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.     

Putative terminase subunits of herpes simplex virus 1 form a complex in the cytoplasm and interact with portal protein in the nucleus

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocates DNA through the portal vertex in the capsid during DNA packaging. The HSV terminase is believed to consist of the UL15, UL28, and UL33 gene products (pUL15, pUL28, and pUL33, respectively), whereas the HSV type 1 portal vertex is encoded by UL6. Immunoprecipitation reactions revealed that pUL15, pUL28, and pUL33 interact in cytoplasmic and nuclear lysates. Deletion of a canonical nuclear localization signal (NLS) from pUL15 generated a dominant-negative protein that, when expressed in an engineered cell line, decreased the replication of wild-type virus up to 80-fold. When engineered into the genome of recombinant HSV, this mutation did not interfere with the coimmunoprecipitation of pUL15, pUL28, and pUL33 from cytoplasmic lysates of infected cells but prevented viral replication, most nuclear import of both pUL15 and pUL28, and coimmunoprecipitation of pUL15, pUL28, and pUL33 from nuclear lysates. When the pUL15/pUL28 interaction was reduced in infected cells by the truncation of the C terminus of pUL28, pUL28 remained in the cytoplasm. Whether putative terminase components localized in the nucleus or cytoplasm, pUL6 localized in infected cell nuclei, as viewed by indirect immunofluorescence. The finding that the portal and terminase do eventually interact was supported by the observation that pUL6 coimmunoprecipitated strongly with pUL15 and weakly with pUL28 from extracts of infected cells in 1.0 M NaCl. These data are consistent with the hypothesis that the pUL15/pUL28/pUL33 complex forms in the cytoplasm and that an NLS in pUL15 is used to import the complex into the nucleus where at least pUL15 and pUL28 interact with the portal to mediate DNA packaging.     

Interaction of the putative human cytomegalovirus portal protein pUL104 with the large terminase subunit pUL56 and its inhibition by benzimidazole-D-ribonucleosides

Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-D-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.     

The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid

During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 +/- 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 +/- 1.1 and 5.0 +/- 0.7 nm, respectively, and a height of 19.5 +/- 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as phi29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.     

A "coiled-coil" motif is important for oligomerization and DNA binding properties of human cytomegalovirus protein UL77

Human cytomegalovirus (HCMV) UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM) at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM) implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE) was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0 ± 0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2 ± 0.41 and 4.9 ± 0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i) could form homodimers, (ii) CCM of pUL77 is crucial for oligomerization and (iii) could bind to dsDNA in a sequence independent manner.     

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28

The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.     

Interactions between proteins encoded within the human cytomegalovirus UL133-UL138 locus

We previously described a novel genetic locus within the ULb' region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as the UL133-UL138 locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of the UL133-UL138 locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by the UL133-UL138 locus and demonstrate an important interaction impacting the outcome of HCMV infection.     

Herpes simplex virus 1 DNA packaging proteins encoded by UL6, UL15, UL17, UL28, and UL33 are located on the external surface of the viral capsid

Studies to localize the herpes simplex virus 1 portal protein encoded by UL6, the putative terminase components encoded by UL15, UL 28, and UL33, the minor capsid proteins encoded by UL17, and the major scaffold protein ICP35 were conducted. ICP35 in B capsids was more resistant to trypsin digestion of intact capsids than pUL6, pUL15, pUL17, pUL28, or pUL33. ICP35 required sectioning of otherwise intact embedded capsids for immunoreactivity, whereas embedding and/or sectioning decreased the immunoreactivities of pUL6, pUL17, pUL28, and pUL33. Epitopes of pUL15 were recognized roughly equally well in both sectioned and unsectioned capsids. These data indicate that pUL6, pUL17, pUL28, pUL33, and at least some portion of pUL15 are located at the external surface of the capsid.     

ATPase activity of the terminase subunit pUL56 of human cytomegalovirus.

Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity.     

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.     

Differing roles of inner tegument proteins pUL36 and pUL37 during entry of herpes simplex virus type 1

Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17⁺ did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.     

伪狂犬病病毒ul24基因表达蛋白的胞内定位研究

根据GenBank已发表的PrVul24基因序列(NC006151),设计并合成一对引物,PCR扩增出ul24基因编码区,克隆于pEGFP-N1载体,得到重组质粒pUL24-GFP。酶切鉴定,测序及WesternBlot验证重组质粒。ul24基因序列测定结果已提交GenBank,登录号DQ226544。Westernblot分析结果表明UL24-GFP融合蛋白为45KD。将pUL24-GFP转染真核细胞,激光共聚焦显微镜观察融合蛋白的细胞内定位,结果表明UL24-GFP融合蛋白定位于细胞核。     

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.     

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.     

Cytomegalovirus pUL96 is critical for the stability of pp150-associated nucleocapsids

Maturation of human cytomegalovirus (HCMV) initiates with nucleocapsids that egress from the nucleus and associate with a juxtanuclear cytoplasmic assembly compartment, where virion envelopment and release are orchestrated. Betaherpesvirus conserved proteins pp150 (encoded by UL32) and pUL96 are critical for HCMV growth in cell culture. pp150 is a capsid-proximal tegument protein that preserves the integrity of nucleocapsids during maturation. pUL96, although expressed as an early protein, acts late during virus maturation, similar to pp150, based on the comparable antigen distribution in UL96, UL32, or UL96/UL32 dual mutant virus-infected cells. pp150 associates with nuclear capsids prior to DNA encapsidation, whereas both pp150 and pUL96 associate with extracellular virus, suggesting that pUL96 is added after pp150. In the absence of pUL96, capsid egress from the nucleus continues; however, unlike wild-type virus infection, pp150 accumulates in the nuclear, as well as in the cytoplasmic, compartment. Ultrastructural evaluation of a UL96 conditional mutant revealed intact nuclear stages but aberrant nucleocapsids accumulating in the cytoplasm comparable to the known phenotype of UL32 mutant virus. In summary, pUL96 preserves the integrity of pp150-associated nucleocapsids during translocation from the nucleus to the cytoplasm.     

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.