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 (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.
Jarvis
Michael A
MA
Vaccine and Gene Therapy Institute, Oregon Health Sciences University, Portland, Oregon, USA. jarvismi@ohsu.edu
Jones
Thomas R
TR
Drummond
Derek D
DD
Smith
Patsy P
PP
Britt
William J
WJ
Nelson
Jay A
JA
Baldick
Carl J
CJ
D019943:Amino Acid Substitution; D047390:Casein Kinase II; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D005347:Fibroblasts / Q000821:virology; D006801:Humans; D016366:Open Reading Frames; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic; D012694:Serine; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D014779:Virus Replication; D021601:trans-Golgi Network / Q000378:metabolism*
D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases