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Cytomegaloviruses (CMVs) transmit via chronic shedding from the salivary glands. How this relates to the broad cell tropism they exhibit in vitro is unclear. Human CMV (HCMV) infection presents only after salivary gland infection is established. Murine CMV (MCMV) is therefore useful to analyse early infection events. It reaches the salivary glands via infected myeloid cells. Three adjacent spliced genes designated as m131/129 (MCK-2), sgg1 and sgg1.1, positional homologues of the HCMV UL128/130/131 tropism determinants, are implicated. We show that a sgg1 null mutant is defective in infected myeloid cell entry into the salivary glands, a phenotype distinct from MCMV lacking MCK-2. These data point to a complex, multi-step process of salivary gland colonization.

CD4 + T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4 + T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4 + T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4 + T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c + cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII + CD11c + antigen-presenting cells is key to the CD4 + T cell/NK cell protection axis. By contrast, CD8 + T cell control of AEC1 infection appeared to operate independently., Importance: CD4 + T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4 + T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c + dendritic cells. This mechanism of CD4 + T cell control releases the need for the direct engagement of CD4 + T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens., Competing Interests: The authors declare no conflict of interest.

Host control of mouse cytomegalovirus (MCMV) infection of MHCII - salivary gland acinar cells is mediated by CD4 + T cells, but how they protect is unclear. Here, we show CD4 + T cells control MCMV indirectly in the salivary gland, via IFNγ engagement with uninfected, but antigen + MHCII + APC and recruitment of NK cells to infected cell foci. This immune mechanism renders direct contact of CD4 + T cells with infected cells unnecessary and may represent a host strategy to overcome viral immune evasion.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world population and causes lifelong latent infection. HCMV has been shown to exacerbate cardiovascular diseases, including myocarditis, vascular sclerosis, and transplant vasculopathy. Recently, we have shown that murine CMV (MCMV) recapitulates the cardiovascular dysfunction observed in patients with HCMV-induced myocarditis. To understand the viral mechanisms involved in CMV-induced heart dysfunction, we further characterized cardiac function in response to MCMV and examined virally encoded G-protein-coupled receptor homologs (vGPCRs) US28 and M33 as potential factors that promote infection in the heart. We hypothesized that the CMV-encoded vGPCRs could exacerbate cardiovascular damage and dysfunction. Three viruses were used to evaluate the role of vGPCRs in cardiac dysfunction: wild-type MCMV, a M33-deficient virus (∆M33), and a virus with the M33 open reading frame (ORF) replaced with US28, an HCMV vGPCR (i.e., US28+). Our in vivo studies revealed that M33 plays a role in promoting cardiac dysfunction by increasing viral load and heart rate during acute infection. During latency, ΔM33-infected mice demonstrated reduced calcification, altered cellular gene expression, and less cardiac hypertrophy compared with wild-type MCMV-infected mice. Ex vivo viral reactivation from hearts was less efficient in ΔM33-infected animals. HCMV protein US28 expression restored the ability of the M33-deficient virus to reactivate from the heart. US28+ MCMV infection caused damage to the heart comparable with wild-type MCMV infection, suggesting that the US28 protein is sufficient to complement the function of M33 in the heart. Altogether, these data suggest a role for vGPCRs in viral pathogenesis in the heart and thus suggest that vGPCRs promote long-term cardiac damage and dysfunction.

Introduction: Human cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8 + T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33 stop ), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation., Methods: Mice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33 stop ., Results: We found that ΔM33 stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33 stop , confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion., Discussion: Our results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic., Competing Interests: Author RC is employed by Pfizer, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 White, Bonavita, Stanfield, Farrell, Davis-Poynter and Cardin.)

Animal models that mimic human infections provide insights in virus-host interplay; knowledge that in vitro approaches cannot readily predict, nor easily reproduce. Human cytomegalovirus (HCMV) infections are acquired asymptomatically, and primary infections are difficult to capture. The gap in our knowledge of the early events of HCMV colonization and spread limits rational design of HCMV antivirals and vaccines. Studies of natural infection with mouse cytomegalovirus (MCMV) have demonstrated the olfactory epithelium as the site of natural colonization. Systemic spread from the olfactory epithelium is facilitated by infected dendritic cells (DC); tracking dissemination uncovered previously unappreciated DC trafficking pathways. The olfactory epithelium also provides a unique niche that supports efficient MCMV superinfection and virus recombination. In this review, we summarize recent advances to our understanding of MCMV infection and spread and the tissue-specific mechanisms utilized by MCMV to modulate DC trafficking. As these mechanisms are likely conserved with HCMV, they may inform new approaches for preventing HCMV infections in humans.

CD4 + T cells are key to controlling cytomegalovirus infections. Salivary gland infection by murine cytomegalovirus (MCMV) provides a way to identify mechanisms. CD11c + dendritic cells (DC) disseminate MCMV to the salivary glands, where they transfer infection to acinar cells. Antiviral CD4 + T cells are often considered to be directly cytotoxic for cells expressing major histocompatibility complex class II (MHCII). However, persistently infected salivary gland acinar cells are MHCII - and are presumably inaccessible to direct CD4 T cell recognition. Here, we show that CD4 + T cell depletion amplified infection of MHCII - acinar cells but not MHCII + cells. MCMV-infected mice with disrupted MHCII on CD11c + cells showed increased MHCII - acinar infection; antiviral CD4 + T cells were still primed, but their recruitment to the salivary glands was reduced, suggesting that engagement with local MHCII + DC is important for antiviral protection. As MCMV downregulates MHCII on infected DC, the DC participating in CD4 protection may thus be uninfected. NK cells and gamma interferon (IFN-γ) may also contribute to CD4 + T cell-dependent virus control: CD4 T cell depletion reduced NK cell recruitment to the salivary glands, and both NK cell and IFN-γ depletion equalized infection between MHCII-disrupted and control mice. Taken together, these results suggest that CD4 + T cells protect indirectly against infected acinar cells in the salivary gland via DC engagement, requiring the recruitment of NK cells and the action of IFN-γ. Congruence of these results with an established CD4 + T cell/NK cell axis of gammaherpesvirus infection control suggests a common mode of defense against evasive viruses. IMPORTANCE Cytomegalovirus infections commonly cause problems in immunocompromised patients and in pregnancy. We lack effective vaccines. CD4 + T cells play an important role in normal infection control, yet how they act has been unknown. Using murine cytomegalovirus as an accessible model, we show that CD4 + T cells are unlikely to recognize infected cells directly. We propose that CD4 + T cells interact with uninfected cells that present viral antigens and recruit other immune cells to attack infected targets. These data present a new outlook on understanding how CD4 + T cell-directed control protects against persistent cytomegalovirus infection.

Common to all cytomegalovirus (CMV) genomes analyzed 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 DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling 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 signaling 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 signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33 ΔC38 -infected DCs 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 life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed 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 the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.

Human cytomegalovirus (HCMV) encodes four homologs of G protein coupled receptors (vGPCRs), of which two, designated UL33 and US28, signal constitutively. UL33 and US28 are also conserved with chemokine receptors: US28 binds numerous chemokine classes, including the membrane bound chemokine, fractalkine; whereas UL33 remains an orphan receptor. There is emerging data that UL33 and US28 each contribute to HCMV associated disease, although no studies to date have reported their potential contribution to aberrant placental physiology that has been detected with HCMV congenital infection. We investigated the signaling repertoire of UL33 and US28 and their potential to enable trophoblast mobilization in vitro. Results demonstrate the constitutive activation of CREB by each vGPCR in ACIM-88 and HTR-8SVneo trophoblasts; constitutive NF-kB activation was detected for US28 only. Constitutive signaling by each vGPCR enabled trophoblast migration. For US28, fractalkine exhibited inverse agonist activity and dampened trophoblast migration. UL33 stimulated expression of both p38 mitogen activated (MAP) and Jun N-terminal (JNK) kinases; while p38 MAP kinase stimulated CREB, JNK was inhibitory, suggesting that UL33 dependent CREB activation was regulated by p38/JNK crosstalk. Given that chemokines and their receptors are important for placental development, these data point to the potential of HCMV UL33 and US28 to interfere with trophoblast responses which are important for normal placental development.

Herpesvirus genomes show abundant evidence of past recombination. Its functional importance is unknown. A key question is whether recombinant viruses can outpace the immunity induced by their parents to reach higher loads. We tested this by coinfecting mice with attenuated mutants of murid herpesvirus 4 (MuHV-4). Infection by the natural olfactory route routinely allowed mutant viruses to reconstitute wild-type genotypes and reach normal viral loads. Lung coinfections rescued much less well. Attenuated murine cytomegalovirus mutants similarly showed recombinational rescue via the nose but not the lungs. These infections spread similarly, so route-specific rescue implied that recombination occurred close to the olfactory entry site. Rescue of replication-deficient MuHV-4 confirmed this, showing that coinfection occurred in the first encountered olfactory cells. This worked even with asynchronous inoculation, implying that a defective virus can wait here for later rescue. Virions entering the nose get caught on respiratory mucus, which the respiratory epithelial cilia push back toward the olfactory surface. Early infection was correspondingly focused on the anterior olfactory edge. Thus, by concentrating incoming infection into a small area, olfactory entry seems to promote functionally significant recombination. IMPORTANCE All organisms depend on genetic diversity to cope with environmental change. Small viruses rely on frequent point mutations. This is harder for herpesviruses because they have larger genomes. Recombination provides another means of genetic optimization. Human herpesviruses often coinfect, and they show evidence of past recombination, but whether this is rare and incidental or functionally important is unknown. We showed that herpesviruses entering mice via the natural olfactory route meet reliably enough for recombination routinely to repair crippling mutations and restore normal viral loads. It appeared to occur in the first encountered olfactory cells and reflected a concentration of infection at the anterior olfactory edge. Thus, natural host entry incorporates a significant capacity for herpesvirus recombination.

Vaccination against the betaherpesvirus, human cytomegalovirus (HCMV) is a public health goal. However, HCMV has proved difficult to vaccinate against. Vaccination against single HCMV determinants has not worked, suggesting that immunity to a wider antigenic profile may be required. Live attenuated vaccines provide the best prospects for protection, but the question remains as to how to balance vaccine virulence with safety. Animal models of HCMV infection provide insights into identifying targets for virus attenuation and understanding how host immunity blocks natural, mucosal infection. Here, we evaluated the vaccine potential of a mouse cytomegalovirus (MCMV) vaccine deleted of a viral G protein-coupled receptor (GPCR), designated M33, that renders it attenuated for systemic spread. A single noninvasive olfactory ΔM33 MCMV vaccine replicated locally, but as a result of the loss of the M33 GPCR, it failed to spread systemically and was attenuated for latent infection. Vaccination did not prevent host entry of a superinfecting MCMV but spread from the mucosa was blocked. This approach to vaccine design may provide a viable alternative for a safe and effective betaherpesvirus vaccine. IMPORTANCE Human cytomegalovirus (HCMV) is the most common cause of congenital infection for which a vaccine is not yet available. Subunit vaccine candidates have failed to achieve licensure. A live HCMV vaccine may prove more efficacious, but it faces safety hurdles which include its propensity to persist and to establish latency. Understanding how pathogens infect guide rational vaccine design. However, HCMV infections are asymptomatic and thus difficult to capture. Animal models of experimental infection provide insight. Here, we investigated the vaccine potential of a mouse cytomegalovirus (MCMV) attenuated for systemic spread and latency. We used olfactory vaccination and virus challenge to mimic its natural acquisition. We provide proof of concept that a single olfactory MCMV that is deficient in systemic spread can protect against wild-type MCMV superinfection and dissemination. This approach of deleting functional counterpart genes in HCMV may provide safe and effective vaccination against congenital HCMV disease.

The cytomegaloviruses (CMVs) spread systemically via myeloid cells and demonstrate broad tissue tropism. Human CMV (HCMV) UL128 encodes a component of the virion pentameric complex (PC) that is important for entry into epithelial cells and cell-cell spread in vitro . It possesses N-terminal amino acid sequences similar to those of CC chemokines. While the species specificity of HCMV precludes confirmation of UL128 function in vivo , UL128-like counterparts in experimental animals have demonstrated a role in salivary gland infection. How they achieve this has not been defined, although effects on monocyte tropism and immune evasion have been proposed. By tracking infected cells following lung infection, we show that although the UL128-like protein in mouse CMV (MCMV) (designated MCK-2) facilitated entry into lung macrophages, it was dispensable for subsequent viremia mediated by CD11c + dendritic cells (DCs) and extravasation to the salivary glands. Notably, MCK-2 was important for the transfer of MCMV infection from DCs to salivary gland acinar epithelial cells. Acinar cell infection of MCMVs deleted of MCK-2 was not rescued by T-cell depletion, arguing against an immune evasion mechanism for MCK-2 in the salivary glands. In contrast to lung infection, peritoneal MCMV inoculation yields mixed monocyte/DC viremia. In this setting, MCK-2 again promoted DC-dependent infection of salivary gland acinar cells, but it was not required for monocyte-dependent spread to the lung. Thus, the action of MCK-2 in MCMV spread was specific to DC-acinar cell interactions. IMPORTANCE Cytomegaloviruses (CMVs) establish myeloid cell-associated viremias and persistent shedding from the salivary glands. In vitro studies with human CMV (HCMV) have implicated HCMV UL128 in epithelial tropism, but its role in vivo is unknown. Here, we analyzed how a murine CMV (MCMV) protein with similar physical properties, designated MCK-2, contributes to host colonization. We demonstrate that MCK-2 is dispensable for initial systemic spread from primary infection sites but within the salivary gland facilitates the transfer of infection from dendritic cells (DCs) to epithelial acinar cells. Virus transfer from extravasated monocytes to the lungs did not require MCK-2, indicating a tissue-specific effect. These results provide new information about how persistent viral tropism determinants operate in vivo .

Cytomegaloviruses (CMVs) use myeloid cells to move within their hosts. Murine CMV (MCMV) colonizes the salivary glands for long-term shedding, and reaches them via CD11c + infected cells. A need to recruit patrolling monocytes for systemic spread has been proposed, based on poor salivary gland infection in fractalkine receptor (CX3CR1)-deficient mice. We found no significant CX3CR1 dependence of salivary gland infection. CCL2 and the viral m131/m129 chemokine homologue were also redundant for acute MCMV spread, arguing against a need for inflammation or infection to recruit additional monocytes to the entry site. M131/m129 promoted salivary gland infection, but only after the initial seeding of infected cells to this site. Our data support the idea that MCMV disseminates by infecting and mobilizing tissue-resident dendritic cells.

Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells. Murine CMV (MCMV) spreads from the lungs via infected CD11c + cells, consistent with an important role for dendritic cells (DC). We show here that MCMV entering via the olfactory epithelium, a natural transmission portal, also spreads via infected DC. They reached lymph nodes, entered the blood via high endothelial venules, and then entered the salivary glands, driven by constitutive signaling of the viral M33 G protein-coupled receptor (GPCR). Intraperitoneal infection also delivered MCMV to the salivary glands via DC. However, it also seeded F4/80 + infected macrophages to the blood; they did not enter the salivary glands or require M33 for extravasation. Instead, they seeded infection to a range of other sites, including brown adipose tissue (BAT). Peritoneal cells infected ex vivo then adoptively transferred showed similar cell type-dependent differences in distribution, with abundant F4/80 + cells in BAT and CD11c + cells in the salivary glands. BAT colonization by CMV-infected cells was insensitive to pertussis toxin inhibition of the GPCR signaling through G i/o substrate, whereas salivary gland colonization was sensitive. Since salivary gland infection required both M33 and G i/o -coupled signaling, whereas BAT infection required neither, these migrations were mechanistically distinct. MCMV spread from the lungs or nose depended on DC, controlled by M33. Infecting other monocyte populations resulted in unpredictable new infections. IMPORTANCE Cytomegaloviruses (CMVs) spread through the blood by infecting monocytes, and this can lead to disease. With murine CMV (MCMV) we can track infected myeloid cells and so understand how CMVs spread. Previous experiments have injected MCMV into the peritoneal cavity. MCMV normally enters mice via the olfactory epithelium. We show that olfactory infection spreads via dendritic cells, which MCMV directs to the salivary glands. Peritoneal infection similarly reached the salivary glands via dendritic cells. However, it also infected other monocyte types, and they spread infection to other tissues. Thus, infecting the "wrong" monocytes altered virus spread, with potential to cause disease. These results provide a basis for understanding how the monocyte types infected by human CMV might promote different infection outcomes., (Copyright © 2019 American Society for Microbiology.)

Cytomegaloviruses (CMVs) are large, complex pathogens that persistently and systemically colonize most mammals. Human cytomegalovirus (HCMV) causes congenital harm, and has proved hard to control. One problem is that key vaccine targets - virus entry and spread in naive hosts - remain ill-defined. As CMVs predate human speciation, those of other mammals can provide new insight. Murine CMV (MCMV) enters new hosts via olfactory neurons. Like HCMV it binds to heparan, which is lacking from most differentiated apical epithelia but is displayed on olfactory neuronal cilia. It then spreads via infected dendritic cells (DCs), which migrate to draining lymph nodes (LNs), rejoin the circulation by entering high endothelial venules (HEVs), and extravasate into other tissues. This migration depends quantitatively on M33, a constitutively active viral G protein-coupled receptor (GPCR). The homologous US28 GPCR of HCMV can substitute for M33 in allowing MCMV-infected DCs to leave LNs via HEVs, so HCMV could potentially use the same route. The capacity of DCs to seed MCMV to tissues, and for other DCs to collect it for redistribution, suggest that DC recirculation chronically maintains and links diverse CMV reservoirs through lytic exchange.

Cytomegaloviruses (CMVs) establish systemic infections across diverse cell types. Glycoproteins that alter tropism can potentially guide their spread. Glycoprotein O (gO) is a nonessential fusion complex component of both human CMV (HCMV) and murine CMV (MCMV). We tested its contribution to MCMV spread from the respiratory tract. In vitro , MCMV lacking gO poorly infected fibroblasts and epithelial cells. Cell binding was intact, but penetration was delayed. In contrast, myeloid infection was preserved, and in the lungs, where myeloid and type 2 alveolar epithelial cells are the main viral targets, MCMV lacking gO showed a marked preference for myeloid infection. Its poor epithelial cell infection was associated with poor primary virus production and reduced virulence. Systemic spread, which proceeds via infected CD11c + myeloid cells, was initially intact but then diminished, because less epithelial infection led ultimately to less myeloid infection. Thus, the tight linkage between peripheral and systemic MCMV infections gave gO-dependent infection a central role in host colonization. IMPORTANCE Human cytomegalovirus is a leading cause of congenital disease. This reflects its capacity for systemic spread. A vaccine is needed, but the best viral targets are unclear. Attention has focused on the virion membrane fusion complex. It has 2 forms, so we need to know what each contributes to host colonization. One includes the virion glycoprotein O. We used murine cytomegalovirus, which has equivalent fusion complexes, to determine the importance of glycoprotein O after mucosal infection. We show that it drives local virus replication in epithelial cells. It was not required to infect myeloid cells, which establish systemic infection, but poor local replication reduced systemic spread as a secondary effect. Therefore, targeting glycoprotein O of human cytomegalovirus has the potential to reduce both local and systemic infections., (Copyright © 2019 American Society for Microbiology.)

Human cytomegalovirus (HCMV) colonizes blood-borne dendritic cells (DCs). They express US28, a viral G protein-coupled receptor (GPCR). In vitro functions have been described for US28, but how it contributes to host colonization has been unclear. The murine CMV (MCMV) M33 GPCR promotes DC recirculation. We show that US28 shares this function. Thus, DC recirculation is also available to HCMV via US28, and inhibiting US28 G protein-dependent signalling has the potential to reduce systemic infection. We show that M33 also promotes systemic infection through infected DC extravasation.

Cytomegaloviruses (CMVs) persistently and systemically infect the myeloid cells of immunocompetent hosts. Persistence implies immune evasion, and CMVs evade CD8+ T cells by inhibiting MHC class I-restricted antigen presentation. Myeloid cells can also interact with CD4+ T cells via MHC class II (MHC II). Human CMV (HCMV) attacks the MHC II presentation pathway in vitro, but what role this evasion might play in host colonization is unknown. We show that Murine CMV (MCMV) down-regulates MHC II via M78, a multi-membrane spanning viral protein that captured MHC II from the cell surface and was necessary although not sufficient for its degradation in low pH endosomes. M78-deficient MCMV down-regulated MHC I but not MHC II. After intranasal inoculation, it showed a severe defect in salivary gland colonization that was associated with increased MHC II expression on infected cells, and was significantly rescued by CD4+ T cell loss. Therefore MCMV requires CD4+ T cell evasion by M78 to colonize the salivary glands, its main site of long-term shedding.

Herpesviruses have coevolved with their hosts over hundreds of millions of years and exploit fundamental features of their biology. Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells, and it has been hypothesized that systemic dissemination arises from infected stem cells in bone marrow. However, poor CMV transfer by stem cell transplantation argues against this being the main reservoir. To identify alternative pathways for CMV spread, we tracked murine CMV (MCMV) colonization after mucosal entry. We show that following intranasal MCMV infection, lung CD11c + dendritic cells (DC) migrated sequentially to lymph nodes (LN), blood, and then salivary glands. Replication-deficient virus followed the same route, and thus, DC infected peripherally traversed LN to enter the blood. Given that DC are thought to die locally following their arrival and integration into LN, recirculation into blood represents a new pathway. We examined host and viral factors that facilitated this LN traverse. We show that MCMV-infected DC exited LN by a distinct route to lymphocytes, entering high endothelial venules and bypassing the efferent lymph. LN exit required CD44 and the viral M33 chemokine receptor, without which infected DC accumulated in LN and systemic spread was greatly reduced. Taken together, our studies provide the first demonstration of virus-driven DC recirculation. As viruses follow host-defined pathways, high endothelial venules may normally allow DC to pass from LN back into blood. IMPORTANCE Human cytomegalovirus (HCMV) causes devastating disease in the unborn fetus and in the immunocompromised. There is no licensed vaccine, and preventive measures are impeded by our poor understanding of early events in host colonization. HCMV and murine CMV (MCMV) both infect blood-borne myeloid cells. HCMV-infected blood cells are thought to derive from infected bone marrow stem cells. However, infected stem cells have not been visualized in vivo nor shown to produce virus ex vivo , and hematopoietic transplants poorly transfer infection. We show that MCMV-infected dendritic cells in the lungs reach the blood via lymph nodes, surprisingly migrating into high endothelial venules. Dissemination did not require viral replication. It depended on the constitutively active viral chemokine receptor M33 and on the host hyaluronan receptor CD44. Thus, viral chemokine receptors are a possible target to limit systemic CMV infections., (Copyright © 2017 Farrell et al.)

Cytomegaloviruses (CMVs) establish chronic, systemic infections. Peripheral infection spreads via lymph nodes, which are also a focus of host defence. Thus, this is a point at which systemic infection spread might be restricted. Subcapsular sinus macrophages (SSM) captured murine CMV (MCMV) from the afferent lymph and poorly supported its replication. Blocking the type I interferon (IFN-I) receptor (IFNAR) increased MCMV infection of SSM and of the fibroblastic reticular cells (FRC) lining the subcapsular sinus, and accelerated viral spread to the spleen. Little splenic virus derived from SSM, arguing that they mainly induce an anti-viral state in the otherwise susceptible FRC. NK cells also limited infection, killing infected FRC and causing tissue damage. They acted independently of IFN-I, as IFNAR blockade increased NK cell recruitment, and NK cell depletion increased infection in IFNAR-blocked mice. Thus SSM restricted MCMV infection primarily though IFN-I, with NK cells providing a second line of defence. The capacity of innate immunity to restrict MCMV escape from the subcapsular sinus suggested that enhancing its recruitment might improve infection control., Competing Interests: The authors have declared no competing interests exist.

Virus homologues of seven-transmembrane receptors (7TMR) are encoded by all beta- and gammaherpesviruses, suggesting important functional roles. M78 of mouse cytomegalovirus (MCMV) is representative of a family of 7TMR conserved in all betaherpesviruses. M78 family members have been found to exhibit cell-type specific effects upon virus replication in tissue culture and to affect virus pathogenesis in vivo. We reported previously that M78, for which no ligands are known, undergoes rapid, constitutive endocytosis. In this study, we have investigated the role of the M78 cytoplasmic C-tail in mediating endocytosis and consequences of C-tail deletion upon replication and pathogenesis. Mutations of M78 (C-tail truncations or point mutations) and CCR5-M78 chimeras identified two distinct regions affecting endocytosis. The first was a classical acidic di-leucine motif (DDxxxLL), located close to the C-terminus. The second region, the activity of which was suppressed by downstream sequences, included the putative 8th helix, located close to the 7th transmembrane domain. A recombinant MCMV expressing an endocytosis-deficient M78, lacking most of the C-tail (M78_CΔ155), had a cell-type specific replication phenotype. M78_CΔ155 had restricted replication in bone marrow macrophages, indistinguishable from an M78-null recombinant. In contrast, M78_CΔ155 replicated normally or with enhanced titres to wild type virus in other tested cell-types, whereas M78-null was attenuated. Distinct phenotypes for M78_CΔ155 and M78-null suggest that the C-tail deletion resulted in M78 dysfunction, rather than complete loss of function; furthermore, they highlight a cell-type specific role of M78 during replication. Infection of mice (intranasal) demonstrated that M78_CΔ155, similar to M78-null, was cleared more rapidly from the lungs than wild type virus and was severely attenuated for replication in salivary glands. It may be speculated that attenuation of both M78_CΔ155 and M78-null for replication in macrophages may have contributed to their similar pathogenic phenotypes., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: Viruses transmit via the environmental and social interactions of their hosts. Herpesviruses have colonized mammals since their earliest origins, suggesting that they exploit ancient, common pathways. Cytomegaloviruses (CMVs) are assumed to enter new hosts orally, but no site has been identified. We show by live imaging that murine CMV (MCMV) infects nasally rather than orally, both after experimental virus uptake and during natural transmission. Replication-deficient virions revealed the primary target as olfactory neurons. Local, nasal replication by wild-type MCMV was not extensive, but there was rapid systemic spread, associated with macrophage infection. A long-term, transmissible infection was then maintained in the salivary glands. The viral m131/m129 chemokine homolog, which influences tropism, promoted salivary gland colonization after nasal entry but was not required for entry per se The capacity of MCMV to transmit via olfaction, together with previous demonstrations of experimental olfactory infection by murid herpesvirus 4 (MuHV-4) and herpes simplex virus 1 (HSV-1), suggest that this is a common, conserved route of mammalian herpesvirus entry., Importance: Cytomegaloviruses (CMVs) infect most mammals. Human CMV (HCMV) harms people with poor immune function and can damage the unborn fetus. It infects approximately 1% of live births. We lack a good vaccine. One problem is that how CMVs first enter new hosts remains unclear. Oral entry is often assumed, but the evidence is indirect, and no infection site is known. The difficulty of analyzing HCMV makes related animal viruses an important source of insights. Murine CMV (MCMV) infected not orally but nasally. Specifically, it targeted olfactory neurons. Viral transmission was also a nasal infection. Like HCMV, MCMV infected cells by binding to heparan, and olfactory surfaces display heparan to incoming viruses, whereas most other mucosal surfaces do not. These data establish a new understanding of CMV infections and a basis for infection control., (Copyright © 2016 Farrell et al.)

Unlabelled: Cytomegaloviruses (CMVs) infect the lungs and cause pathological damage there in immunocompromised hosts. How lung infection starts is unknown. Inhaled murine CMV (MCMV) directly infected alveolar macrophages (AMs) and type 2 alveolar epithelial cells (AEC2s) but not type 1 alveolar epithelial cells (AEC1s). In contrast, herpes simplex virus 1 infected AEC1s and murid herpesvirus 4 (MuHV-4) infected AEC1s via AMs. MCMV-infected AMs prominently expressed viral reporter genes from a human CMV IE1 promoter; but most IE1-positive cells were AEC2s, and CD11c-cre mice, which express cre in AMs, switched the fluorochrome expression of <5% of floxed MCMV in the lungs. In contrast, CD11C-cre mice exhibited fluorochrome switching in >90% of floxed MuHV-4 in the lungs and 50% of floxed MCMV in the blood. AM depletion increased MCMV titers in the lung during the acute phase of infection. Thus, the influence of AMs was more restrictive than permissive. Circulating monocytes entered infected lungs in large numbers and became infected, but not directly; infection occurred mainly via AEC2s. Mice infected with an MCMV mutant lacking its m131/m129 chemokine homolog, which promotes macrophage infection, showed levels of lung infection equivalent to those of wild-type MCMV-infected mice. The level of lung infiltration by Gr-1-positive cells infected with the MCMV m131/m129-null mutant was modestly different from that for wild-type MCMV-infected lungs. These results are consistent with myeloid cells mainly disseminating MCMV from the lungs, whereas AEC2s provide local amplification., Importance: Cytomegaloviruses (CMVs) chronically and systemically infect most mammals. Human CMV infection is usually asymptomatic but causes lung disease in people with poor immune function. As human infection is hard to analyze, studies with related animal viruses provide important insights. We show that murine CMV has two targets in the lungs: macrophages and surfactant-secreting epithelial cells. Acute virus replication occurred largely in epithelial cells. Macrophages had an important defensive role, as their removal increased the level of infection. These results establish the dual nature of lung infection, with local virus replication occurring in epithelial cells and spread occurring via quiescently infected macrophages. Distinct therapies may be needed to target these contrasting events., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Cytomegaloviruses (CMVs) establish chronic infections that spread from a primary entry site to secondary vascular sites, such as the spleen, and then to tertiary shedding sites, such as the salivary glands. Human CMV (HCMV) is difficult to analyze, because its spread precedes clinical presentation. Murine CMV (MCMV) offers a tractable model. It is hypothesized to spread from peripheral sites via vascular endothelial cells and associated monocytes. However, viral luciferase imaging showed footpad-inoculated MCMV first reaching the popliteal lymph nodes (PLN). PLN colonization was rapid and further spread was slow, implying that LN infection can be a significant bottleneck. Most acutely infected PLN cells were CD169(+) subcapsular sinus macrophages (SSM). Replication-deficient MCMV also reached them, indicating direct infection. Many SSM expressed viral reporter genes, but few expressed lytic genes. SSM expressed CD11c, and MCMV with a cre-sensitive fluorochrome switch showed switched infected cells in PLN of CD11c-cre mice but yielded little switched virus. SSM depletion with liposomal clodronate or via a CD169-diphtheria toxin receptor transgene shifted infection to ER-TR7(+) stromal cells, increased virus production, and accelerated its spread to the spleen. Therefore, MCMV disseminated via LN, and SSM slowed this spread by shielding permissive fibroblasts and poorly supporting viral lytic replication., Importance: HCMV chronically infects most people, and it can cause congenital disability and harm the immunocompromised. A major goal of vaccination is to prevent systemic infection. How this is established is unclear. Restriction to humans makes HCMV difficult to analyze. We show that peripheral MCMV infection spreads via lymph nodes. Here, MCMV infected filtering macrophages, which supported virus replication poorly. When these macrophages were depleted, MCMV infected susceptible fibroblasts and spread faster. The capacity of filtering macrophages to limit MCMV spread argued that their infection is an important bottleneck in host colonization and might be a good vaccine target., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Herpesviruses are an ancient group which have exploited gene capture of multiple cellular modulators of the immune response. Viral homologues of 7 transmembrane receptors (v7TMRs) are a consistent feature of beta- and gammaherpesviruses; the majority of the v7TMRs are homologous to cellular chemokine receptors (CKRs). Conserved families of v7TMRs distinguish between beta- versus gammaherpesviruses; furthermore, significant divisions within these subfamilies, such as between genera of the gammaherpesviruses or between the primate and rodent cytomegaloviruses, coincide with specific v7TMR gene families. Divergence of functional properties between the viral 7TMR and their cellular counterparts is likely, therefore, to reflect adaptation supporting various aspects of the viral lifecycle with concomitant effects upon viral pathogenesis. Consistent with their long evolutionary history, the v7TMRs have acquired a range of distinctive characteristics. This chapter reviews key features of the v7TMRs which are likely to impact upon their functional roles: trafficking properties, ligand specificity, and signaling capacity. Rapid, constitutive endocytosis, reminiscent of cellular "scavenger" receptors, may provide a mechanism for immune evasion, or alternatively relate to virion assembly, including incorporation of v7TMRs within the virion envelope. Some v7TMRs display relatively broad chemokine-binding specificity, whereas others remain "orphan" and may be completely independent of ligand activation. Indeed, many of the v7TMRs have been shown to signal constitutively, associated in some cases with notable divergence of highly conserved regulatory elements such as the "DRY" motif of TMIII. The availability of rodent models for v7TMR functional studies has provided evidence for important biological roles, including cellular transformation, tissue tropism, and viral persistence. Recent studies addressing signaling pathways critical to these phenotypes will be discussed, with reference to both beta- and gammaherpesviruses., (© 2015 Elsevier Inc. All rights reserved.)

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. Herein, we demonstrate that complementation of salivary gland tropism is mediated predominantly by G protein-dependent signaling conserved with that of M33; in contrast, both G protein-dependent and -independent pathways contribute to the latency phenotypes. A novel M33-dependent replication phenotype in cultured bone marrow macrophages is also described.

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.

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, DeltaM33B(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, DeltaM33B(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 DeltaM33B(T2) viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent DeltaM33B(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 DeltaM33B(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.

Murine cytomegalovirus (MCMV) M78 is a member of the betaherpesvirus 'UL78 family' of seven transmembrane receptor (7TMR) genes. Previous studies of M78 and its counterpart in rat cytomegalovirus (RCMV) have suggested that these genes are required for efficient cell-cell spread of their respective viruses in tissue culture and demonstrated that gene knockout viruses are significantly attenuated for replication in vivo. However, in comparison with other CMV 7TMRs, relatively little is known about the basic biochemical properties and subcellular trafficking of the UL78 family members. We have characterized MCMV M78 in both transiently transfected and MCMV-infected cells to determine whether M78 exhibits features in common with cellular 7TMR. We obtained preliminary evidence that M78 formed dimers, a property that has been reported for several cellular 7TMR. M78 traffics to the cell surface, but was rapidly and constitutively endocytosed. Antibody feeding experiments demonstrated co-localization of M78 with markers for both the clathrin-dependent and lipid raft/caveolae-mediated internalization pathways. In MCMV-infected cells, the subcellular localization of M78 was modified during the course of infection, which may be related to the incorporation of M78 into the virion envelope during the course of virion maturation.

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.

Equid herpesvirus 2 (EHV-2), in common with other members of the subfamily Gammaherpesvirinae, encodes homologues of cellular seven-transmembrane receptors (7TMR), namely open reading frames (ORFs) E1, 74 and E6, which each show some similarity to cellular chemokine receptors. Whereas ORF74 and E6 are members of gammaherpesvirus-conserved 7TMR gene families, E1 is currently unique to EHV-2. To investigate their genetic variability, EHV-2 7TMRs from a panel of equine gammaherpesvirus isolates were sequenced. A region of gB was sequenced to provide comparative sequence data. Phylogenetic analysis revealed six 'genogroups' for E1 and four for ORF74, which exhibited approximately 10-38 and 11-27 % amino acid difference between groups, respectively. In contrast, E6 was highly conserved, with two genogroups identified. The greatest variation was observed within the N-terminal domains and other extracellular regions. Nevertheless, analysis of the number of non-synonymous (d(N)) and synonymous (d(S)) substitutions per site generally supported the hypothesis that the 7TMRs are under negative selective pressure to retain functionally important residues, although some site-specific positive selection (d(N)>d(S)) was also observed. Collectively, these data are consistent with transmembrane and cytoplasmic domains being less tolerant of mutations with adverse effects upon function. Finally, there was no evidence for genetic linkage between the different gB, E1, ORF74 and E6 genotypes, suggesting frequent intergenic recombination between different EHV-2 strains.

Homologues of MHC class I proteins have been identified in the genomes of human, murine and rat cytomegaloviruses (CMVs). Given the pivotal role of the MHC class I protein in cellular immunity, it has been postulated that the viral homologues subvert the normal antiviral immune response of the host, thus promoting virus replication and dissemination in an otherwise hostile environment. This review focuses on recent studies of the CMV MHC class I homologues at the molecular, cellular and whole animal level and presents current hypotheses for their roles in the CMV life cycle.

NK cells mediate early host defense against viral infection. In murine CMV (MCMV) infection NK cells play a critical role in controlling viral replication in target organs, such as spleen and liver. Until now it has not been possible to directly examine the role of NK cells in MCMV-induced inflammation in situ due to the inability to stain specifically for NK cells in infected tissues. In this study, we describe a method of in vivo fixation, resulting in the first identification of NK cells in situ using NK1.1 as the marker. Using this method, we characterize the NK1.1(+) cellular component of the inflammatory response to wild-type MCMV in the spleen, liver, and lung of genetically susceptible and resistant mice following i.p. infection. This study provides the first in situ description of the cellular response mediated specifically by NK cells following MCMV infection.

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Cytomegaloviruses likely encode numerous gene products involved in regulating virus-host cell interactions and pathogenesis. We previously identified a region of murine cytomegalovirus (MCMV) within HindIII-J and -I that regulates pathogenesis of the virus [open reading frames (ORFs) M139-M141] or is likely required for MCMV replication (ORFs m142 and m143). As a prerequisite for further studies on the structure and function of this gene region, we mapped the transcripts encoded within MCMV HindIII-I. Probes for ORFs M140 and M141 hybridized to 5.4- and 7.0-kb RNA, respectively, which were transcribed with early kinetics and were 3' coterminal with HindIII-J ORF M139. Probes representing ORFs m142, m143, or m144 hybridized to 3' coterminal transcripts of 1.8, 3.8, and 5.1 kb, respectively. ORFs m142 and m143 were transcribed with immediate-early kinetics but were most abundantly expressed at early times. Probes for the rightmost end of HindIII-I hybridized to a 5. 1-kb early/late RNA corresponding to m144 and to a 1.8-kb early RNA transcribed from m145. All of the major transcripts were polyadenylated and therefore are likely coding. Additional minor transcripts of intermediate sizes were also detected. ORFs M139-m143 showed homology to the betaherpesvirus-specific HCMV US22 gene family. Because deletion of these viral genes results in attenuated or helper-dependent phenotypes, this conserved region of US22 family genes may have a role in virus replication as well as in the pathogenesis of betaherpesviruses in their natural hosts., (Copyright 1999 Academic Press.)

Natural killer (NK) cells are an important component of the innate cellular immune system. They are particularly important during the early immune responses following virus infection, prior to the induction of cytotoxic T cells (CTL). Unlike CTL, which recognize specific peptides displayed on the surface of cells by class I MHC, NK cells respond to aberrant expression of cell surface molecules, in particular class I MHC, in a non-specific manner. Thus, cells expressing low levels of surface class I MHC are susceptible to recognition by NK cells, with concomitant triggering of cytolytic and cytokine-mediated responses. Many viruses, including the cytomegaloviruses, downregulate cell surface MHC class I: this is likely to provide protection against CTL-mediated clearance of infected cells, but may also render infected cells sensitive to NK-cell attack. This review focuses upon cytomegalovirus-encoded proteins that are believed to promote evasion of NK-cell-mediated immunity. The class I MHC homologues, encoded by all cytomegaloviruses characterised to date, have been implicated as molecular 'decoys', which may mimic the ability of cellular MHC class I to inhibit NK-cell functions. Results from studies in vitro are not uniform, but in general they support the proposal that the class I homologues engage inhibitory receptors from NK cells and other cell types that normally interact with cellular class I. Consistent with this, in vivo studies of murine cytomegalovirus indicate that the class I homologue is required for efficient evasion of NK-cell-mediated clearance. Recently a second murine cytomegalovirus protein, a C-C chemokine homologue, has been implicated as promoting evasion of NK and T-cell-mediated clearance in vivo.

Murine cytomegalovirus (CMV)-encoded protein m144 is homologous to class I MHC heavy-chain and is thought to regulate NK-cell-mediated immune responses in vivo. To examine the effects of m144 on NK cytotoxicity in vitro, various cell lines were transfected with wild-type m144 or a chimeric construct in which the cytoplasmic domain of m144 was replaced with green fluorescence protein. Burkitt lymphoma line Raji expressed a significant level of m144 as determined by anti-m144 mAb binding or the green fluorescence of the fusion protein. The level of m144 expression was relatively low compared with that of transfected murine class I MHC Dd. However, m144 on Raji cells partially inhibited antibody-dependent cell-mediated cytotoxicity of IL-2-activated NK cells. NK cells from the CMV-susceptible BALB/c as well as those from the resistant C57BL/6 mice were inhibited by m144. Antibodies against the known murine NK inhibitory receptors Ly-49A, C, G, and I did not affect the inhibitory effect of m144. These results suggest that the murine CMV class I MHC homologue m144 partially inhibits NK cells by interacting with a novel inhibitory receptor., (Copyright 1999 Academic Press.)

The study of 'molecular mimicry' or 'genetic piracy', with respect to the utilisation of cellular genes captured and modified during the course of virus evolution, has been an area of increasing research with the expansion in virus genome sequencing. Examples of cellular immunomodulatory genes which have been captured from hosts have been identified in a number of viruses. This review concentrates upon studies of murine cytomegalovirus (MCMV), investigating the functions of viral genes homologous to G protein-coupled receptors, MHC class I and chemokines. The study of recombinant MCMV engineered with specific disruptions of these genes has revealed their significance during virus replication and dissemination within the host. In the case of the latter two classes of genes, evidence suggests they interfere with cellular immune responses, although the detailed mechanisms underlying this interference have yet to be delineated., (Copyright 2000 S. Karger AG, Basel.)

The outcome of a virus infection is strongly influenced by interactions between host immune defences and virus 'antidefence' mechanisms. For many viruses, their continued survival depends on the speed of their attack:their capacity to replicate and transmit to uninfected hosts prior to their elimination by an effective immune response. In contrast, the success of persistent viruses lies in their capacity for immunological subterfuge: the evasion of host defence mechanism by either mutation (covered elsewhere in this issue, by Gould and Bangham, pp. 331-338) or interference with the action of host cellular proteins that are important components of the immune response. This review will focus on the strategies employed by persistent viruses against two formidable host defences against virus infection: the CD8+ cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses.

The murine cytomegalovirus (MCMV) M97 gene is homologous with both eukaryotic protein kinases and the phosphotransferases of herpesviruses. The gene conserves the domain structure of protein kinases and of the human cytomegalovirus UL97 (phosphotransferase) gene. An M97 transcript of 2.5 kb is present predominantly at late times, and much smaller quantities of the transcript are detected at early times postinfection. Comparison of the DNA sequences of the complete M97 genes from 12 ganciclovir-sensitive and aciclovir-sensitive strains of MCMV showed that the sensitive isolates strongly conserve the sequence of the catalytic domains, but have only moderate conservation of the sequence of the amino-terminal (regulatory) region. MCMV provides a useful model for studying the in vivo function of the phosphotransferase genes of the betatherpesviruses and has potential for use in studies of antiviral resistance.

Herpesviruses, such as murine and human cytomegalovirus (MCMV and HCMV), can establish a persistent infection within the host and have diverse mechanisms as protection from host immune defences. Several herpesvirus genes that are homologous to host immune modulators have been identified, and are implicated in viral evasion of the host immune response. The discovery of a viral major histocompatibility complex (MHC) class I homologue, encoded by HCMV, led to speculation that it might function as an immune modulator and disrupt presentation of peptides by MHC class I to cytotoxic T cells. However, there is no evidence concerning the biological significance of this gene during viral infection. Recent analysis of the MCMV genome has also demonstrated the presence of a MHC class I homologue. Here we show that a recombinant MCMV, in which the gene encoding the class I homologue has been disrupted, has severely restricted replication during the acute stage of infection compared with wild-type MCMV. We demonstrate by in vivo depletion studies that natural killer (NK) cells are responsible for the attenuated phenotype of the mutant. Thus the viral MHC class I homologue contributes to immune evasion through interference with NK cell-mediated clearance.

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

The complete DNA sequence of the Smith strain of murine cytomegalovirus (MCMV) was determined from virion DNA by using a whole-genome shotgun approach. The genome has an overall G+C content of 58.7%, consists of 230,278 bp, and is arranged as a single unique sequence with short (31-bp) terminal direct repeats and several short internal repeats. Significant similarity to the genome of the sequenced human cytomegalovirus (HCMV) strain AD169 is evident, particularly for 78 open reading frames encoded by the central part of the genome. There is a very similar distribution of G+C content across the two genomes. Sequences toward the ends of the MCMV genome encode tandem arrays of homologous glycoproteins (gps) arranged as two gene families. The left end encodes 15 gps that represent one family, and the right end encodes a different family of 11 gps. A homolog (m144) of cellular major histocompatibility complex (MHC) class I genes is located at the end of the genome opposite the HCMV MHC class I homolog (UL18). G protein-coupled receptor (GCR) homologs (M33 and M78) occur in positions congruent with two (UL33 and UL78) of the four putative HCMV GCR homologs. Counterparts of all of the known enzyme homologs in HCMV are present in the MCMV genome, including the phosphotransferase gene (M97), whose product phosphorylates ganciclovir in HCMV-infected cells, and the assembly protein (M80).

Herpesviruses have acquired a variety of different mechanisms to avoid the damaging effects of host immunity. Frequently, these viruses subvert normal immune regulatory functions utilized by the host. The focus of this review is upon herpesvirus genes encoding known or potential immunomodulatory proteins. Areas covered include inhibition of complement and antibody function, herpesvirus-encoded homologues of cytokines and chemokine receptors, and potential disruption of cellular recognition of virally infected targets.

We have identified three open reading frames (ORFs) in murine cytomegalovirus (MCMV), designated M82, M83, and M84, which likely encode homologs of the human cytomegalovirus (HCMV) UL82 and UL83 matrix phosphoproteins. These ORFs, in the HindIII C fragment of MCMV, are colinear with the UL82, UL83, and UL84 ORFs of HCMV. M82 encodes a 598-amino-acid (aa) protein with homology to UL82, M83 encodes an 809-aa protein with homology to UL82 and UL83, and M84 encodes a 587-aa protein with homology to UL83 and UL84. Analysis of transcription by Northern (RNA) blotting indicated that the M82 and M83 ORFs are transcribed as 2.2- and 5-kb mRNAs, respectively, at 24 to 48 h postinfection (p.i.), while M84 is transcribed as a 6.9-kb mRNA only at 8 h p.i. All transcripts appear to terminate at the same position 3' of the M82 ORF. Of the products of the three ORFs, only M83 is strongly recognized by hyperimmune mouse serum. The M83 protein is a virion-associated phosphoprotein with an apparent molecular mass of 125 kDa. In MCMV-infected cells, it is detectable by Western blotting (immunoblotting) only at 48 h p.i. in the absence of phosphonoacetic acid, consistent with late gene expression. The M83 ORF is also expressed at high levels in cells infected by a recombinant vaccinia virus and yields a protein which is serologically cross-reactive and comigrates with the authentic MCMV protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.

We have characterized the gene encoding the murine cytomegalovirus (MCMV) homologue of the human cytomegalovirus (HCMV) UL100 open reading frame (ORF) that encodes the HCMV glycoprotein M (gM) molecule. It was identified based on its collinearity with MCMV homologues of the HCMV UL99, UL102, UL103 and UL104 ORFs which lie in the HindIII G fragment of the K181 strain of MCMV. Sequencing of a 2.3 kb EcoRI-BamHI subfragment of the EcoRI G fragment adjacent to the EcoRI A fragment revealed the presence of the complete MCMV gM ORF and two incomplete ORFs, which corresponded to homologues of HCMV UL99 and UL102. The MCMV gM ORF consists of 1059 nucleotides and is expressed as a 1.2 kb transcript at late times post-infection. To precisely characterize the gM transcript, the 5' and 3' ends were mapped. It was found that the transcript initiates at nucleotides 740 or 745, and that the site of polyadenylation at nucleotide 1961 occurs downstream of the second potential polyadenylation signal located at nucleotide 1934. Based on these findings the MCMV gM is predicted to consist of 353 residues and when compared with HCMV gM has a 47% level of identity. Of great interest is the finding that the MCMV gM amino acid sequence is completely conserved among six isolates of MCMV that had been shown to exhibit considerable variation both in the MCMV glycoprotein B and the immediate-early 1 gene-encoded pp89 molecule. Thus, this glycoprotein appears to be antigenically conserved.

DNA sequence analysis of the genome of the Smith strain of murine cytomegalovirus (MCMV) revealed an open reading frame (ORF) with amino acid sequence identity to glycoprotein L (gL) of other herpesviruses. The ORF is 822 bp in size and has the capacity to encode a protein of 274 amino acids. It has significant identity with the gL genes of human CMV and human herpesvirus 6. The coding sequence of the gL gene of MCMV strain K181 was also determined, and expressed in Escherichia coli as a fusion protein with glutathione S-transferase using the pGEX expression system. Two antibody-binding regions were identified on the basis of the reactivity of a series of truncated gL constructs with anti-MCMV antibodies. One was mapped to residues 1 to 38 and the other between residues 230 and 274. Polyclonal antibodies specific to gL were raised against the full-length gL fusion protein. The antisera were shown to react with a 46K protein present in purified virions by Western blotting. Treatment of purified virions with endoglycosidase-H or -F resulted in reductions in M(r) of the 46K species to 42K and 31K, respectively. The antisera did not exhibit any neutralizing activity in a plaque reduction assay.