Partial Functional Complementation between Human and Mouse Cytomegalovirus Chemokine Receptor Homologues

Abstract

The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33-mediated G protein-coupled signaling is critical for the salivary gland phenotype. In this report, we demonstrate that US28 and (to a lesser degree) UL33 restore reactivation from tissue explants and partially restore replication in salivary glands (compared to a signaling-deficient M33 mutant). These studies provide a novel small animal model for evaluation of therapies targeting the human CMV CKRs.Chemokine receptor (CKR) homologues and other seventransmembrane receptor (7TMR) homologues are characteristic of beta-and gammaherpesviruses and are potential therapeutic targets (7,14,16). Human cytomegalovirus (HCMV) encodes four 7TMRs: UL33 and UL78 are conserved in all betaherpesviruses, whereas US28/US27 homologues are restricted to primate betaherpesviruses.Clues to the roles of HCMV 7TMRs have come from in vivo studies of mouse and rat CMVs (MCMV and RCMV, respectively). Acute-phase replication in primary organs is followed by dissemination to secondary sites, such as salivary glands, where virus may replicate for several days, attaining high titers. Following immune clearance of productive infection, latently infected cells remain distributed in a number of organs. In the absence of M33 (UL33 homologue), MCMV failed to attain detectable levels of infectious virus in salivary glands, with similar observations for R33 of RCMV (1, 5), suggesting evolutionarily conserved functions. The mechanisms underlying salivary gland attenuation may include defects in dissemination to, initial infection of, or maintenance of productive infection within that organ. Definitive studies are lacking, but there is some evidence for attenuation at a postdissemination step; the M33-null mutant was defective for replication following direct intrasalivary gland inoculation, and the R33-null mutant was detectable in salivary glands (by PCR), similar to the wild type at early times postinfection (1, 5). We and others have demonstrated the importance of constitutive M33-induced signaling for the salivary gland phenotype (4, 15). We further demonstrated that an M33-null mutant was deficient for reactivation in an ex vivo tissue explant model (2), although whether M33 plays a specific role during the establishment of, maintenance of, or reactivation from latency is not yet known.

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.…”

“…This mutant was found to be attenuated for replication in the pancreas and spleen (Cardin, 2009). However, attenuation in the pancreas was not observed for either a signalling defective mutant [M33(R131Q)] or a lacZ negative deletion mutant (premature stop codon within M33), suggesting that lacZ expression, rather than disruption of M33, resulted in defective replication in the pancreas (Farrell et al 2011). For the spleen, however, there was a consistent phenotype for M33 disrupted mutants, apparent as early clearance compared with wild type.…”

“…With regard to complementation of the latency reactivation phenotype (spleen and lung explant), US28 restored to wild type efficiency and UL33 resulted in partial rescue, compared with a signalling deficient M33 mutant (Farrell et al, 2011). In some respects, signalling by US28 is more similar to M33 than UL33, which may have influenced the outcomes (Sherrill & Miller, 2006;Waldhoer et al, 2002).…”

“…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. This included the spleen, suggesting that UL33 and US28 were competent for rescue of the 'early clearance' phenotype observed for M33 null (Farrell et al, 2011). UL33 and US28 also partially rescued replication in salivary glands, although the titres were substantially lower than wild-type.…”

“…It is noteworthy that US28 only partially restored salivary gland replication (which may contribute to seeding to sites of latency) but fully restored explant reactivation rates, in recombinant MCMV where the M33 coding sequence was replaced by US28 (Farrell et al, 2011).…”