Functional Analysis of the Murine Cytomegalovirus Chemokine Receptor Homologue M33: Ablation of Constitutive Signaling Is Associated with an Attenuated Phenotype In Vivo

Abstract

The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within the salivary glands. In this study, we probed N-and C-terminal regions of M33 as well as known 7TMR signature motifs in transmembrane (TM) II and TM III to determine the impact on cell surface expression, constitutive signaling, and in vivo phenotype. The region between amino acids R 340 and A 353 of the C terminus was found to be important for CREB-and NFAT-mediated signaling, although not essential for phosphatidylinositol turnover. Tagging or truncation of the N terminus of M33 resulted in loss of cell surface expression. Within TM II, an F79D mutation abolished constitutive signaling, demonstrating a role, as in other cellular and viral 7TMR, of TM II in receptor activation. In TM III, the arginine (but not the asparagine) residue of the NRY motif (the counterpart of the common DRY motif in cellular 7TMR) was found to be essential for constitutive signaling. Selected mutations incorporated into recombinant MCMV showed that disruption of constitutive signaling for a viral 7TMR homologue resulted in a reduced capacity to disseminate to or replicate in the salivary glands. In addition, HCMV UL33 was found to partially compensate for the lack of M33 in vivo, suggesting conserved biological roles of the UL33 gene family.

Capripoxvirus G-protein-coupled chemokine receptor: a host-range gene suitable for virus animal origin discrimination

“…In this 8-TM domain model for the CaPV GPCR, the preferential prediction for the short N-terminal region was intracytoplasmic (data not shown) and the following long loop was extracellular. This is in clear contrast with the predicted structure of the prototype monkey CCR8 with its 7-TM domains (Case et al, 2008;Oppermann, 2004;Schoneberg et al, 1999). Although shorter than in other known GPCRs, the C-tail of CaPV GPCR is still rich in serine residues, which, upon phosphorylation, is involved in the clathrin-mediated endocytosis of the receptor (Fraile-Ramos et al, 2001).…”

Cell-type specific activities of cytomegalovirus GPCR homologues

“…When M33 was replaced by either UL33 or US28, a functional complementation was observed in vivo, consistent with some conservation of function among the receptors (Case et al, 2008;Farrell et al, 2011). With the exception of salivary glands, replication titres in different sites of infection were similar when comparing MCMV expressing either M33 or US28 (in place of M33) with wild type virus.…”

“…Nevertheless, UL33, similar to its murine counterparts M33 and R33, has been shown to signal in a constitutive manner, altering transcriptional pathways Case et al, 2008;Gruijthuijsen et al, 2004;Gruijthuijsen et al, 2002;Waldhoer et al, 2002). Upregulation of transcription factors vary among the homologues.…”

“…Analysis in vivo of mutants that preserved cell-surface trafficking highlighted the importance of signalling to biological function. Single amino acid substitutions in either TMII or TMIII that abolished signalling were defective for salivary gland replication; mutations where signalling was not reduced replicated normally and a C-tail truncated mutant with an intermediate signalling profile was partially attenuated (Case et al, 2008). Mutagenic analysis of UL33 and R33 has also been conducted and highlighted differences in signalling associated with structural differences.…”

“…Whilst mutation of the arginine abrogates constitutive G protein-coupled signalling for all three receptors, changing the NRY motif of M33 to DRY motif (the UL33 motif) greatly enhanced the CREB and NFAT signalling (Case et al, 2008;Gruijthuijsen et al, 2004;Waldhoer et al, 2002).…”