The mouse cytomegalovirus chemokine receptor homologue (CKR) M33 is required for salivary gland tropism and efficient reactivation from latency phenotypes partially rescued by the human cytomegalovirus CKR US28. and gammaherpesviruses express one or more 7-transmembrane receptor homologues (7TMR) several of which have been implicated in viral pathogenesis and A-966492 are thereby regarded as potential therapeutic targets (1). Human cytomegalovirus (HCMV) encodes four 7TMRs: UL33 UL78 (conserved in all betaherpesviruses) and US27 and US28 which are encoded by tandem genes (restricted to primate CMV). Of these US28 a CC chemokine receptor homologue (CKR) has been the most thoroughly characterized. Unlike most cellular CKR US28 exhibits promiscuous binding of CC chemokines and the membrane-tethered CX3CL1 A-966492 chemokine fractalkine. As A-966492 these chemokines elicit chemotactic responses of monocytes and vascular endothelial cells US28 has been implicated in virus dissemination. US28 has been shown to signal constitutively via Gαq (2) and the mitogen-activated protein kinase (MAPK) pathways (3-5) and invoke activation of transcription factors including NF-κB CREB NFAT and SRE (2-4 6 7 Potential consequences of the diverse signaling cascade elicited by US28 include modulation of the expression of host genes involved in pathogenesis enhancement of replication in particular cell types and triggering reactivation from latency (8 9 Similar to several other viral CKRs US28 is rapidly and constitutively endocytosed providing a mechanism for A-966492 both regulation of G protein-dependent signaling and initiation of G protein-independent signaling (3 5 10 11 Previous characterization of N- and C-terminal US28 mutations demonstrated the importance of particular US28 domains in receptor signaling and endocytosis. The US28 C-tail is necessary and sufficient to confer efficient endocytosis to US28 and heterologous CKR (3). C-terminal truncations FIGF of US28 (ΔC36 ΔC40 and ΔC54) have revealed modulatory effects A-966492 on either classical G protein-mediated phospholipase C-β (PLC-β) signaling engagement of the p38 MAP kinase pathway or activation of NF-κB and CREB transcription factors (3 5 12 Mutation of the highly conserved arginine within the transmembrane III DRY motif abolished constitutive G protein-mediated signaling but the mutant protein retained constitutive endocytosis (3). Models for the function of HCMV-encoded CKRs have utilized mouse and rat CMVs (MCMV and RCMV respectively). We previously established that the MCMV homologue of HCMV UL33 (M33) is important for salivary gland tropism and establishment of and/or reactivation from latency (13-15). Mutagenesis of M33 demonstrated that while salivary gland tropism was partly preserved in the absence of the M33 C tail it was highly dependent on M33 G protein coupling (14). In contrast MCMV latency and/or reactivation was substantially reduced with mutation of either the DRY motif or the M33 C tail suggesting that both G protein-dependent and -independent mechanisms are important for the latency phenotype (16; A-966492 H. E. Farrell and N. Davis-Poynter unpublished observations). Notably we demonstrated that wild-type (wt) HCMV US28 can partially substitute for the role of M33 < 0.05) was observed in the dual RQ/ΔC54 US28 mutant whereas the single mutants were not significantly different from the wild type consistent with previous studies showing that p38 MAPK is induced by both G protein-dependent and -independent mechanisms (13 22 25 In contrast p-ERK1/2 and p-JNK MAPK induction was diminished (< 0.001 and < 0.01 respectively) only in the absence of G protein coupling (mutants R129Q and RQ/ΔC54) suggesting that the US28 C tail was dispensable for their induction. None of the mutations including RQ/ΔC54 reduced MAPK activation to control levels (pcDNA3). Given that US28ΔC54 and RQ/ΔC54 exhibited low-level endocytosis (compared with CCR5) these mutants may retain the capacity to initiate MAPK or additional signaling pathways via scaffold interactions despite the absence of the C tail. Fig 3 HEK293 cells were transfected with 20 μg of the indicated HA-tagged US28 constructs or the pcDNA3 vector using calcium chloride precipitation. At 48 h after transfection the cells were lysed on ice using radioimmunoprecipitation assay (RIPA) ... US28 constitutively activates multiple transcription factors including CREB NF-κβ and to a lesser extent NFAT (2 4 24 G protein coupling appears to be the predominant mechanism since the DRY motif mutants.