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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

Michael A. Jarvis,^1* Thomas R. Jones,² Derek D. Drummond,¹ Patsy P. Smith,³ William J. Britt,⁴ Jay A. Nelson,^1,3 and Carl J. Baldick^2,

Vaccine and Gene Therapy Institute,¹ Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon,³ Infectious Disease Section, Wyeth Research, Pearl River, New York,² Department of Pediatrics and Microbiology, University of Alabama at Birmingham, Birmingham, Alabama⁴

Received 17 June 2003/ Accepted 15 September 2003

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 (Ser₉₀₀) 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.

Present address: Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Conn.

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