The human cytomegalovirus (HCMV)-encoded viral G protein-coupled receptor pUS28 contributes to an array of biological effects, including cell migration and proliferation. Using FIX-BAC (bacterial artificial chromosome, derived from the HCMV clinical isolate VR1814) and lambda red recombination techniques, we generated HCMV recombinants expressing amino-terminally FLAG-tagged versions of wild-type pUS28 (FLAG–US28/WT), G-protein coupling deficient pUS28 (FLAG–US28/R129A) and chemokine-binding domain deficient pUS28 (FLAG–US28/ΔN). Infection with the FLAG–US28/R129A virus failed to induce inositol phosphate accumulation, indicating that G-protein coupling is essential for pUS28 signalling to phospholipase C-β (PLC-β) during HCMV infection. The FLAG–US28/ΔN virus induced about 80 % of the level of PLC-β signalling induced by the FLAG–US28/WT virus, demonstrating that the N-terminal chemokine-binding domain is not required for pUS28-induced PLC-β signalling in infected cells. The data presented here are the first to describe the functional analyses of several key pUS28 mutants in HCMV-infected cells. Elucidating the mechanisms by which pUS28 signals during infection will provide important insights into HCMV pathogenesis.
BeisserP. S.,
VinkC.,
Van DamJ. G.,
GraulsG.,
VanherleS. J.,
BruggemanC. A.1998; The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection. J Virol 72:2352–2363
BennettT. A.,
MaestasD. C.,
ProssnitzE. R.2000; Arrestin binding to the G protein-coupled N-formyl peptide receptor is regulated by the conserved “DRY” sequence. J Biol Chem 275:24590–24594[CrossRef]
BillstromM. A.,
JohnsonG. L.,
AvdiN. J.,
WorthenG. S.1998; Intracellular signaling by the chemokine receptor US28 during human cytomegalovirus infection. J Virol 72:5535–5544
BodaghiB.,
JonesT. R.,
ZipetoD.,
VitaC.,
SunL.,
LaurentL.,
Arenzana-SeisdedosF.,
VirelizierJ. L.,
MichelsonS.1998; Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J Exp Med 188:855–866[CrossRef]
BoomkerJ. M.,
VerschuurenE. A.,
BrinkerM. G.,
de LeijL. F.,
TheT. H.,
HarmsenM. C.2006; Kinetics of US28 gene expression during active human cytomegalovirus infection in lung-transplant recipients. J Infect Dis 193:1552–1556[CrossRef]
BorstE. M.,
HahnG.,
KoszinowskiU. H.,
MesserleM.1999; Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli : a new approach for construction of HCMV mutants. J Virol 73:8320–8329
BrittW. J.,
JarvisM.,
SeoJ. Y.,
DrummondD.,
NelsonJ.2004; Rapid genetic engineering of human cytomegalovirus by using a lambda phage linear recombination system: demonstration that pp28 (UL99) is essential for production of infectious virus. J Virol 78:539–543[CrossRef]
BrowneH.,
ChurcherM.,
MinsonA.1992; Identification, characterization and deletion analysis of HCMV gene products with homology to G protein-coupled receptors. The Seventeenth Annual International Herpesvirus Workshop 275
CasarosaP.,
MengeW. M.,
MinisiniR.,
OttoC.,
van HeterenJ.,
JongejanA.,
TimmermanH.,
MoeppsB.,
KirchhoffF.other authors2003; Identification of the first nonpeptidergic inverse agonist for a constitutively active viral-encoded G protein-coupled receptor. J Biol Chem 278:5172–5178[CrossRef]
CasarosaP.,
WaldhoerM.,
LiWangP. J.,
VischerH. F.,
KledalT.,
TimmermanH.,
SchwartzT. W.,
SmitM. J.,
LeursR.2005; CC and CX3C chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor. J Biol Chem 280:3275–3285[CrossRef]
CheeM. S.,
SatchwellS. C.,
PreddieE.,
WestonK. M.,
BarrellB. G.1990; Human cytomegalovirus encodes three G protein-coupled receptor homologues. Nature 344:774–777[CrossRef]
ChungD. A.,
WadeS. M.,
FowlerC. B.,
WoodsD. D.,
AbadaP. B.,
MosbergH. I.,
NeubigR. R.2002; Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors. Biochem Biophys Res Commun 293:1233–1241[CrossRef]
DatsenkoK. A.,
WannerB. L.2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645[CrossRef]
Davis-PoynterN. J.,
LynchD. M.,
VallyH.,
ShellamG. R.,
RawlinsonW. D.,
BarrellB. G.,
FarrellH. E.1997; Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus. J Virol 71:1521–1529
HahnG.,
KhanH.,
BaldantiF.,
KoszinowskiU. H.,
RevelloM. G.,
GernaG.2002; The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics. J Virol 76:9551–9555[CrossRef]
KledalT. N.,
RosenkildeM. M.,
SchwartzT. W.1998; Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28. FEBS Lett 441:209–214[CrossRef]
KuhnD. E.,
BeallC. J.,
KolattukudyP. E.1995; The cytomegalovirus US28 protein binds multiple CC chemokines with high affinity. Biochem Biophys Res Commun 211:325–330[CrossRef]
LohL. C.,
KeelerV. D.,
ShanleyJ. D.1999; Sequence requirements for the nuclear localization of the murine cytomegalovirus M44 gene product pp50. Virology 259:43–59[CrossRef]
MaussangD.,
VerzijlD.,
van WalsumM.,
LeursR.,
HollJ.,
PleskoffO.,
MichelD.,
van DongenG. A.,
SmitM. J.2006; Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis. Proc Natl Acad Sci U S A 103:13068–13073[CrossRef]
McLeanK. A.,
HolstP. J.,
MartiniL.,
SchwartzT. W.,
RosenkildeM. M.2004; Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74. Virology 325:241–251[CrossRef]
MichelsonS.,
Dal MonteP.,
ZipetoD.,
BodaghiB.,
LaurentL.,
OberlinE.,
Arenzana-SeisdedosF.,
VirelizierJ. L.,
LandiniM. P.1997; Modulation of RANTES production by human cytomegalovirus infection of fibroblasts. J Virol 71:6495–6500
MillerW. E.,
HoutzD. A.,
NelsonC. D.,
KolattukudyP. E.,
LefkowitzR. J.2003; G-protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomegalovirus US28 GPCR. J Biol Chem 278:21663–21671[CrossRef]
MinisiniR.,
TuloneC.,
LuskeA.,
MichelD.,
MertensT.,
GierschikP.,
MoeppsB.2003; Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28. J Virol 77:4489–4501[CrossRef]
MokrosT.,
RehmA.,
DroeseJ.,
OppermannM.,
LippM.,
HopkenU. E.2002; Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by agonist-independent phosphorylation. J Biol Chem 277:45122–45128[CrossRef]
OppenheimA. B.,
RattrayA. J.,
BubunenkoM.,
ThomasonL. C.,
CourtD. L.2004; In vivo recombineering of bacteriophage λ by PCR fragments and single-strand oligonucleotides. Virology 319:185–189[CrossRef]
Randolph-HabeckerJ. R.,
RahillB.,
Torok-StorbB.,
VieiraJ.,
KolattukudyP. E.,
RovinB. H.,
SedmakD. D.2002; The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production. Cytokine 19:37–46[CrossRef]
RheeM. H.,
NevoI.,
LevyR.,
VogelZ.2000; Role of the highly conserved Asp-Arg-Tyr motif in signal transduction of the CB2 cannabinoid receptor. FEBS Lett 466:300–304[CrossRef]
SeifertR.,
Wenzel-SeifertK.2002; Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors. Naunyn Schmiedebergs Arch Pharmacol 366:381–416[CrossRef]
VieiraJ.,
SchallT. J.,
CoreyL.,
GeballeA. P.1998; Functional analysis of the human cytomegalovirus US28 gene by insertion mutagenesis with the green fluorescent protein gene. J Virol 72:8158–8165
ZipetoD.,
BodaghiB.,
LaurentL.,
VirelizierJ. L.,
MichelsonS.1999; Kinetics of transcription of human cytomegalovirus chemokine receptor US28 in different cell types. J Gen Virol 80:543–547