Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice

Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, Toledo(BAC) virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. Toledo(BAC), like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (Toledo(BAC)) retained its virulence. The 15-kb segment was then deleted from Toledo(BAC), and the resulting virus, Toledo(Delta15kb), was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. Toledo(Delta15kb) had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the Toledo(Delta15kb) genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.     

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains.

Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (UL)/b' (IRL) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b' sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the UL/b' region during laboratory passage.     

Human cytomegalovirus binding to fibroblasts is receptor mediated.

The binding of radiolabeled human cytomegalovirus (HCMV) strain AD169 to human lymphocytes, lymphoblastoid cell lines, monocytes, and fibroblasts varied over a 20-fold range. Since maximum binding was observed with human foreskin fibroblasts (HFF), interactions of radiolabeled HCMV with this cell type were analyzed quantitatively. Binding of HCMV to HFF at 4 degrees C was specific and saturable; at low viral inputs specific binding averaged 16.4% of input and nonspecific binding was less than 1% of input. Binding curves yielded single-component linear Scatchard plots indicating an average Kd of 1.1 nM and 5,262 available virus-binding sites per cell. A two-component Scatchard curve was obtained at 37 degrees C and reflected viral internalization, since it could be converted to a single-component curve by the use of paraformaldehyde-fixed cells. HCMV strain Towne was found to bind to the receptor used by HCMV strain AD169 with similar affinity. HCMV failed to bind to protease-treated HFF or to HFF grown in the presence of inhibitors of glycosylation. Sialic acid residues, however, were not found to be important in binding. These data indicate that a single type of molecule, likely a glycoprotein, on the surface of HFF serves as a specific receptor for the virus.     

Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1 alpha, and the CX(3)C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca(2+). Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G(q/11)-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5'-O-(3-[(35)S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.     

人巨细胞病毒miR-UL22A的重组载体构建以及表达分析

优化构建人巨细胞病毒(human cytomegalovirus,HCMV)miR-UL22A的真核表达载体;明确HCMV临床株及实验室株感染过程中miR-UL22A-5p及miR-UL22A-3p的表达情况。扩增HCMV UL22A编码区不同长度(975、494、209及140 bp)的序列,构建表达miR-UL22A的重组p Silencer(p S)载体,转染人胚肾293细胞后提取总RNA;收集HCMV临床株Han株以及实验室株Towne株感染人胚肺成纤维细胞后6、12、24、48和72h以及不同感染时相(即刻早期、早期、晚期)的总RNA标本;应用Taq Man颈环-实时荧光定量PCR技术检测上述标本miR-UL22A-5p及miR-UL22A-3p的表达量。成功构建了包含上述不同长度miR-UL22A编码序列的重组载体;侧翼序列长度各为40 bp左右的pre-miRNA(即插入140 bp的编码序列)表达成熟miRNA的效果最佳;在病毒的一个复制周期内,HCMV临床株和实验室株miR-UL22A的表达趋势无明显差异,均在感染后6h即检测到表达,72 h达到峰值,且在即刻早期即有miRNA的明确表达;无论是在重组载体表达状态下还是自然感染状态下,miR-UL22A-5p始终为miR-UL22A前体优势表达的miRNA。结果表明,有140 bp片段miRUL22A编码序列的重组载体能够高效地异源表达miR-UL22A-5p及miR-UL22A-3p;miR-UL22A-5p始终为miR-UL22A前体优势表达的miRNA。为进一步研究miRNA转录后调控作用提供了参考。     

人类巨细胞病毒UL133基因在临床低传代株中的多态性研究

人类巨细胞病毒在多次传代后,会表现出不同的毒力水平.与临床低传代株Toledo相比,实验室高传代株AD169缺失了19个开放阅读框(ORF).这19个基因被认为是与HCMV致病性最可能相关的一组基因,研究这些基因的多态性对揭示HCMV致病性的遗传基础具有指导意义.UL133基因是这19个ORF中的一个.以临床低传代株Toledo和Merlin为对照,分析了23个临床病毒株UL133基因的遗传多态性.序列分析表明,UL133基因具有一定的多态性,Toledo株、Merlin株与我们分离到的临床株一起可分为3个基因型:G1、G2和G3.G2、G3型毒株均能导致先天性感染.没有发现UL133基因型与患儿临床疾病的必然关联.     

Viral regulation of RANTES expression during human cytomegalovirus infection of endothelial cells

Human cytomegalovirus (HCMV) evades healthy immune responses during infection, and this evasion may allow HCMV to establish latency in the host. The human vasculature has been recognized as a site of HCMV infection and may also be a site of latent HCMV infection. As the interface between circulating cells and underlying parenchymal cells, the vascular endothelium provides signals for local reaction of inflammatory cells. We propose that HCMV down-regulates expression of the proinflammatory chemokine RANTES from the infected endothelium, which may result in reduced recruitment of mononuclear cells to the site of infection. Abortive HCMV infection of primary endothelial cells with the clinical isolate HCMV 4010, under conditions in which viral gene expression could not occur, induced high levels of RANTES expression. Replicative HCMV infection, however, induced cells in parallel cultures to express significantly lower levels of RANTES. Expression of the chemokines interleukin 8 and MCP-1 by endothelial cells was found to be unaffected by replicative HCMV infection and thus may not play an important role during early HCMV infection of the endothelium. HCMV may regulate RANTES expression from endothelial cells as a mechanism to evade the local immune response to infection.     

Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism

Epithelial cells are one of the prominent cell types infected by human cytomegalovirus (HCMV) within its host. However, many cultured epithelial cells, such as ARPE-19 retinal pigmented epithelial cells, are poorly infected by laboratory-adapted strains in cell culture, and little is known about the viral factors that determine HCMV epithelial cell tropism. In this report, we demonstrate that the UL131 open reading frame (ORF), and likely the entire UL131-128 locus, is required for efficient infection of epithelial cells. Repair of the mutated UL131 gene in the AD169 laboratory strain of HCMV restored its ability to infect both epithelial and endothelial cells while compromising its ability to replicate in fibroblasts. ARPE-19 epithelial cells support replication of the repaired AD169 virus as well as clinical isolates of HCMV. Productive infection of cultured epithelial cells, endothelial cells, and fibroblasts with the repaired AD169 virus leads to extensive membrane fusion and syncytium formation, suggesting that the virus may spread through cell-cell fusion.     

Human Cytomegalovirus miR-UL112-3p Targets TLR2 and Modulates the TLR2/IRAK1/NFκB Signaling Pathway

Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1β, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.     

Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.     

Human cytomegalovirus subverts the functions of monocytes, impairing chemokine-mediated migration and leukocyte recruitment

Despite their role in innate and adaptive immunity, during human cytomegalovirus (HCMV) infection, monocytes are considered to be an important target of infection, a site of latency, and vehicles for virus dissemination. Since chemokine receptors play crucial roles in monocyte activation and trafficking, we investigated the effects of HCMV on their expression and function. By using endotheliotropic strains of HCMV, we obtained high rates (roughly 50%) of in vitro-infected monocytes but only restricted viral gene expression. At 24 h after infection, while the chemokine receptors CX3CR and CCR7 were unaffected, CCR1, CCR2, CCR5, and CXCR4 were downmodulated on the cell surface and retained intracellularly. Structural components of the viral particles, but not viral gene expression or soluble factors released from infected cells, accounted for the changed localization of the receptor molecules and for the block of chemokine-driven migration. HCMV-infected monocytes indeed became unresponsive to inflammatory and homeostatic chemokines, although the basal cell motility and responsiveness to N-formyl-Met-Leu-Phe were unaffected or slightly increased. The production of inflammatory mediators responsible for the recruitment of other immune cells was also hampered by HCMV. Whereas endothelial and fibroblast cells infected by HCMV efficiently recruited leukocytes, infected monocytes were unable to recruit lymphocytes, monocytes, and neutrophils. Our data further highlight the complex level of interference exerted by HCMV on the host immune system.     

Human Cytomegalovirus UL36 Protein Is Dispensable for Viral Replication in Cultured Cells

Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.     

Human cytomegalovirus blocks canonical TGFβ signaling during lytic infection to limit induction of type I interferons

Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFβ interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.     

Human cytomegalovirus infection reduces surface CCR5 expression in human microglial cells,astrocytes and monocyte-derived macrophages

Within the brain, glial cells are target cells for human cytomegalovirus (HCMV) and HIV. We infected cultures of unstimulated human microglial cells and astrocytes of embryonic origin and of monocyte-derived macrophages (MDM) with HCMV strain AD169 and observed down-regulation of the plasma membrane expression of CCR5 in the three cell types, and of CXCR4 and CD4 in microglial cells only. Cells were then coinfected simultaneously or at a 24-h interval with both AD169 and two different HIV-1 monocytotropic strains. HCMV late antigens and HIV-1 tat protein colocalized in the cytoplasm of 5-10% of microglia and MDM. p24 antigen levels decreased 10- to 40-fold in supernatants of MDM and the reduction was greater when HCMV infection was performed 24 h before HIV-1 infection. These data suggest that HCMV-induced reduction in the cell-surface expression of the primary co-receptor of HIV-1 monocytotropic strains may impair the ability of HIV to infect these cells.