Helen E. Farrell

A Mouse Cytomegalovirus Glycoprotein Retains MHC Class I Complexes in the ERGIC/cis-Golgi Compartments

The principle by which mouse cytomegalovirus blocks antigen presentation in the MHC class I pathway was investigated. The responsible gene m152, encoding a type I transmembrane glycoprotein of 40 kDa, is a member of a gene family located in the right-hand terminal region of the 230 kb virus genome. Expression of m152 in murine and human cells arrested the export of mouse class I complexes from the ER-Golgi intermediate compartment/cis-Golgi compartment and inhibited lysis by cytotoxic T cells. The plasma membrane transport of human MHC class I molecules was not affected. The deletion of the cytoplasmic tail of gp40 did not lift its effect on class I molecule export, indicating that this protein differs in its functions from known immunosubversive viral gene products and represents a novel principle by which a herpesvirus shuts off MHC class I function.

A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface

Murine cytomegalovirus (MCMV) interferes with antigen presentation by means of retaining major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER). Here we identify and characterize an MCMV‐encoded glycoprotein, gp34, which tightly associates with properly conformed MHC class I molecules in the ER. Gp34 is synthesized in large quantities during MCMV infection and it leaves the ER only in association with MHC class I complexes. Many but not all class I molecules are retained in the ER during the early phase of MCMV infection, and we observe an inverse correlation between amounts of gp34 synthesized during the course of infection and class I retention. An MCMV deletion mutant lacking several genes, including the gene encoding gp34, shows increased class I retention. Thus, MCMV gp34 may counteract class I retention, perhaps to decrease susceptibility of infected cells to recognition by natural killer cells.

Murine Cytomegalovirus (CMV) M33 and Human CMV US28 Receptors Exhibit Similar Constitutive Signaling Activities

ABSTRACT Cellular infection by cytomegalovirus (CMV) is associated with very early G-protein-mediated signal transduction and reprogramming of gene expression. Here we investigated the involvement of human CMV (HCMV)-encoded US27, US28, and UL33 receptors as well as murine CMV-encoded M33 transmembrane (7TM) receptors in host cell signaling mechanisms. HCMV-encoded US27 did not show any constitutive activity in any of the studied signaling pathways; in contrast, US28 and M33 displayed ligand-independent, constitutive signaling through the G protein q (Gq)/phospholipase C pathway. In addition, M33 and US28 also activated the transcription factor NF-κB as well as the cyclic AMP response element binding protein (CREB) in a ligand-independent, constitutive manner. The use of specific inhibitors indicated that the p38 mitogen-activated protein (MAP) kinase but not the extracellular signal-regulated kinase 1/2-MAP kinase pathway is involved in M33- and US28-mediated CREB activation but not NF-κB activation. Interestingly, UL33—the HCMV-encoded structural homologue of M33—was only marginally constitutively active in the Gq/phospholipase C turnover and CREB activation assays and did not show any constitutive activity in the NF-κB pathway, where M33 and US28 were highly active. Hence, CMVs appear to have conserved mechanisms for regulating host gene transcription, i.e., constitutive activation of certain kinases and transcription factors through the constitutive activities of 7TM proteins. These data, together with the previous identification of the incorporation of such proteins in the viral envelope, suggest that these proteins could be involved in the very early reprogramming of the host cell during viral infection.

Masters of deception: A review of herpesvirus immune evasion strategies

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.

Protection Against Murine Cytomegalovirus Infection by Passive Transfer of Neutralizing and Non-Neutralizing Monoclonal Antibodies

The ability of eight neutralizing monoclonal antibodies (MAbs) specific for structural proteins of murine cytomegalovirus (MCMV) to protect mice passively against MCMV infection was examined to determine firstly whether a correlation existed between the neutralization titres of the MAbs in vitro and the protection afforded by the MAbs in vivo and, secondly, the contribution of the host towards neutralization by the MAbs in vivo. The reduction in MCMV titre in the livers of BALB/c and C57BL/10 mice by the MAbs closely correlated with their neutralization titres in vitro. However, in the spleens of BALB/c mice, in which MCMV replicates to high titre, almost all of the MAbs tested were ineffective in reducing MCMV replication. Indeed, a significant increase in splenic MCMV replication was observed in mice treated 24 h prior to MCMV replication with either neutralizing MAbs or polyclonal Ig. Each of six MAbs prophylactically protected between 66 and 100% of mice from an intraperitoneal challenge with 4 LD50 MCMV regardless of their neutralization titre in vitro. The persistence of MCMV replication in the salivary gland was not prevented by either polyclonal Ig or MAbs. Despite the absolute requirement for complement for the neutralization of MCMV in vitro, both polyclonal Ig and MAb 4F9 protected A/J mice, which are deficient in the fifth component of complement, as efficiently as they did complement competent BALB/c mice. These results demonstrate that MAbs specific for single MCMV polypeptides are protective in vivo. In addition, the extent to which the MAbs protected against MCMV could not be predicted from their immunoreactive or neutralizing titres in vitro or by their effect on splenic MCMV replication in vivo. Furthermore, these studies suggest that the mechanism(s) of neutralization of MCMV in vitro are different to those which act in vivo.

NK1.1+ Cells and Murine Cytomegalovirus Infection: What Happens In Situ?

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.

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 R340 and A353 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.

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, ΔM33BT2, 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, ΔM33BT2 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 ΔM33BT2 viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent ΔM33BT2 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 ΔM33BT2 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.

The genetic background modulates the effect of the beige gene on susceptibility to cytomegalovirus infection in mice.

Homozygous beige (bg/bg) mice were more susceptible to the development of fatal disease induced by murine cytomegalovirus (MCMV) than their bg/+ littermates. However, the increase in susceptibility depended on the genetic background of the strain carrying the bg gene. C57BL/6, SB/Le, DBA/2, and CBA bg/bg mice showed, respectively, 2.5‐, 3.2‐, 9.5‐, and 18.6‐fold increases in susceptibility compared with the corresponding bg/+ animals. Beige mice showed higher liver titres of MCMV than bg/+ by the 2nd or 3rd day after infection, and tissue damage was also greater. Splenic NK cells were not detected in uninfected bg/bg mice, and after virus inoculation the increment in cytotoxicity was greater in bg/+ than in bg/bg mice. However, cytotoxicity towards WEH1‐164 cells was not impaired in bg/bg mice and was not augmented by MCMV. Interferon titres were also not impaired by the beige mutation. Of the strains examined, CBA had the highest endogenous levels of NK cells and were most genetically resistant to MCMV. Thus, our observation that the beige gene had the greatest effect on susceptibilty in this strain suggests that NK cells are important mediators of genetically determined resistance to MCMV.

Lymph Node Macrophages Restrict Murine Cytomegalovirus Dissemination

ABSTRACT 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.