Phosphorylation of Human Cytomegalovirus Glycoprotein B (gB) at the Acidic Cluster Casein Kinase 2 Site (Ser₉₀₀) Is Required for Localization of gB to the trans-Golgi Network and Efficient Virus Replication

Human cytomegalovirus (HCMV) glycoprotein B (gB), encoded by the UL55 open reading frame, is an essential envelope glycoprotein involved in cell attachment and entry. Previously, we identified residue serine 900 (Ser ₉₀₀) as a unique site of reversible casein kinase 2 phosphorylation in the cytoplasmic domain of HCMV gB. We have also recently shown that gB is localized to the trans-Golgi network (TGN) in HCMV-permissive cells, thereby identifying the TGN as a possible site of virus envelopment. The aim of the current study was to determine the role of Ser₉₀₀ phosphorylation in transport of gB to the TGN and in HCMV biogenesis. Recombinant HCMV strains were constructed that expressed gB molecules containing either an aspartic acid (gBAsp₉₀₀) or alanine residue (gBAla₉₀₀) substitution at Ser₉₀₀ to mimic the phosphorylated or nonphosphorylated form, respectively. Immunofluorescence analysis of the trafficking of gB mutant molecules in fibroblasts infected with the HCMV recombinants revealed that gBAsp₉₀₀ was localized to the TGN. In contrast, gBAla₉₀₀ was partially mislocalized from the TGN, indicating that phosphorylation of gB at Ser₉₀₀ was necessary for TGN localization. The increased TGN localization of gBAsp₉₀₀ was due to a decreased transport of the molecule to post-TGN compartments. Remarkably, the substitution of an aspartic acid residue for Ser₉₀₀ also resulted in an increase in levels of progeny virus production during HCMV infection of fibroblasts. Together, these results demonstrate that phosphorylation of gB at Ser₉₀₀ is necessary for gB localization to the TGN, as well as for efficient viral replication, and further support the TGN as a site of HCMV envelopment.