巨细胞病毒编码的趋化因子及其受体同源物

巨细胞病毒(CMV)属β疱疹病毒亚科,普遍存在于自然界.人巨细胞病毒(HCMV)是对人类具有致病性的CMV,在人群中的感染非常普遍,但大多数呈临床不显性感染或潜伏感染.CMV感染机体后,可调动多种机制逃逸正常机体的免疫监视,建立潜伏感染[1,2],这些机制包括下调MHC-Ⅰ和MHC-Ⅱ类基因的表达,编码MHC-Ⅰ同源物抑制NK细胞活性,以及编码趋化因子(chemokines,CKs)及其受体(chemokine receptors,CKPs)同源物干扰机体正常的免疫应答等.     

In Vivo Replication, Latency, and Immunogenicity of Murine Cytomegalovirus Mutants with Deletions in the M83 and M84 Genes, the Putative Homologs of Human Cytomegalovirus pp65 (UL83)

We previously identified two open reading frames (ORFs) of murine cytomegalovirus (MCMV), M83 and M84, which are putative homologs of the human cytomegalovirus (HCMV) UL83 tegument phosphoprotein pp65 (L. D. Cranmer, C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector, J. Virol. 70:7929-7939, 1996). In this report, we show that unlike the M83 gene product, the M84 protein is expressed at early times in the infection and cannot be detected in the virion. To elucidate the functional differences between the two pp65 homologs in acute and latent MCMV infections, we constructed two MCMV K181 mutants in which either the M83 or M84 ORF was deleted. The resultant viruses, designated DeltaM83 and DeltaM84, respectively, were found to replicate in NIH 3T3 cells with kinetics identical to those of the parent strain. Western blot analysis demonstrated that except for the absence of M83 or M84 protein expression in the respective mutants, no global perturbations of protein expression were detected. When DeltaM83 and DeltaM84 were inoculated intraperitoneally (i.p.) into BALB/c mice, both viruses showed similar attenuated growth in the spleen, liver, and kidney. However, only DeltaM83 was severely growth restricted in the salivary glands, a phenotype that was abolished upon restoration of the M83 ORF. DeltaM83's growth was similarly restricted in the salivary glands of the resistant C3H/HeN or highly sensitive 129/J strain, as well as in the lungs of all three strains following intranasal inoculation. Using a nested-PCR assay, we found that both DeltaM83 and DeltaM84 established latency in BALB/c mice, with slightly decreased levels of DeltaM83 and DeltaM84 genomic DNAs, relative to K181, observed in the salivary glands and lungs. Immunization of BALB/c mice with 10(5) PFU of K181, DeltaM83, or DeltaM84 i.p. provided similar levels of protection against lethal challenge. Although immunization with 200 PFU of DeltaM83 also provided complete protection, this dose allowed both the immunizing and challenge viruses to establish latency in the spleen. Our results show that the two MCMV pp65 homologs differ in their expression kinetics, virion association, and influence on viral tropism and/or dissemination.     

Knockout of the Host Resistance Gene Pkr Fully Restores Replication of Murine Cytomegalovirus m142 and m143 Mutants In Vivo

Murine cytomegalovirus (MCMV) proteins m142 and m143 are essential for viral replication. They bind double-stranded RNA and prevent protein kinase R-induced protein synthesis shutoff. Whether the two viral proteins have additional functions such as their homologs in human cytomegalovirus do remained unknown. We show that MCMV m142 and m143 knockout mutants attain organ titers equivalent to those attained by wild-type MCMV in Pkr knockout mice, suggesting that these viral proteins do not encode additional PKR-independent functions relevant for pathogenesis in vivo.     

The Murine CAR Homolog Is a Receptor for Coxsackie B Viruses and Adenoviruses

Complementary DNA clones encoding the murine homolog (mCAR) of the human coxsackievirus and adenovirus receptor (CAR) were isolated. Nonpermissive CHO cells transfected with mCAR cDNA became susceptible to infection by coxsackieviruses B3 and B4 and showed increased susceptibility to adenovirus-mediated gene transfer. These results indicate that the same receptor is responsible for virus interactions with both murine and human cells. Analysis of receptor expression in human and murine tissues should be useful in defining factors governing virus tropism in vivo.     

Liver Sinusoidal Endothelial Cells Are a Site of Murine Cytomegalovirus Latency and Reactivation

Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients'' tissues. The viral genome was found to localize primarily to sry-negative CD11b⁻ CD11c⁻ CD31⁺ CD146⁺ cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146⁺ cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10⁴ LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients'' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.In human cytomegalovirus (hCMV) infection, hematopoietic progenitor cells of the myeloid differentiation lineage are a recognized cellular site of virus latency (for more-recent reviews, see references 75 and 94), and cell differentiation-dependent as well as cytokine-mediated viral gene desilencing by chromatin remodeling is discussed as the triggering event leading to virus reactivation (for a review, see reference 7). Although hematopoietic stem cell or bone marrow transplantation (BMT) is frequently associated with hCMV reactivation and recurrence in recipients after hematoablative leukemia/lymphoma therapy, the incidence of virus recurrence and disease is highest in the combination of an hCMV-negative donor (D⁻) and an hCMV-positive recipient (R⁺) (D⁻R⁺ > D⁺R⁺ > D⁺R⁻), indicating that donor hematopoietic cells are not the only source of latent hCMV and actually not the predominant source (34). Rather, the recipients experience reactivation of their own virus. Just the opposite is true in the case of solid organ transplantation, where the reactivating virus is mostly transmitted with the transplanted organ (D⁺R⁻ > D⁺R⁺ > D⁻R⁺) (34). Collectively, these risk assessments support the suggestion that reactivation, in both instances, occurs in latently infected tissue cells, that is, within the recipient''s organs and the transplanted donor organ, respectively. Although tissue-resident cells of hematopoietic origin remain candidates, stromal and parenchymal tissue cells come into consideration as additional sites of CMV latency.Longitudinal analysis of viral genome load in the latency models of murine CMV (mCMV) infection of neonatal mice (9, 91) as well as of adult mice after experimental BMT (8, 62, 64) has demonstrated a high viral latency burden in multiple organs long after clearance of viral DNA from bone marrow and blood (reviewed in reference 92). These findings support the suggestion that there exist two types of latency, namely, a temporary latency in hematopoietic cells and a latency in tissue cells that lasts through life. Accordingly, both types of latency may coexist early after primary infection, while “late latency” is restricted to organ sites. As we have shown previously in a sex-mismatched murine BMT model, bone marrow cells (BMCs) derived from latently infected donors in the phase of organ-restricted “late latency” cannot transmit latent or reactivated infection to naïve recipients upon intravenous cell transfer (99).A first hint for mCMV latency in stromal or reticular cells was presented long ago by Mercer and colleagues (73), who showed that infected cells during acute infection of the spleen are predominantly sinusoidal lining cells and that latent mCMV can be recovered from a major histocompatibility complex class II (MHC-II) antigen-negative and Thy-1 (CD90)-negative “stromal” cell fraction, which includes endothelial cells (ECs). These findings strongly argued against T and B lymphocytes, macrophages, and dendritic cells (DCs) being major reservoirs of latent mCMV in the spleen, a conclusion supported by later work of Pomeroy and colleagues (86). Similarly, Klotman and colleagues (54) as well as Hamilton and Seaworth (44) concluded that in kidney transplantation, donor kidney is the source of latent mCMV and that the latent viral genome is harbored by renal peritubular epithelial cells (53). A first hint for mCMV latency in ECs within the liver was provided by in situ PCR images presented by Koffron and colleagues (59) showing nuclear staining in cells with a microanatomical localization suspicious of liver sinusoidal ECs (LSECs). For hCMV, ECs, in particular those in arterial vessel walls, are regarded as a site of latency on the basis of the presence of viral DNA in cells expressing an EC marker (81; for a review, see reference 48), although other authors did not detect viral DNA in venous vessel walls (95). As discussed by Jarvis and Nelson (48), these data are not necessarily conflicting but may rather reflect the diversity of EC subsets at different anatomical locations (21, 27). As far as we know, and reactivation of productive infection from ECs that carry latent viral DNA is not yet formally proven for any type of EC.Hepatitis is a relevant organ manifestation of CMV disease in immunocompromised hosts (65), and hCMV reactivation has been reported to be the most common cause of infection following liver transplantation, in particular in a D⁺R⁻ combination (34, 76). In the murine model of immunocompromised hosts, viral histopathology in the liver is dominated by the cytopathogenic infection of hepatocytes, leading to extended plaque-like tissue lesions (41, 84; reviewed in reference 45). Occasionally, however, in these studies, infected hepatic ECs as well as Kupffer macrophages were detected by virus-specific immunohistology or by in situ virus-specific DNA hybridization.Using cell-type-specific conditional recombination of a fluorescence-tagged reporter virus in Cre-transgenic mice expressing Cre selectively in hepatocytes under the control of the albumin promoter, hepatocytes were recently identified as the main virus-producing cell type during mCMV infection. In Cre-transgenic mice expressing Cre selectively in vascular ECs under the control of the Tie2 promoter, the reporter virus was found to recombine also in LSECs, which released an amount of virus sufficient for virus spread to neighboring hepatocytes, although the virus productivity of LSECs was low and contributed little to the overall virus load in the liver (97).LSECs represent a unique liver-resident population of antigen-presenting cells that bear the capacity to cross-present antigens to naïve CD8 T cells (68, 69; reviewed in reference 57). They constitute the fenestrated endothelial lining of the hepatic sinusoids (15). By separating the sinusoidal compartment of the liver from the space of Disse and the liver parenchyma, LSECs form a boundary surface for sensing of pathogens and interaction with passenger lymphocytes. They perform a scavenger function contributing to hepatic clearance of bacterial degradation products derived from the gastrointestinal tract (103; reviewed in reference 57). According to this physiological role, antigen presentation by LSECs is associated with tolerance induction rather than with triggering an inflammatory immune response (29, 56, 68, 104).Here we provide evidence to support the suggestion that mCMV has chosen the tolerogenic and long-lived LSECs as an immunoprivileged niche for establishing viral latency in the liver.     

Products of US22 Genes M140 and M141 Confer Efficient Replication of Murine Cytomegalovirus in Macrophages and Spleen

Efficient replication of murine cytomegalovirus (MCMV) in macrophages is a prerequisite for optimal growth and spread of the virus in its natural host. Simultaneous deletion of US22 gene family members M139, M140, and M141 results in impaired replication of MCMV in macrophages and mice. In this study, we characterized the proteins derived from these three genes and examined the impact of individual gene deletions on viral pathogenesis. The M139, M140, and M141 gene products were identified as early proteins that localize to both the nucleus and cytoplasm in infected cells. Gene M139 encodes two proteins, of 72 and 61 kDa, while M140 and M141 each encode a single protein of 56 (pM140) and 52 (pM141) kDa, respectively. No role for the M139 proteins in MCMV replication in macrophages or mice was determined in these studies. In contrast, deletion of either M140 or M141 resulted in impaired MCMV replication in macrophages and spleen tissue. Replication of the M140 deletion mutant was significantly more impaired than that of the virus lacking M141. Further analyses revealed that the absence of the pM140 adversely affected pM141 levels by rendering the latter protein unstable. Since the replication defect due to deletion of M140 was more profound than could be explained by the reduced half-life of pM141, pM140 must exert an additional, independent function in mediating efficient replication of MCMV in macrophages and spleen tissue. These data indicate that the US22 genes M140 and M141 function both cooperatively and independently to regulate MCMV replication in a cell type-specific manner and, thus, to influence viral pathogenesis.     

Ablation of the Regulatory IE1 Protein of Murine Cytomegalovirus Alters In Vivo Pro-inflammatory TNF-alpha Production during Acute Infection

Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNβ, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo.     

CCL18 Exhibits a Regulatory Role through Inhibition of Receptor and Glycosaminoglycan Binding

CCL18 has been reported to be present constitutively at high levels in the circulation, and is further elevated during inflammatory diseases. Since it is a rather poor chemoattractant, we wondered if it may have a regulatory role. CCL18 has been reported to inhibit cellular recruitment mediated by CCR3, and we have shown that whilst it is a competitive functional antagonist as assessed by Schild plot analysis, it only binds to a subset of CCR3 receptor populations. We have extended this inhibitory activity to other receptors and have shown that CCL18 is able to inhibit CCR1, CCR2, CCR4 and CCR5 mediated chemotaxis, but has no effect on CCR7 and CCR9, nor the CXC receptors that we have tested. Whilst CCL18 is able to bind to CCR3, it does not bind to the other receptors that it inhibits. We therefore tested the hypothesis that it may displace glycosaminoglycan (GAG) chemokines bound either in cis- on the leukocyte, or in trans-presentation on the endothelial surface, thereby inhibiting the recruitment of leukocytes into the site of inflammation. We show that CCL18 selectivity displaces heparin bound chemokines, and that chemokines from all four chemokine sub-classes displace cell bound CCL18. We propose that CCL18 has regulatory properties inhibiting chemokine function when GAG-mediated presentation plays a role in receptor activation.     

The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.     

An In Vivo EGF Receptor Localization Screen in C. elegans Identifies the Ezrin Homolog ERM-1 as a Temporal Regulator of Signaling

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.