The tegument protein UL94 of human cytomegalovirus as a binding partner for tegument protein pp28 identified by intracellular imaging

The tegument protein pp28 of human cytomegalovirus (HCMV) is essential for the assembly of infectious HCMV virions, but how it functions during the process of HCMV tegumentation and envelopment remains unclear. By using live cell fluorescence resonance energy transfer (FRET) microscopy and yeast two-hybrid assays, we found that another HCMV tegument protein, UL94, was a specific binding partner for pp28. The interaction between pp28 and UL94 was imaged in a punctuate, juxtanuclear compartment, previously designated as the virus assembly compartment (AC). Amino acids 22-43 of pp28 were identified as being responsible for its binding with UL94, while no linear binding site could be found within UL94. The interaction between pp28 and UL94 may serve as a link in the sequential processes of HCMV capsidation, tegumentation and envelopment. This study provides a foundation for further studies into how the HCMV tegument proteins act in the assembly of HCMV virions.     

Immunological analysis of the tegument phosphoprotein ppUL83 of human cytomegalovirus

Immunological properties of the tegument phosphoprotein, ppUL83, of human cytomegalovirus (HCMV), expressed using a replication deficient recombinant adenovirus vector (RAd83) are described. The initial characterisation of this protein was carried out by immunofluorescence (IF), immunoprecipitation (RIP) and immunoblotting using nine mouse monoclonal antibodies (Mabs) directed against five linear and four conformational epitopes of ppUL83. The reactivity of the recombinant protein with the Mabs was similar to that observed with native ppUL83, although, the kinetics of its expression was in agreement with expression derived from the HCMV major immediate early promoter (MIEP). The recombinant antigen was used successfully in an Enzyme Immunoassay (EIA) for the detection of IgG class antibodies in 171 sequential sera taken from 21 heart transplant recipients. Comparison of HCMV-infected and RAd83-infected cell extracts in this experiment showed that recombinant antigen could substitute whole virus extracts as a single well-characterised protein in EIA. Serum IgG avidity measurements, using the recombinant ppUL83, differentiated between primary and past HCMV infections in the population studied.     

人巨细胞病毒皮层蛋白pUL23与宿主蛋白IGFBP4相互作用的鉴定

目的:利用蛋白质相互作用的技术筛选与pUL23相互作用宿主蛋白,为研究pUL23蛋白对人巨细胞病毒繁殖的影响提供线索。方法:通过酵母双杂交系统从人胚肾cDNA文库筛选与pUL23相互作用的宿主蛋白;通过GST-pu ll-down技术研究二者体外物理性相互作用;免疫共沉淀技术进一步研究二者在胞内相互作用。结果:Pu ll-down技术、免疫共沉淀技术确定了宿主蛋白IGFBP4与pUL23具有相互作用。结论:上述结果为研究pUL23蛋白调节病毒自身繁殖功能提供重要依据。     

人疱疹病毒7型被膜蛋白pp85的原核表达及应用

目的构建人疱疹病毒7型(HHV7)被膜蛋白pp85编码基因(ORF U14)的原核表达载体,并进行原核表达。方法应用HHV7标准株Glasgow感染SupT1细胞,PCR技术扩增HHV7ORFU14基因的主要抗原决定簇编码区(328~533AA),目的基因与原核表达载体pThioHis A连接后,转化宿主菌E.coliBL21,IPTG诱导融合蛋白表达,经镍-螯合物琼脂糖树脂柱亲和层析纯化获得重组抗原。重组蛋白电泳后转至PVDF膜,与HHV7阳性血清进行免疫印迹反应。结果DNA序列分析表明,目的基因序列与HHV7标准毒株Glasgow相应序列完全一致,SDS-PAGE可观察到相对分子质量(Mr)为35.7×103融合蛋白的表达,免疫印迹反应显示重组抗原具有较高的特异性。结论HHV7重组抗原具有较好的抗原性,进一步完善后可用于HHV7抗体的检测。     

Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1

Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ((51)RRPR(54)) or pat7 ((48)PRVRRPR(54)) NLS2 abrogated nuclear accumulation, whereas deletion of (48)PRV(50) did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ((11)RRPRR(15)), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids (485)LSAYLTLFVAL(495). This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm.     

Detection of human cytomegalovirus (HCMV) pp67-mRNA and pp65 antigenemia in relation to development of clinical HCMV disease in renal transplant recipients

Objective   To evaluate the performance of the recently introduced method based on detection of human cytomegalovirus (HCMV) pp67 mRNA in blood by the nucleic acid sequence-based amplification (NucliSens), in comparison to semiquantitative detection of pp65 HCMV antigen in white blood cells, in relation to development of clinical HCMV disease.
Methods   Thirty patients, recipients of renal transplants, were monitored prospectively for the presence of pp67 mRNA, the presence and level of pp65 antigenemia, IgG and IgM antibodies, and the development of clinical HCMV disease. A total of 148 samples were examined during the observation period.
Results   Twenty-five samples were positive for pp67-mRNA and 45 samples contained at least one pp65 positive cell, with 68% agreement between the two assays. Both assays predicted correctly the development of clinical disease in five patients, giving a sensitivity of 100%. However, the specificity of the pp67-mRNA test was 72%, and of the pp65 antigenemia test from 20 to 64%, depending on the level of antigenemia chosen for cut-off. pp67-RNA appeared somewhat earlier than pp65 antigenemia, and responded earlier to treatment. Sero-conversion and appearance of IgM antibodies were of very little clinical value.
Conclusion   Both the pp67-mRNA and the pp65 antigenemia assay predicted correctly the development of clinical HCMV disease in renal transplant recipients. However, the specificity of both tests with respect to development of HCMV disease, especially the pp65 antigen test was moderate. Significantly positive tests not necessarily prove the development of clinical disease. Testing for pp67-mRNA may improve the diagnosis and management of HCMV disease in renal transplant patients.     

Proteasome-dependent, ubiquitin-independent degradation of Daxx by the viral pp71 protein in human cytomegalovirus-infected cells

The cellular Daxx protein represses human cytomegalovirus (HCMV) gene expression from the major immediate early promoter. HCMV prevents Daxx-mediated silencing during lytic infection by delivering the viral pp71 tegument protein to the nucleus, where pp71 binds to and induces the proteasomal degradation of Daxx. In this study, we show that a functional ubiquitin pathway is not required for the proteasomal degradation of the endogenous Daxx protein by tegument-delivered pp71 in HCMV-infected cells, demonstrating that the pp71-mediated degradation of Daxx occurs through a proteasome-dependent, ubiquitin-independent pathway.     

HCMVpp65抗原、HCMV mRNA和IgM抗体检测在诊断HCMV活动性感染中的比较

目的比较人巨细胞病毒（HCMV）pp65抗原、HCMV mRNA和血清HCMV—IgM抗体检测3种方法在诊断HCMV活动性感染中的实用意义。方法采集医院TORCH检查HCMV—IgM阳性的病人外周血（60份）。将标本分2份2ml和3ml,别用于HCMV mRNA和pp65检测。将三者结果进行比较。结果pp65抗原检测的结果与IgM抗体检测的阳性符合率为81．67％。与HCMV mRNA检测相比pp65抗原检测法的符合率、特异度和敏感度分别为81．67％,81．81％和81．63％。而且高pp65抗原血症与患者的临床症状密切相关。结论pp65抗原血症反映该病毒活动状况,可监测HCMV活动性感染,联合HCMV—IgM的检测可以提高临床的诊断率并可用于指导临床用药及监测药物疗效。     

Residues F593 and E596 of HSV-1 tegument protein pUL36 (VP1/2) mediate binding of tegument protein pUL37

The herpes simplex virus type 1 (HSV-1) structural tegument proteins pUL36 (VP1/2) and pUL37 are essential for secondary envelopment during the egress of viral particles. Our laboratory has previously shown that HSV-1 pUL36(512-767) fragment interacts with full-length pUL37. A number of single and double amino acid changes of conserved residues in the pUL36(512-767) fragment were generated using alanine-scanning site-directed mutagenesis. The interaction of pUL36(512-767) and pUL37 was then assessed using a combination of yeast two-hybrid and coimmunoprecipitation assays. Single changes to alanine of pUL36 residues F593 and E596 impaired binding of pUL37 with the greatest effect observed for the substitution E596A. Double mutations involving either of these residues in combination with the substitution E580A essentially blocked binding of pUL37. This information will provide the basis for generation of viral mutants to further define the importance of the pUL36/pUL37 interaction in assembly of HSV-1.     

Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44

The processivity factor of human cytomegalovirus DNA polymerase, phosphoprotein ppUL44, is essential for viral replication. During viral infection ppUL44 is phosphorylated by the viral kinase pUL97, but neither the target residues on ppUL44 nor the effect of phosphorylation on ppUL44's activity are known. We report here that ppUL44 is phosphorylated when transiently expressed in mammalian cells and coimmunoprecipitates with cellular kinases. Of three potential phosphorylation sites (S413, S415, S418) located upstream of ppUL44's nuclear localization signal (NLS) and one (T427) within the NLS itself, protein kinase CK2 (CK2) specifically phosphorylates S413, to trigger a cascade of phosphorylation of S418 and S415 by CK1 and CK2, respectively. Negative charge at the CK2/CK1 target serine residues facilitates optimal nuclear accumulation of ppUL44, whereas negative charge on T427, a potential cyclin-dependent 1 phosphorylation site, strongly decreases nuclear accumulation. Thus, nuclear transport of ppUL44 is finely tuned during viral infection through complex phosphorylation events.     

Expression of tegument protein pp65 of human cytomegalovirus (CMV) and its application to the analysis of viral-specific cellular immunity in CMV-infected individuals

Summary.  Investigations into human cytomegalovirus (CMV)-specific cellular immunity are important to better understand and manage CMV infections. CMV phosphoprotein pp65 is thought to be a major antigen for CMV-specific cellular immunity. We newly synthesized protein pp65 with a baculovirus expression system and purified it via metal affinity chromatography in a soluble form. The recombinant protein pp65 was antigenic in an enzyme immuno-linked assay for pp65-specific IgG in sera from 196 children. Traditional lymphoproliferation assays have shown that pp65 protein promotes specific lymphoproliferation in CMV-seropositive donors. Using an intracellular cytokine detection system, we showed that this recombinant protein stimulated CD4-positive T cells to express interferon-γ. The results of these assays using protein pp65 were comparable with the use of CMV whole antigen. pp65- and CMV-specific cellular immunity, and CMV DNA load were also compared in four recipients of unrelated cord blood transplantation. The delay in re-constitution in CMV-specific cellular immunity was associated with reactivation of CMV. These results indicated that the recombinant protein pp65 can be used to study specific immunity in CMV infections. Received March 1, 2002; accepted May 28, 2002     

Circulating cytomegalic endothelial cells are associated with high human cytomegalovirus (HCMV) load in AIDS patients with late-stage disseminated HCMV disease

The prevalence of circulating cytomegalic endothelial cells, detected currently by the pp65-antigenemia assay and described previously in blood of transplanted and AIDS patients with disseminated human cytomegalovirus (HCMV) infection, was found to be 2.9% in the AIDS population and 6.5% in the fraction of the AIDS population with HCMV in blood. Cytomegalic endothelial cells increased to 39.7% and 48.4%, respectively, in AIDS patients with very high levels of antigenemia and viremia, while an end organ disease reached an incidence of 76.4%. Positive and negative predictive values of cytomegalic endothelial cell detection for diagnosis of HCMV end organ disease were 73.1% and 21.4% with antigenemia levels >1,000, respectively. On the other hand, in a selected group of 38 cytomegalic endothelial cell-positive AIDS patients with <50 CD4+ T cells/μl and late-stage HCMV disease, who were followed-up for variable periods of time, the prevalence of high level antigenemia was 95.3%, that of viremia 86.0% and that of L-DNAemia 92.7%, while the incidence of HCMV end organ disease was 84.2%. In this population, it was shown that cytomegalic endothelial cell presence was associated with lack of (56.0% of episodes) or insufficient (4.0%) anti-HCMV treatment or emergence of HCMV drug-resistant strains (17.3%) or short-term response to antiviral treatment (22.7%); was determined in the same patient by different conditions during follow-up. Longitudinal observations indicated that cytomegalic endothelial cells were detected often in blood at least 3 months later than end organ disease suggesting that the duration of end organ disease was a cofactor associated with the appearance of cytomegalic endothelial cells. J. Med. Virol. 55:64–74, 1998. © 1998 Wiley-Liss, Inc.     

Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35

The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.     

Sites and roles of phosphorylation of the human cytomegalovirus DNA polymerase subunit UL44

The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides. Viruses containing alanine substitutions of most UL44 residues that are phosphorylated in infected cells exhibited at most modest effects on viral DNA synthesis and yield. However, substitution of highly phosphorylated sites adjacent to the nuclear localization signal abolished viral replication. The results taken together are consistent with UL44 being phosphorylated directly by UL97 during infection, and a crucial role for phosphorylation-mediated nuclear localization of UL44 for viral replication, but lend little support to the widely held hypothesis that UL97-mediated phosphorylation of UL44 is crucial for viral DNA synthesis.     

人巨细胞病毒pp65核酸疫苗的构建、表达与动物免疫效应

构建真核重组表达质粒PVAX1-pp65,通过对其表达产物的鉴定和免疫小鼠实验,探讨PVAX1-pp65载体对诱导免疫应答的效果及方式。用已构建的原核表达载体pp65-pet32a,经酶切后与DNA疫苗载体PVAX1连接,构建HCMVpp65核酸疫苗(PVAX1-pp65)。用脂质体法将其转染293细胞,经间接免疫荧光、免疫印迹试验来验证其表达的产物。同时,免疫小鼠后取其脾脏细胞,经流式细胞仪检测其CD4+和CD8+T细胞;并利用MTT法测定免疫小鼠的T细胞对ConA和重组pp65蛋白刺激后的增殖活性。结果显示构建的真核表达载体PVAX1-pp65,经测序验证其序列正确。体外转染实验结果表明转染重组质粒的细胞胞浆内呈现与特异性pp65单克隆抗体反应的颗粒状荧光产物,免疫印迹试验也显示重组质粒转染的细胞裂解物中有pp65蛋白条带。另外,免疫小鼠脾CD8+T细胞的含量明显高于对照组;免疫鼠脾细胞对重组pp65抗原蛋白的刺激后增殖反应明显。综上结果证实,成功地构建了HCMVpp65核酸疫苗,并能有效地表达,表达的蛋白具有良好的免疫原性和免疫反应性。HCMVpp65DNA疫苗可诱导小鼠产生针对HCMV的特异性细胞免疫应答,可作为一种有应用前景的核酸疫苗进一步深入研究。     

Procaryotic expression of phosphorylated tegument protein pp65 of human cytomegalovirus and application of recombinant peptides for immunoblot analyses.

The tegument of human cytomegalovirus (HCMV) contains a phosphorylated protein of 65 kilodaltons, termed pp65, which was reported to carry significant epitopes for the stimulation of the humoral immune response during natural infection. A monoclonal antibody directed against this protein was used to screen a lambda gt11 cDNA library for recombinant polypeptides. Two DNA fragments from purified lambda clones and one fragment from genomic DNA were used for cloning in a bacterial high-level expression vector. The resulting fusion proteins were tested for their reactivity with a panel of monoclonal antibodies directed against pp65 and with polyspecific anti-HCMV rabbit antisera. The binding site for all the monoclonal antibodies tested was found to be contained in one of the recombinant proteins with a viral portion of 26 amino acids. Immunoblot analyses with HCMV-positive human sera revealed that pp65 alone is not a reliable antigen for serodiagnosis but may be very useful in combination with other HCMV proteins.     

Visualization of the dynamic multimerization of human Cytomegalovirus pp65 in punctuate nuclear foci

The phosphorylated protein pp65 of human Cytomegalovirus (HCMV) is the predominant virion protein and the major tegument constituent. It plays important roles in HCMV infection and virion assembly. Live cell imaging and fluorescence recovery after photobleaching (FRAP) analysis showed that HCMV pp65 accumulated dynamically in punctuate nuclear foci when transiently expressed in mammalian cells. Fluorescence resonance energy transfer (FRET) imaging disclosed that pp65 can self-interact in its localization foci. Yeast two-hybrid assay verified that pp65 is a self-associating protein, and the N-terminal amino acids 14–22 were determined to be essential for pp65 self-association. However, these amino acids were not related to pp65 localization in the specific nuclear foci. The interaction of pp65 and ppUL97 was also studied by FRET microscopy, and the result suggested that there is another signal sequence in pp65, being the ppUL97 phosphorylation site, that is responsible for localization of pp65 in nuclear foci. These results help to understand the function of pp65 in HCMV infection and virion morphogenesis.     

Identification of a monoclonal antibody from Peromyscus maniculatus (deer mouse) cytomegalovirus (PCMV) which binds to a protein with homology to the human CMV matrix protein HCMV pp71

In this study we identified and characterized a monoclonal antibody against the matrix protein of a cytomegalovirus isolated from the common deer mouse (Peromyscus maniculatus) (PCMV). The monoclonal antibody was isolated using previously described technology which could be applied to the production of monoclonal antibodies against zoonotic disease. The antibody was found to react with a protein homologous to the human cytomegalovirus (HCMV) matrix protein (pp71), the product of the UL82 open reading frame (ORF). mAbs were generated from heterologous fusion of spleen cells from PCMV-positive mice and Balb/C P3X63-Ag8.653 myeloma cells. Using this approach, four monoclonal antibodies: B8C4, C12E8, G6A2 and P4E5 were generated. Antibody G6A2 reacted strongly with PCMV-infected cells as well as purified virions on ELISA and immunofluorescence. Western blot analysis, using sucrose gradient-purified virions, demonstrated that this mAb reacted specifically to a single protein with an apparent molecular weight of 71 kDa. The protein band was excised from the gel, purified and subjected to trypsin digestion followed by mass spectrometry. The protein sequences obtained were found to have identity to HCMV UL82 gene product. Sequence analysis indicated that it is the putative HCMV pp71 protein homolog of PCMV. G6A2 mAb did not cross-react with either human or murine recombinant pp71 proteins expressed in mammalian cells.     

Antibody response to human cytomegalovirus (HCMV) glycoprotein B (gB) in AIDS patients with HCMV end-organ disease

Human cytomegalovirus (HCMV)-specific antibody responses in HIV-1 infected individuals either with or without HCMV end-organ disease were examined to determine the whether development of HCMV disease was associated with a particular deficit in the antibody response. Anti-whole HCMV, anti-glycoprotein B (gB), and neutralizing antibody levels were higher in HIV-1 infected individuals than in healthy immunocompetent subjects, particularly in patients with AIDS either with or without HCMV-associated disease. Irrespective of location and spread of HCMV disease, patients who had received anti-HCMV therapy prior to sampling exhibited significantly higher anti-gB and neutralizing antibody titers than those who remained untreated. Likewise, patients with HCMV disease who were antigenemic or viremic had significantly lower anti-gB and neutralizing antibody titers than those who tested negative in either assay. Patients with untreated HCMV disease had significantly lower antibody titers than AIDS patients without disease. Analysis of the IgG subclass antibody responses to gB revealed no significant differences among HIV-1 infected individuals. These results suggest that levels of detectable anti-gB and HCMV neutralizing antibodies are inversely related to systemic viral load. Thus, antibodies with such specificities may be relevant in preventing the establishment of HCMV-associated disease or in modulating its progression. J. Med. Virol. 55:272–280, 1998. © 1998 Wiley-Liss, Inc.     

人巨细胞病毒感染对宿主细胞周期蛋白表达影响的实验研究

目的观察人巨细胞病毒（HCMV）感染对宿主细胞周期蛋白（Cyclins）表达的影响。方法用HCMV感染同步化于G0/G1期的人胚肺成纤维细胞（HEL）,分别于感染后0 h、3 h、6 h、12 h、24 h、48 h、72 h、96 h终止培养。用免疫蛋白印迹法（Western Blot）检测CyclinE、CyclinA、CyclinD1蛋白的表达。结果HCMV感染接触抑制细胞12 h时CyclinE蛋白被诱导,感染后24 h出现CyclinE峰值;HCMV不能诱导CyclinA蛋白表达;CyclinD1没有被诱导,且在感染后24 h开始下降。结论HCMV感染同步化于G0/G1期的细胞后,诱导CyclinE蛋白明显升高,激活CyclinE/Cdk2激酶,使细胞周期越过G1/S限制点,将细胞周期阻止于晚G1期。