The M33 Chemokine Receptor Homolog of Murine Cytomegalovirus Exhibits a Differential Tissue-Specific Role during In Vivo Replication and Latency

Abstract

M33, encoded by murine cytomegalovirus (MCMV), is a member of the UL33 homolog G-protein-coupled receptor (GPCR) family and is conserved across all the betaherpesviruses. Infection of mice with recombinant viruses lacking M33 or containing specific signaling domain mutations in M33 results in significantly diminished MCMV infection of the salivary glands. To determine the role of M33 in viral dissemination and/or infection in other tissues, viral infection with wild-type K181 virus and an M33 mutant virus, ⌬M33B T2 , was characterized using two different routes of inoculation. Following both intraperitoneal (i.p.) and intranasal (i.n.) inoculation, M33 was attenuated for infection of the spleen and pancreas as early as 7 days after infection. Following i.p. inoculation, ⌬M33B T2 exhibited a severe defect in latency as measured by a diminished capacity to reactivate from spleens and lungs in reactivation assays (P < 0.001). Subsequent PCR analysis revealed markedly reduced ⌬M33B T2 viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent ⌬M33B T2 viral DNA was significantly reduced in the spleen and, in agreement with results from i.p. inoculation, did not reactivate from the spleen (P < 0.001). Furthermore, in vivo complementation of ⌬M33B T2 virus replication and/or dissemination to the salivary glands and pancreas was achieved by coinfection with wild-type virus. Overall, our data suggest a critical tissue-specific role for M33 during infection in the salivary glands, spleen, and pancreas but not the lungs. Our data suggest that M33 contributes to the efficient establishment or maintenance of long-term latent MCMV infection.

Cell-type specific activities of cytomegalovirus GPCR homologues

“…This phenotype was associated with a lower DNA viral load in both organs and with a lower number of infected cells in the spleen. It also showed that M33 G protein signalling was involved with reactivation in both organs, as signalling mutant viruses (R131Q) present a reduced reactivation rate (Cardin et al, 2009;Farrell et al, 2011). Taken together, these data suggest that the vGPCR could play a part in colonization and/or replication of specific cell types during viral dissemination.…”

“…Although not tested here, previous reports (Cardin et al, 2009) showed that M33 was attenuated in spread to the spleen following i.n. infection, demonstrating a M33-dependent defect in virus colonisation beyond the viraemic phase of infection.…”

“…Disruption of M33 has been found to suppress reactivation (explant culture) from several latency sites (salivary glands, lungs and bone marrow) (Cardin et al, 2009;Farrell et al, 2013). There was a tendency for disrupted M33 to result in lower levels of viral DNA in tissues at the time of explant, suggesting that that M33 plays a role in effectively establishing or maintaining MCMV latency.…”

“…These results are consistent with previous studies that showed improved clearance of M33 MCMV from these organs beyond day 3 p.i. (Cardin et al, 2009). Whilst wt-MCMV-LUC presented low titres at day 5 p.i.…”

“…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).…”