The chemokine receptor homologue encoded by US27 of human cytomegalovirus is heavily glycosylated and is present in infected human foreskin fibroblasts and enveloped virus particles

Abstract

Human cytomegalovirus (HCMV), a member of the β-herpesvirus family, encodes four homologues of cellular G protein-coupled receptors (GPCRs). One of these, the protein product of HCMV open reading frame (ORF) UL33, has been identified in HCMV-infected cells and virus particles and shown to be heat-aggregatable and N-glycosylated. Another, the product of ORF US28, has been functionally characterized as a β-chemokine receptor. Here we report the use of RT-PCR, coupled in vitro transcription-translation, immunoprecipitation, and Western immunoassays to (i) show that RNA from the open reading frame US27 appears predominantly during the late phase of replication; (ii) identify the protein encoded by HCMV US27 in infected cells and enveloped virus particles; (iii) demonstrate that the US27-encoded protein is heterogeneously N-glycosylated and resolves as two species following treatment with peptide N-glycosidase F; and (iv) show that both the recombinant and deglycoylated infected cell US27 protein aggregate when heated in the presence of SDS prior to electrophoresis in polyacrylamide gels, a property which is abrogated with the addition of urea to sample buffer.

Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells

“…In our analysis, we have probed for US28 via its triple FLAG C-terminal epitope tag, thus increasing the sensitivity for this protein. It is also not surprising to find that US28 copurifies with mature virions, as US27 (53,70,73), UL33 (74), and UL78 (55) have been identified as components of the viral particle.…”

Recovery of an HMWP/hmwBP (pUL48/pUL47) Complex from Virions of Human Cytomegalovirus: Subunit Interactions, Oligomer Composition, and Deubiquitylase Activity

“…Complex III is of particular interest as it appears to be comprised of at least four proteins (a, d, g, and i), all of which show size heterogeneity and at least three of which (a, d, and i) aggregate when heated-properties consistent with hydrophobic glycoproteins such as the G-protein-coupled receptor (GCR) proteins (pUS27, pUS28, and pUL33) (75,76) and envelope glycoproteins gM and gN (77). Some upper matrix protein (UM, pp71, pUL82) appears to cosediment with these proteins as part of the complex.…”

Desensitization of herpesvirus-encoded G protein-coupled receptors

“…HCMV GPCR US28-Of the approximately 200kb within the HCMV genome there are fourteen ORFs which encode seven-pass transmembrane proteins: US27, US28, UL33, UL78, and the US12 family (US12-US21) (Chee et al, 1990;Gao and Murphy, 1994;Margulies and Gibson, 2007;Rigoutsos et al, 2003). US28 shares sequence identity with human CC and CXC chemokine receptors and is able to bind a number of CC chemokines including RANTES, MIP-1α/β, and MCP-1 (Gao and Murphy, 1994;Kuhn et al, 1995;Vieira et al, 1998).…”

“…Beta-HCMV US27 (Chee et al, 1990), (Margulies and Gibson, 2007) US28 (Chee et al, 1990), (Gao and Murphy, 1994) UL33 (Chee et al, 1990), (Margulies et al, 1996 (Rawlinson et al, 1996) M78 (Oliveira and Shenk, 2001), (Rawlinson et al, 1996) RCMV R33 (Beisser et al, 1998), (Vink et al, 2000) R78 (Beisser et al, 1999), (Vink et al, 2000) HHV-6 U12 (Gompels et al, 1995), (Isegawa et al, 1998) U51 (Gompels et al, 1995), (Milne et al, 2000) HHV-7 U12 (Nakano et al, 2003), (Nicholas, 1996) U51 (Nicholas, 1996), (Tadagaki et al, 2005) Gamma-KSHV ORF74 (Arvanitakis et al, 1997) MHV-68 ORF74 (Virgin et al, 1997), (Wakeling et al, 2001) EBV BILF1 (Paulsen et al, 2005) HVS ECRF3 (Nicholas et al, 1992) …”