Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4⁺ T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.     

Suppression of rat cytomegalovirus replication by antibodies against gamma interferon.

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma.     

Human cytomegalovirus expresses novel microRNAs during productive viral infection

MicroRNAs (miRNAs) are a large class of approximately 22-nucleotide non-coding RNAs that facilitate mRNA cleavage and translation repression through the RNA interference pathway. Until recently, miRNAs have been exclusively found in eukaryotic organisms. A non-immunogenic molecule requiring minimal genomic investment, these RNAs may offer an efficient means for viruses to modulate both their own and the host's gene expression during a productive viral infection. In this study we report that human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of four clinically relevant human cell types: fibroblast, endothelial, epithelial and astrocyte cells. The sequences of the miRNAs, expressed from the UL23 and US24 loci of the viral genome, were conserved among all HCMV strains examined and in chimpanzee cytomegalovirus. Furthermore, their expression was detected from both a laboratory-adapted strain and a clinical isolate of HCMV. The conservation of these miRNAs and their expression in different cell types suggests that they represent an evolutionarily primitive feature in the viral genome, and that virus-encoded miRNAs may be more common than previously believed.     

Competitive virus and host RNAs: the interplay of a hidden virus and host interaction

During virus infection, viral RNAs and mRNAs function as blueprints for viral protein synthesis and possibly as pathogen-associated molecular patterns (PAMPs) in innate immunity. Here, considering recent research progress in microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs), we speculate that viral RNAs act as sponges and can sequester endogenous miRNAs within infected cells, thus cross-regulating the stability and translational efficiency of host mRNAs with shared miRNA response elements. This cross-talk and these reciprocal interactions between viral RNAs and host mRNAs are termed “competitive viral and host RNAs” (cvhRNAs). We further provide recent experimental evidence for the existence of cvhRNAs networks in hepatitis B virus (HBV), as well as Herpesvirus saimiri (HVS), lytic murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) infections. In addition, the cvhRNA hypothesis also predicts possible cross-regulation between host and other viruses, such as hepatitis C virus (HCV), HIV, influenza virus, human papillomaviruses (HPV). Since the interaction between miRNAs and viral RNAs also inevitably leads to repression of viral RNA function, we speculate that virus may evolve either to employ cvhRNA networks or to avoid miRNA targeting for optimal fitness within the host. CvhRNA networks may therefore play a fundamental role in the regulation of viral replication, infection establishment, and viral pathogenesis.     

人巨细胞病毒编码miRNA功能及其作用机制

人巨细胞病毒（human cytomegalovirus, HCMV）是疱疹病毒β亚科中的代表成员之一,是一种具有囊膜包裹的DNA双链病毒,对免疫耐受群体和先天性感染的婴幼儿具有很高的发病率。HCMV具有潜伏感染和裂解感染两种感染状态。这两种感染过程中均有不同的miRNA表达模式。这些miRNA不仅参与胞内宿主或病毒自身基因表达调控与病毒复制,也能调节胞内物质的转运和病毒感染状态的转变等过程。本文就HCMV编码的miRNA,其生物合成机制和生物学功能进行简要综述,为深入研究其生物功能和作用机制奠定基础。     

The r144 major histocompatibility complex class I-like gene of rat cytomegalovirus is dispensable for both acute and long-term infection in the immunocompromised host

The rat cytomegalovirus (RCMV) r144 gene encodes a polypeptide homologous to major histocompatibility complex class I heavy chains. To study the role of r144 in virus replication, an RCMV r144 null mutant strain (RCMVDeltar144) was generated. This strain replicated with efficiency similar to that of wild-type (WT) RCMV in vitro. Additionally, WT RCMV and RCMVDeltar144 were found not to differ in their replication characteristics in vivo. First, the survival rate was similar among groups of immunosuppressed rats infected with either RCMVDeltar144 or WT RCMV. Second, the dissemination of virus did not differ in either RCMVDeltar144- or WT RCMV-infected, immunosuppressed rats, either in the acute phase of infection or approximately 1 year after infection. These data indicate that the RCMV r144 gene is essential neither for virus replication in the acute phase of infection nor for long-term infection in immunocompromised rats. Interestingly, in a local infection model in which footpads of immunosuppressed rats were inoculated with virus, a significantly higher number of infiltrating macrophage cells as well as of CD8(+) T cells was observed in WT RCMV-infected paws than in RCMVDeltar144-infected paws. This suggests that r144 might function in the interaction with these leukocytes in vivo.     

Structure of the rat cytomegalovirus genome termini.

The lytic replication cycle of herpesviruses can be divided into the following three steps: (i) circularization, in which, after infection, the termini of the linear double-stranded viral genome are fused; (ii) replication, in which the circular DNA serves as template for DNA replication, which generates large DNA concatemers; and (iii) maturation, in which the concatemeric viral DNA is processed into unit-length genomes, which are packaged into capsids. Sequences at the termini of the linear virion DNA are thought to play a key role in both genome circularization and maturation. To investigate the mechanism of these processes in the replication of rat cytomegalovirus (RCMV), we cloned, sequenced, and characterized the genomic termini of this betaherpesvirus. Both RCMV genomic termini were found to contain a single copy of a direct terminal repeat (TR). The TR sequence is 504 bp in length, has a high GC content (76%), and is not repeated at internal sites within the RCMV genome. The TR comprises several small internal direct repeats as well as two sequences which are homologous to herpesvirus pac-1 and pac-2 sites, respectively. The organization of the RCMV TR is unique among cytomegaloviruses with respect to the position of the pac sequences: pac-1 is located near the left end of the TR, whereas pac-2 is present near the right end. Both RCMV DNA termini carry an extension of a single nucleotide at the 3' end. Since these nucleotides are complementary, circularization of the viral genome is likely to occur via a simple ligation reaction.     

The effect of biological response modifiers on chronic and latent murine cytomegalovirus infections

Host-mediated antiviral effect of 2 biological response modifiers (BRM), OK-432, and PS-K, against murine cytomegalovirus (MCMV) was evaluated in chronically or latently infected mice. In the early stage of chronic MCMV infection, the BRM-induced resistance was evidenced by decrease in infectious viruses replicated in the salivary glands and by augmented cytotoxic activity of the spleen cells against YAC-1 cells and MCMV-infected mouse embryonic fibroblasts (MEF). In the late stage of chronic MCMV infection, the BRM treatment did not eliminate MCMV from the mice, but did prevent exacerbation of MCMV infection in the salivary glands induced by administration of cyclophosphamide (CY). In mice latently infected by MCMV, BRM treatment suppressed CY-induced reactivation of MCMV in the salivary glands. It was suggested that the antiviral effect of BRM against MCMV in chronically or latently infected mice was based on activation of natural killer (NK) cells and cytotoxic T lymphocytes (CTL).     

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.     

Murine cytomegalovirus with a transposon insertional mutation at open reading frame M35 is defective in growth in vivo

We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn3-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.     

Discrete clusters of virus-encoded micrornas are associated with complementary strands of the genome and the 7.2-kilobase stable intron in murine cytomegalovirus

The prevalence and importance of microRNAs (miRNAs) in viral infection are increasingly relevant. Eleven miRNAs were previously identified in human cytomegalovirus (HCMV); however, miRNA content in murine CMV (MCMV), which serves as an important in vivo model for CMV infection, has not previously been examined. We have cloned and characterized 17 novel miRNAs that originate from at least 12 precursor miRNAs in MCMV and are not homologous to HCMV miRNAs. In parallel, we applied a computational analysis, using a support vector machine approach, to identify potential precursor miRNAs in MCMV. Four of the top 10 predicted precursor sequences were cloned in this study, and the combination of computational and cloning analysis demonstrates that MCMV has the capacity to encode miRNAs clustered throughout the genome. On the basis of drug sensitivity experiments for resolving the kinetic class of expression, we show that the MCMV miRNAs are both early and late gene products. Notably, the MCMV miRNAs occur on complementary strands of the genome in specific regions, a feature which has not previously been observed for viral miRNAs. One cluster of miRNAs occurs in close proximity to the 5' splice site of the previously identified 7.2-kb stable intron, implying a variety of potential regulatory mechanisms for MCMV miRNAs.