The mouse cytomegalovirus G protein-coupled receptor homolog, M33, coordinates key features of in vivo infection via distinct components of its signalling repertoire.

Common to all cytomegalovirus (CMV) genomes analysed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DC to the salivary gland and the frequency of reactivation events from latently-infected tissue explants. Constitutive G protein-coupled M33 signalling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages G q/11 to constitutively activate phospholipase C β (PLCβ) and downstream cyclic AMP response-element binding protein (CREB) in vitro . Identification of a MCMV M33 mutant (M33 ΔC38 ) for which CREB signalling was disabled, but PLCβ activation was preserved, provided the opportunity to investigate their relevance in vivo . Following intranasal infection with MCMV M33 ΔC38 , the absence of M33 CREB G q/11 -dependent signalling correlated with reduced mobilisation of lytically-infected DC to draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33 ΔC38 -infected DC within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, G i/o -dependent and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33 ΔC38 -infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV lifecycle are coordinated in diverse tissues by distinct pathways of the M33 signalling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular “switches” which regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analysed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and for the establishment and/or reactivation of latent MCMV infection. The signalling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signalling pathways for in vivo M33 function. In this report, we show temporal and tissue-specific M33 signalling is required facilitating in vivo infection. Understanding the relevance of the viral GPCR signalling profiles for in vivo function will provide opportunities for future targeted interventions.

Tegument Protein pp150 Sequence-Specific Peptide Blocks Cytomegalovirus Infection

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 μM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150–capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.

Mouse Cytomegalovirus Differentially Exploits Cell Surface Glycosaminoglycans in a Cell Type-Dependent and MCK-2-Independent Manner

Many viruses initiate interaction with target cells by binding to cell surface glycosaminoglycans (GAGs). Heparan sulfate (HS) appears to be particularly important in fibroblasts, epithelial cells and endothelial cells, where it represents the dominant GAG. How GAGs influence viral infectivity in HS-poor target cells such as macrophages has not been clearly defined. Here, we show that mouse cytomegalovirus (MCMV) targets HS in susceptible fibroblasts and cultured salivary gland acinar cells (SGACs), but not in macrophage cell lines and primary bone marrow-derived macrophages, where chondroitin sulfate was the dominant virus-binding GAG. MCK-2, an MCMV-encoded GAG-binding chemokine that promotes infection of macrophages as part of a gH/gL/MCK-2 entry complex, was dispensable for MCMV attachment to the cell surface and for direct infection of SGACs. Thus, MCMV tropism for target cells is markedly influenced by differential GAG expression, suggesting that the specificity of anti-GAG peptides now under development as HCMV therapeutics may need to be broadened for effective application as anti-viral agents.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b

ABSTRACT Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression. IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells (“missing-self”). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.