Human Cytomegalovirus Clinical Strain-Specific microRNA miR-UL148D Targets the Human Chemokine RANTES during Infection

The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b'' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.     

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.     

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.     

Monocyte-derived inhibitor of interleukin 1 induced by human cytomegalovirus.

It has previously been shown that human cytomegalovirus (HCMV) can exert immunosuppressive effects, and it has been suggested that these may be mediated by monocytes, although the mechanism is unclear. We showed that infection of human monocytes with the AD169 strain of HCMV abrogates their production of interleukin 1 (IL-1) activity. This was associated with the release from infected monocytes of an inhibitor of IL-1 activity which was also released after HCMV infection of the U937 macrophage-like cell line. The inhibitor of IL-1 activity is a protein with an apparent molecular weight of ca. 95,000. This action of HCMV strain AD169 was virus specific and required infectious virus but occurred without virus replication or detectable expression of viral proteins. This effect may account, at least in part, for the previously observed immunosuppressive properties of HCMV.     

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.     

Insertion of an EYFP-pp71 (UL82) coding sequence into the human cytomegalovirus genome results in a recombinant virus with enhanced viral growth

The human cytomegalovirus (HCMV) UL82-encoded tegument protein pp71 has recently been shown to activate viral immediate-early (IE) gene expression by neutralizing a cellular intrinsic immune defense instituted by the ND10 protein hDaxx. Pp71 localizes to ND10 upon infection and induces the degradation of hDaxx. Here, we report the successful generation of a recombinant HCMV expressing enhanced yellow fluorescent protein (EYFP) fused to the N terminus of pp71. Intriguingly, insertion of the EYFP-UL82 coding sequence into the HCMV AD169 genome gave rise to a recombinant virus, termed AD169/EYFP-pp71, that replicates to significantly higher titers than wild-type AD169. In particular, we noticed strongly increased protein levels of pp71 after AD169/EYFP-pp71 inoculation. Although the high abundance of pp71 resulted in augmented packaging of the tegument protein into viral particles, no increased hDaxx degradation was detectable upon AD169/EYFP-pp71 infection. In contrast, further investigation revealed a significantly enhanced viral DNA replication compared to wild-type AD169. Thus, we hypothesize that an as-yet-unidentified function of pp71 contributes to the enhanced infectivity of AD169/EYFP-pp71. This assumption is additionally supported by the observation that increased early and late gene expression after AD169/EYFP-pp71 infection occurs independent of elevated IE protein levels. Finally, immunofluorescence analyses confirmed that hDaxx determines the ND10-localization of pp71 upon infection, since pp71 exhibited a nucleolar distribution in the absence of hDaxx. Taken together, we generated a recombinant HCMV that constitutes a useful tool not only to dissect the in vivo dynamics of pp71 subnuclear localization more precisely but also to explore new features of this viral transactivator.     

Human cytomegalovirus-induced upregulation of intercellular cell adhesion molecule-1 on villous syncytiotrophoblasts

Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF)alpha and, to a lesser extent, interleukin (IL)1 beta inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNF alpha and IL1 beta, as well as by a direct effect on infected cells.     

Production of infectious human cytomegalovirus virions is inhibited by drugs that disrupt calcium homeostasis in the endoplasmic reticulum

We previously reported that human cytomegalovirus (HCMV) infection induces endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR). Although some normal consequences of UPR activation (e.g., translation attenuation) are detrimental to viral infection, we have previously shown that HCMV infection adapts the UPR to benefit the viral infection (14). For example, UPR-induced translation attenuation is inhibited by viral infection, while potentially beneficial aspects of the UPR are maintained. In the present work, we tested the ability of HCMV to overcome a robust induction of the UPR by the drugs thapsigargin and clotrimazole (CLT), which disrupt ER calcium homeostasis. A 24-h treatment with these drugs beginning at 48, 72, or 96 h postinfection (hpi) completely inhibited further production of infectious virions. HCMV could not overcome the inhibition of global translation by CLT; however, between 48 and 72 hpi, HCMV overcame translational inhibition caused by thapsigargin. Despite the restoration of translation in thapsigargin, the accumulation of immediate-early and early gene products was modestly retarded (50% or less), whereas the accumulation of an early-late and late gene product was significantly retarded. Electron microscopic analysis shows that the drugs severely disrupt the maturation of HCMV virions. This can be accounted for by both the retarded accumulation of late gene products and the drug-induced depletion of ER calcium, which disrupts critical cellular functions needed for maturation.     

HCMV大小鼠眼组织损伤模型的初步研究

目的探讨接种人巨细胞病毒(Humancytomegalovirus,HCMV)是否引起Wistar大鼠、昆明种小鼠眼组织损伤.方法将HCMVAD169毒株经静脉接种Wistar大鼠和昆明种小鼠,观察动物眼部发病情况,原位杂交检测动物眼组织中的HCMVDNA片段.结果接种病毒后部分动物缓慢出现眼部发病,局部分泌物增多、浑浊甚至失明;经原位杂交于视锥、视杆细胞和角膜内皮细胞中检出HCMVDNA片段.结论HCMV可以感染动物眼组织,并引起动物发生眼病.     

HCMV基因在人和小鼠胚胎成纤维细胞中表达的差异性研究

人巨细胞病毒（human cytomegalovirus, HCMV）种属特异性机制尚不清楚。研究通过检测HCMVADl69体外感染人胚胎成纤维细胞（Human embryo fibroblast, HEF）和小鼠胚胎成纤维细胞（mouse embryo fibroblast, MEF）后病毒基因的表达情况,探讨HCMV种属特异性的可能分子机制。首先用HCMV AD169（MOI=5）分别感染HEF和MEF,相差显微镜逐日观察细胞的形态学变化;RT—PCR检测HCMV即刻早期（IE1、IE2）、早期（uL84）和晚期基因（UL83）的表达情况;Western—blot和免疫荧光检测病毒基因编码蛋白表达的情况。形态学观察发现HEF感染HCMV后逐渐变大变圆并相互融合,第4天可见典型的HCMV特征性病变效应,而MEF则未出现上述的变化;RT-PCR和Western—blot表明HEF组表达即刻早期基因IE1和IE2、早期基因uL84和晚期基因UL83 mRNA以及各基因所编码的蛋白,且相对表达量显著高于模拟感染组（P〈0．01）;而MEF组仅IEl和IE2mRNA和蛋白相对表达量显著低于HEF组（P〈0．05）,而高于模拟感染组（P〈0．01）。免疫荧光检测发现HEF感染72h表达IE和UL83蛋白,而MEF则无明显表达。以上结果表明,HC—MV不能在MEF中复制并产生完整子代病毒颗粒,且病毒基因表达阻止在IE2基因表达之后和UL84基因表达之前,其种属特异性可能与即刻早期蛋白低水平的表达量有关。     

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.     

Integrating actin dynamics,mechanotransduction and integrin activation: The multiple functions of actin binding proteins in focal adhesions

Focal adhesions are clusters of integrin transmembrane receptors that mechanically couple the extracellular matrix to the actin cytoskeleton during cell migration. Focal adhesions sense and respond to variations in force transmission along a chain of protein-protein interactions linking successively actin filaments, actin binding proteins, integrins and the extracellular matrix to adapt cell-matrix adhesion to the composition and mechanical properties of the extracellular matrix. This review focuses on the molecular mechanisms by which actin binding proteins integrate actin dynamics, mechanotransduction and integrin activation to control force transmission in focal adhesions.     

Functional interaction of nuclear domain 10 and its components with cytomegalovirus after infections: cross-species host cells versus native cells

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection.     

Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα.     

HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的组装

本文利用Bac-to-Bac杆状病毒表达系统构建了含有丙型肝炎病毒(Hepatitis C Virus,HCV)结构蛋白编码基因的重组杆状病毒vAcHCVspl,并获得了HCV结构蛋白在昆虫细胞Sf21中的表达。HCV mRNA转录和蛋白质表达时相分析表明,感染后16h HCV结构蛋白编码基因开始转录,72h达最高峰;蛋白质表达则是在感染后48h开始,72h达到高峰。电镜观察表明vAcHCVspl感染的Sf21细胞96h时在细胞质中可见很多空泡,空泡中可见50nm的球形颗粒,为HCV结构蛋白组装的病毒样颗粒。     

HCMV感染对胶质瘤细胞U87自噬的影响

研究人巨细胞病毒(HCMV)感染对神经胶质瘤U87细胞自噬的影响。通过观察微管相关蛋白1轻链3(LC3)、自噬相关基因Beclin1及其蛋白表达的变化,从而探讨HCMV与神经胶质瘤发生、发展的关系及意义。用HCMV AD169(MOI=5)感染神经胶质瘤U87细胞,同时将未感染HCMV的U87细胞作为对照组。分别在6、12、24、48 h用RT-PCR检测Beclin1的表达,Western-blot和免疫荧光检测Beclin1和LC3编码蛋白的表达,最后用CCK-8检测细胞的增殖活性。结果显示,HCMV感染的U87细胞LC3-II蛋白表达水平逐渐下降(P<0.05);同时,HCMV感染的U87细胞Beclin1基因及蛋白的表达水平也逐渐下降(P<0.01),且HCMV感染U87细胞增殖显著(P<0.01)。以上结果表明,HCMV感染抑制胶质瘤U87细胞自噬,并会引起Beclin1表达水平下调,进而导致胶质瘤细胞增殖。