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.     

Tet-On3G系统调控人巨细胞病毒蛋白pUL23稳定表达的人胚肺成纤维细胞(HELF)系的建立

人巨细胞病毒(Human cytomegalovirus,HCMV)为疱疹病毒家族一员,易导致免疫力低下或缺陷的人群严重疾病,目前对HCMV编码的皮层蛋白pUL23功能的相关报道很少。本研究应用Tet-On3G诱导表达系统,在人胚肺成纤维细胞(Human embryonic lung fibroblast,HELF)建立诱导表达HCMV病毒蛋白pUL23细胞模型。将病毒基因UL23和阳性对照基因EGFP分别定向插入应答慢病毒载体pLVX-TRE3G中,获得pLVX-TRE3GUL23-3×Flag、pLVX-TRE3G-EGFP重组慢病毒载体。运用二代慢病毒包装系统与Lenti-X293T包装细胞系,制备收获慢病毒后感染人胚肺成纤维细胞HELF。遗传霉素(Geneticin,G418)和嘌呤霉素(Puromycin,Puro)抗性筛选后多西环素(Doxycycline,Dox)诱导外源基因表达。感染后的细胞在96孔板有限稀释法成单克隆,分别采用RT-PCR与Western blot技术检测病毒UL23基因在mRNA水平与蛋白水平的表达量,筛选出当诱导时高效表达且未诱导时低表达的单克隆细胞;并评估Dox诱导剂量与诱导时间对外源蛋白pUL23表达的影响。限制性内切酶与测序显示重组质粒pLVX-TRE3G-UL23-3×Flag、pLVX-TRE3G-EGFP序列和方向正确。病毒蛋白pUL23在感染细胞中能够被诱导表达;当Dox浓度高于400ng/mL时pUL23蛋白表达量不再随Dox剂量增高而增高。在Dox诱导2h后,RT-PCR结果表明在921bp处有特定条带(UL23-3×Flag基因)与此同时,Western blot实验也检测到pUL23蛋白。这些结果表明,成功构建重组慢病毒载体,包装成慢病毒感染后,病毒基因UL23能够在人胚肺成纤维细胞中诱导表达。Dox的最佳工作浓度为400ng/mL,最佳诱导时间为12h至24h之间。这将为进一步研究病毒蛋白pU23的功能奠定基础。     

人巨细胞病毒编码蛋白pUL23在E.coli BL21(DE3)中的表达和纯化

大肠杆菌中高效表达携带组氨酸标签的人巨细胞病毒皮层蛋白pUL23,并进行纯化以及鉴定.提取感染HCMV Towne病毒株的HFF细胞的总RNA,逆转录为cDNA作为模板,经PCR获得UL23的基因片段,将此片段插入表达载体pET-28a(+),构建pET28a(+)-UL23重组质粒.将pET28a(+)-UL23转化至大肠杆菌BL21( DE3),进行IPTG诱导表达.表达产物经Western blotting分析后进行发酵,再用Ni sepharose亲和层析纯化,纯化产物进行SDS-PAGE和Western blotting检测.结果表明,成功构建pET28a(+)-UL23原核表达载体,表达及纯化了His-pUL23融合蛋白.为进一步研究pUL23奠定基础.     

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。     

人巨细胞病毒IE1蛋白功能研究进展

人巨细胞病毒(HCMV)在人群中多数呈隐性感染,但对免疫功能低下者可引起严重的疾病甚至死亡。HC-MV IE1蛋白为一种多功能调节蛋白,是HCMV感染后表达最早且表达量最丰富的蛋白,它可影响病毒和细胞启动子的转录活性,调控HCMV基因的时序性表达,并通过参与不同的信号转导通路,在细胞中发挥促进凋亡或抑制凋亡作用,IE1蛋白与HCMV在体内持续性感染以及肿瘤的形成密切相关。     

用酵母双杂交体系确定病毒特异性蛋白与宿主蛋白的相互作用

用B型流行性感冒病毒BM2蛋白开放阅读框架片段,构建酵母双杂交体系中的"饵”载体,明确其在酵母细胞中的表达,排除自身激活作用,并用酵母双杂交法从人cDNA基因库中筛选出与BM2蛋白相互作用的蛋白.结果表明B型流感病毒BM2蛋白与宿主细胞的某一蛋白有相互作用.应用酵母双杂交体系,可以非常有效地从人cDNA基因库中确定与"饵”蛋白相互作用的蛋白基因.     

基于Red重组系统构建含Flag标签标记UL23基因的重组人巨细胞病毒

利用来源于λ噬菌体的Red系统,将Flag标签及两侧带有FRT位点的卡那霉素抗性基因片段插入原HCMV TowneBAC中UL23基因3 '末端区域,通过卡那抗性筛选带有抗性标记的重组菌株,并通过表达重组酶FLP的质粒pCP20去除卡那霉素抗性基因,得到带有Flag标签标记UL23基因和单一FRT位点的突变BAC.重组后的BAC分子同质粒pcDNA3.1(+)-pUL82共转染HFF细胞后重建重组HCMV.Western blotting检测证实所构建重组病毒能够表达含Flag标签标记的pUL23蛋白.此含有Flag标签标记UL23基因的重组HCMV的成功构建为了进一步研究人巨细胞病毒UL23基因及其产物的功能提供依据.     

人巨细胞病毒的免疫逃避策略

人巨细胞病毒(HCMV)能导致人类的隐性、持续性感染,为了对付宿主免疫所形成的不良环境,HCMV具有一系列不同的防护策略.本文综述了此病毒通过对多种免疫途径进行修饰达到免疫逃避目的的一些研究新进展.     

人巨细胞病毒感染对树突细胞功能的影响

人巨细胞病毒(HCMV)对人多为潜伏感染,但在免疫受损人群中,该病毒感染后会引起严重的后果.人巨细胞病毒感染人体后,可从多条途径干扰宿主细胞的生长、分化,诱导细胞恶变;人巨细胞病毒的IE基因具有抑制凋亡的功能.树突细胞是机体内功能最强的抗原呈递细胞,在免疫应答中发挥重要作用.HCMV感染后,能下调树突细胞表面的主要组织相容性复合物Ⅰ、Ⅱ类分子,CD80,CD86和CD40的表达,也使黏附分子如ICAM-3、ICAM-2等的表达发生改变.由此,人巨细胞病毒逃避了细胞免疫应答,引起持续感染或潜伏感染.     

控制人巨细胞病毒感染的策略

人巨细胞病毒（HCMV）感染对免疫受损人群危害极大,本综述了目前控制HCMV感染的策略,其中包括药物控制和免疫控制。阐述了使用药物控制HCMV感染的效果,疫苗在控制HCMV感染及引发疾病方面的作用及通过被动免疫控制HCMV感染的现状与进展。对这些策略的了解对控制HCMV感染具有重要意义。     

The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host

The human herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), HHV-6B, HHV-7 and HHV-8, establish persistent infections with possible recurrence during immunosuppression. HCMV replication is inhibited by the nucleoside analogue ganciclovir (GCV), the compound of choice for the treatment of HCMV diseases and preemptive treatment of infections. The viral UL97 protein (pUL97) which shares homologies with protein kinases and bacterial phosphotransferases is able to monophosphorylate GCV. Homologues of pUL97 are found in HSV (UL13), VZV (ORF47), EBV (BGLF4), HHV-6 (U69), HHV-8 (ORF36) as well as in murine CMV (M97) or rat CMV (R97). Several indolocarbazoles have been reported to be specific inhibitors of pUL97. The protein is important for efficient replication of the virus. Autophosphorylation of pUL97 was observed using different experimental systems. Most recently, it has been shown that pUL97 interacts with the DNA polymerase processivity factor pUL44. Indolocarbazole protein kinase inhibitors are promising lead compounds for the development of more specific inhibitors of HCMV.     

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.     

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.     

Proteasome-Dependent Disruption of the E3 Ubiquitin Ligase Anaphase-Promoting Complex by HCMV Protein pUL21a

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109–110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity.     

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.     

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.