An Endothelial Cell-Specific Requirement for the UL133-UL138 Locus of Human Cytomegalovirus for Efficient Virus Maturation

Human cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb′ region of the HCMV genome as important for the latent infection in CD34⁺ hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138_NULL virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138_NULL virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138_NULL virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138_NULL virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138_NULL virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation.     

Protein pUL128 of human cytomegalovirus is necessary for monocyte infection and blocking of migration

We have previously shown that only endotheliotropic strains of human cytomegalovirus (HCMV), such as TB40E, infect monocytes and impair their chemokine-driven migration. The proteins encoded by the UL128-131A region (UL128, UL130, and UL131A) of the HCMV genome, which assemble into a pentameric gH-gL-UL128-UL130-UL131A envelope complex, have been recognized as determinants for HCMV endothelial cell tropism. The genes for these proteins are typically inactivated by mutations in all fibroblast-adapted strains that have lost the diversified tropism of clinical isolates. By using mutant HCMV reconstituted from TB40E-derived bacterial artificial chromosomes (BAC) encoding a wild-type (wt) or mutated form of UL128, we show here that UL128-131A products are essential determinants of infection in monocytes and that pUL128, in particular, can block chemokine-driven motility. The virus BAC4, encoding wt UL128, established infection in monocytes, induced the intracellular retention of several chemokine receptors, and rendered monocytes unresponsive to different chemokines. In contrast, the virus BAC1, encoding a mutated UL128, failed to infect monocytes and to downregulate chemokine receptors. BAC1-exposed monocytes did not express immediate-early (IE) products, retained virions in cytoplasmic vesicles, and exhibited normal chemokine responsiveness. A potential role of second-site mutations in the observed phenotype was excluded by using the revertant viruses BAC1rep and BAC4mut. By incubating noninfected monocytes with soluble recombinant pUL128, we observed both the block of migration and the chemokine receptor internalization. We propose that among the gH-gL-UL128-UL130-UL131A complex subunits, the UL128 protein is the one that triggers monocyte paralysis.     

利用酵母双杂交系统筛选与人巨细胞病毒皮层蛋白pUL23相互作用的病毒编码蛋白

人巨细胞病毒(HCMV) UL23基因编码病毒皮层蛋白,该基因缺失时,病毒在人包皮成纤维细胞(HFF)中的繁殖速度加快.为进一步阐述HCMV UL23基因编码产物 pUL23的功能及调控机制,采用鸟枪法构建了融合于GAL4活性区域的HCMV Towne株 基因组随机表达文库.利用酵母双杂交技术,以pGBKT7 -UL23为诱饵质粒,从构建 的HCMV基因组表达文库中筛选到与pUL23相互作用的病毒编码蛋白pUL24. GST-pull down实验和免疫共沉淀实验进一步确认两种病毒蛋白之间的相互作用.结果 表明,构建的HCMV基因组表达文库能够用于GAL4酵母双杂交系统筛选与诱饵蛋白相互作用的病毒自身编码蛋白.病毒蛋白pUL23和pUL24之间具有相互作用,这为进一 步阐述pUL23在HCMV感染过程中的功能提供依据.该研究为揭示HCMV病毒感染机制奠定了基础.     

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.     

人巨细胞病毒UL55编码蛋白结合蛋白的筛选与鉴定

从人胎脑c DNA文库中筛选和鉴定出与人巨细胞病毒(Human cytomegalovirus,HCMV)UL55编码蛋白结合的蛋白。将UL55基因编码区克隆到诱饵载体p GBKT7中,在证实UL55蛋白不具有自激活作用的前提下,采用Match-maker GAL酵母双杂交系统筛选人胎脑c DNA文库中与UL55蛋白结合的宿主蛋白,用酵母双杂交回转实验验证UL55蛋白与获得的蛋白结合的可靠性。将酵母双杂交筛选出的文库蛋白烯醇化酶1(enolase1,ENO 1)构建到p GEX-4T-2载体上,利用GST pull-down技术体外验证ENO 1与HCMV UL55蛋白的结合。并依据所筛选出蛋白的生物学功能分析UL55蛋白可能的生物学功能。结果显示有10种蛋白与HCMV UL55编码蛋白结合。应用GST pull-down技术检测到ENO 1与HCMV UL55相互结合的蛋白条带。成功地筛选出10种与UL55蛋白相互结合的宿主蛋白,GST pull-down实验进一步表明ENO 1可以与HCMV UL55蛋白直接结合,为进一步研究UL55蛋白的功能提供了新的线索。     

Determination of interactions between tegument proteins of herpes simplex virus type 1

The aim of this study was to elucidate protein-protein interactions between tegument proteins of herpes simplex virus type 1 (HSV-1). To do so, we have cloned and expressed in the LexA yeast (Saccharomyces cerevisiae) two-hybrid system, 13 of the 21 currently known tegument proteins of HSV-1. These included the tegument proteins essential for replication in cell lines, UL17, UL36, UL37, UL48, and UL49, and the nonessential tegument proteins US11, UL11, UL14, UL16, UL21, UL41, UL46, and UL47. A total of 104 combinations were screened in the yeast two-hybrid assay, with 9 interactions identified. These included: UL11-UL16, UL36-UL37, UL36-UL48, UL46-UL48, UL47-UL48, and UL48-UL49. The remaining interactions consisted of self-associations that were observed for US11, UL37, and UL49. The interactions UL36-UL37, UL36-UL48, UL37-UL37, UL46-UL48, and UL47-UL48 have not been previously reported for HSV-1. The interaction of UL46-UL48 was verified using an in vitro pull-down assay. The interactions of UL36-UL37 and UL37-UL37 were verified with a coimmunoprecipitation assay. Knowledge of HSV-1 tegument protein-protein interactions will provide insights into the pathways of tegument assembly, and the identified interactions are potential targets for new antiviral drugs.     

The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.     

Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.     

Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.     

Cyclin A Degradation by Primate Cytomegalovirus Protein pUL21a Counters Its Innate Restriction of Virus Replication

Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV) can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC), but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL) cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus'' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.     

The human cytomegalovirus-specific UL1 gene encodes a late-phase glycoprotein incorporated in the virion envelope

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.     

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.