title: Human cytomegalovirus pUS27 G protein-coupled receptor homologue is required for efficient spread by the extracellular route but not for direct cell-to-cell spread.
abstract: Human cytomegalovirus (HCMV) encodes multiple G protein-coupled receptor (GPCR) homologues, including pUS27, pUS28, pUL33, and pUL78. To explore the function of pUS27, we constructed pUS27-deficient derivates of two clinical isolates of HCMV. BFX-GFPstopUS27 is a FIX variant with a single base pair change in the US27 open reading frame, generating a stop codon that ablates accumulation of the GPCR homologue, and TB40/E-mCherrydlUS27 lacks the entire US27 coding region. BFX-GFPstopUS27 generated 10-fold less extracellular progeny in fibroblasts, and TB40/E-mCherrydlUS27 exhibited a similar defect in endothelial cells. The pUS27-deficient FIX derivative produced normal quantities of viral DNA and viral proteins tested, and a late virion protein was appropriately localized to the cytoplasmic assembly zone. After infection at a low multiplicity with wild-type FIX virus, neutralizing antibody reduced the accumulation of intracellular viral DNA and intracellular virions, as would be expected if the virus is limited to direct cell-to-cell spread by neutralization of extracellular virus. In contrast, the antibody had little effect on the spread of the BFX-GFPstopUS27 virus. Further, after infection at a low multiplicity, the pUS27-deficient TB40/E virus exhibited a growth defect in endothelial cells, where the clinical isolate normally generates extracellular virus, but the TB40/E derivative exhibited little defect in epithelial cells, where the wild-type virus does not produce extracellular virus. Thus, mutants lacking pUS27 rely primarily on direct cell-to-cell spread, and we conclude that the viral GCPR homologue acts at a late stage of the HCMV replication cycle to support spread of virus by the extracellular route.
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA.
authors.forename: Christine M
authors.initials: CM
authors.lastname: O'Connor
authors.forename: Thomas
authors.initials: T
authors.lastname: Shenk
mesh_terms: D002478:Cells, Cultured; D018389:Codon, Nonsense; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D003586:Cytomegalovirus Infections / Q000821:virology; D004279:DNA, Viral / Q000096:biosynthesis; D004847:Epithelial Cells / Q000821:virology; D005347:Fibroblasts / Q000821:virology; D017353:Gene Deletion; D006801:Humans; D019707:Receptors, Chemokine / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D018389:Codon, Nonsense; D004279:DNA, Viral; D019707:Receptors, Chemokine; C461644:US27 protein, human cytomegalovirus; D014764:Viral Proteins; D037521:Virulence Factors