herpes simplex viruses 1 and 2 (HSV-1 and -2, respectively), and with the nonhuman alphaherpesviruses. However, VZV spreads only via cell-to-cell infection in culture and is more akin to the betaherpesviruses (i.e., human herpesviruses 6 and 7) in its apparent T-cell-tropism (1). The VZV genome is approximately 125 kb and contains at least 70 unique open reading frames (ORFs), and it is the smallest genome in terms of length and gene set among human herpesviruses (1,C3). Of the 70 identified ORFs, 44 are core genes that are conserved among all human herpesvirus subfamilies (4). Recent genome-wide mutagenesis analysis showed that 34 ORFs among the core genes are essential Isoshaftoside for virus reconstitution in cell culture, whereas deletion of seven ORFs results in viral growth defects, and three ORFs are dispensable in cell culture or skin organ culture (5). Eight core genes encode tegument proteins, which are the structural components of the virion and are located between the nucleocapsid and the envelope. VZV ORF49 encodes a nonessential tegument protein that functions as a cell-tropic factor in cell culture via an unknown mechanism (6). VZV ORF49 is the homolog of HSV-1 UL11 and human cytomegalovirus (HCMV) UL99, which are among the most extensively studied tegument protein-encoding genes. The UL11 and UL99 gene products, pUL11 and pp28, function in secondary envelopment (7,C9), but they have different roles in the viral life cycle. HSV-1 UL11 is not essential for the viral life cycle; however, the UL11 deletion mutant forms small plaques, and the final titers are reduced to 80 to 95% of wild-type levels (10). In contrast, HCMV UL99 Isoshaftoside is an essential gene, and pp28-deficient mutants show extremely impaired growth in Isoshaftoside normal fibroblasts and produce no detectable infectious progeny (9). However, this mutant spreads from cell to cell via an unknown mechanism (11). Several recent reports, beginning with one on HSV-1 UL16, which is a core gene within the intron of a conserved herpesvirus spliced gene, (12), showed that interactions between pUL11 and pUL16 homologs were conserved beyond the host species and herpesvirus subfamilies (13,C15). HSV pUL16 localizes to the nucleus and the cytoplasm of infected cells and functions in virus entry and in nuclear and cytoplasmic egress (16,C19); pUL16 homologs may function in secondary envelopment, as reviewed in reference 20. As described for UL11 homologs, whether UL16 homologs are required for the viral life cycle differs among viruses (13, 15, 21,C25). In a genome-wide mutagenesis analysis, deletion of the entire gene region from the viral genome of VZV ORF44, the UL16 homolog, showed that it is an essential gene by loss-of-function analysis in the MeWo cell line (5), although this Isoshaftoside was not confirmed by a revertant virus generated for gain-of-function analysis. The LI motif and the conserved acidic cluster of pUL11 are essential for Isoshaftoside its interaction with pUL16 of HSV-1, whereas the critical sequences in pUL16 have not been determined because it is highly sensitive to deletions. Its short N-terminal 75-amino-acid (aa) fragment was recently shown to include the pUL11 binding site, and its C-terminal region functions as the binding regulatory domain (26), although this has not been confirmed in the context of HSV-1-infection. HCMV pUL94 directs pp28 to the assembly compartment, where it plays a role in secondary envelopment. Amino acids 37 to 39, near the acidic cluster of pp28, and one of the conserved cysteine residues of pUL94 are involved in binding in the context of infection (24, 27). In VZV, potential ORF49 protein (ORF49p)-binding proteins, including the pUL16 homolog ORF44 protein (ORF44p), were identified by global screening using the yeast two-hybrid system (28, 29), although.