应用外部引导序列切割人巨细胞病毒UL148 RNA的研究CSCD

目的 研究人巨细胞病毒（Human cytomegalovirus,HCMV）临床病毒株UL/b＆#39;区域UL148基因功能及抗HCMV治疗新策略,应用DNA性质外部引导序列（External guide sequences,EGS）在体外抑制UL148 RNA的表达.方法 应用RNA structure生物软件模拟UL148 RNA二级结构,选择二级结构相对简单的区域设计EGS,并合成EGS核苷酸序列.应用PCR方法分别扩增UL148及M1RNA基因,克隆至T7启动子下游,在T7 RNA聚合酶的作用下,体外转录UL148 RNA及M1RNA,其中UL148以32p标记并进行体外转录.混合EGS、M1RNA及UL148 RNA于切割反应液中,反应1h后,进行尿素变性聚丙烯酰胺凝胶电泳分离,Typhoon扫描仪观察结果.结果 根据UL148 RNA二级结构选取第58 ～72位碱基作为EGS结合区域,其切割位点位于57位,针对此区域设计EGS57,并在体外进行EGS57与UL148 RNA结合的二级结构的模拟,可形成M1RNA天然作用底物类似的茎环结构.在T7 RNA聚合酶的作用下,分别在体外转录M1RNA及UL148 RNA,其大小均与预期相符.经切割反应,可获得与预期切割位点相符的切割条带.结论 EGS57可切割UL148RNA,其切割位点准确,与预期相符.该研究为进一步探讨UL148基因功能提供有效基因沉默工具.     

一种HCV靶向性M1GS核酶的构建及其体外活性

目的构建对HCV基因组具有特异切割作用的新型靶向性核酶-M1GS。方法针对HCV基因组的保守区(5′UTR)设计并合成一段引导序列,通过PCR扩增直接将该引导序列连接于大肠杆菌核糖核酸酶P的催化亚基(M1 RNA)的3′末端,从而构建一类靶向性M1GS核酶。结果体外切割实验表明,所构建的核酶(M1GS-HCV/C67)对HCV5′UTR具有明显的切割作用,切割的位点在靶序列67~68 nt之间,属于特异性切割。结论构建了一种对HCV5′UTR具有靶向切割活性的M1GS核酶,为胞内反义效应及动物模型内抗病毒效应的评价提供了实验材料,为新型抗HCV药物的研究奠定了基础。     

PDGF受体β亚单位2个不同切割位点核酶的体外切割效率的比较

目的研究针对大鼠PDGF受体β亚单位基因2个不同切割位点核酶的体外切割活性并加以比较。方法应用计算机对大鼠PDGF受体β亚单位mRNA二级结构进行模拟,设计针对PDGF受体β亚单位mRNA45位CUU和252位CUU的锤头状(Hammerhead)核酶(Ribozyme)基因RZ1和RZ2,定点克隆于自我切割型核酶载体P1．5的5'-cis核酶和3’-cis核酶之间;将大鼠PDGF受体β亚单位cDNA 608 bp的PCR片段克隆于pGEM-T载体T7启动子下游,在T7启动子作用下分别体外转录成RZ1、RZ2和706nt的PDGF受体β亚单位mRNA,比较RZ1和RZ2对706 nt的PDGF受体β亚单位mRNA的切割活性的差异。结果RZ1在体外有较高的切割活性,而RZ2在体外无切割活性。结论提示在应用核酶技术进行基因治疗时,应选择有效的核酶切割位点,以达到有效的治疗目的。     

人工核酶M1GS结构与功能相互关系的研究

目的：研究核酶M₁GS其二级结构与体外切 割活性之间的关系。方法：以人巨细胞病毒（HCMV）DNA聚合酶基因UL54 为靶基因，构建了人工核酶M₁GS-T₇。通过软件RNA structure对M1GS在3个具有相对 稳定结构的温度（20 ℃、37 ℃、55 ℃）的空间构象进行模拟,然后通过体外切割实验来 检测不同温度下核酶M1GS体外切割活性的变化。为一步研究核酶M₁GS二级结构与体外切 割活性之间的关系，参照温度变化实验结果及RNA二级结构的模拟结果，引入突变位点，构 建了在37 ℃与55 ℃时M₁GS-T₇具有相同二级结构的突变型核酶mM₁GS-T₇，并通 过体外切割实验对两者的活性进行比较。结果：在温度变化实验中，55 ℃核酶的体外切割活性最高。而在突变实验中，37 ℃ mM₁GS-T₇比M₁GS-T₇的活 性略高。结论：具有某种特定二级结构的M₁GS-T₇有相对较高的体外 切割活性，核酶的结构与其功能之间存在一定的对应关系。     

HCV5’UTR靶向性人工M1GS核酶的胞内抗病毒活性

目的基于大肠埃希菌核糖核酸酶P的催化性RNA亚基(M1 RNA),人工构建一种抗丙肝病毒(HCV)的新型靶向性核酶,并进一步鉴定其胞外靶向切割活性及胞内抗病毒作用。方法首先采用计算机软件对HCV基因组保守区———5’非翻译区(5’UTR)的序列与结构进行分析,以确定候选靶位。针对靶位的侧翼序列,设计与之互补的引导序列(GS),然后通过PCR将其共价连接至M1 RNA的3’末端,从而构建靶向性的M1GS核酶。结果针对HCV 5’UTR第67位的胞嘧啶成功构建了一种M1GS核酶———M1GS-HCV/C67。该人工核酶不仅在体外可对靶RNA片段产生特异性切割,在HCV感染的宿主细胞内也能够显著抑制HCV核心蛋白的表达(P<0.05),并使上清液HCV RNA的拷贝数减少约800倍(P<0.01)。结论 M1GS-HCV/C67这种新型靶向性核酶具有良好的体外抗HCV活性,其成功构建为HCV感染的治疗研究提供了另一种潜在途径。     

丁型肝炎病毒核酶反式切割HBV mRNA片段的实验研究

目的　探讨反式作用丁型肝炎病毒 (HDV)核酶体外切割乙型肝炎病毒 (HBV)mRNA片段的可行性。方法　将化学合成的核酶cDNA克隆到含有T7启动子的载体PGEM 4Z中。利用体外转录技术转录出核酶及底物 ,研究其体外切割活性。利用E H作图法进行核酶的酶促动力学研究。结果　在体外实验中显示两酶均能成功的将底物切割 ,37℃温浴 90min的切割百分率为 5 0 %和5 1%。利用E H作图法进行的酶促动力学研究中求得Rc1、Rc2的Km值分别为 0 6 1μmol L、0 5 8μmol L,Kcat值分别为 :0 6 4·min 1 、0 6 0·min 1 。结论　反式作用HDV核酶对非HDV底物 HBVmRNA片段的成功切割为寻找新的HBV的反义抑制手段开辟了途径。     

抗HPV16E_6核酶的原核表达与体外活性研究

目的　获得一种特异性抗人乳头瘤病毒(HPV)16型E6基因的核酶,用以在基因调控水平预防、治疗HPV相关性肿瘤。方法　以核酶(ribozyme,Rz)的锤头结构为模型,采用计算机软件针对HPV16E6基因,设计相应的Rz;体外合成其基因后,克隆于原核表达质粒中。将HPV16E6基因片段也克隆于原核表达质粒中,通过体外转录而得到核酶和病毒mRNA,进行体外切割试验以鉴定核酶的活性。结果　抗HPV16E6核酶(简称抗16HRz)被装入pRG523中而构成pRG16HRz,体外转录后能将核酶独立地释放出来。HPV16E6mRNA转录质粒pTZ16E6含有HPV16E6基因片段( 94位～ 296位),体外切割实验证明抗16HRz具备切割活性,在体外能准确、有效地识别和切割HPV16E6mRNA片段,得到82nt和132nt的切割产物。结论　所获得的抗HPV16E6核酶能特异性切割HPV16E6mRNA,可作为对HPV相关性肿瘤进行基因治疗的工具。     

脱氧核酶及其应用进展

脱氧核酶是利用体外分子进化技术合成的一种具有催化功能的单链DNA片段 ,具有高效的催化活性和结构识别能力。迄今为止 ,已发现大量具有催化功能的脱氧核酶。其中具有RNA切割活性的脱氧核酶 ,能催化RNA特定部位的切割反应 ,从mRNA水平对基因灭活 ,从而调控蛋白质的表达 ,可能成为治疗肿瘤、病毒感染性疾病以及其它相关疾病 ,基因功能研究 ,核酸突变分析等的新型工具。     

CⅡTA锤头状核酶的克隆及其体外活性

通过CⅡTA核酶抑制HeLa细胞表面MHC Ⅱ类分子的表达.设计并合成针对人类CⅡTA的核酶Rz464,通过体外转录和切割实验鉴定其活性.将Rz464亚克隆到真核表达载体pIRES2-EGFP(pRz464),并稳定转染HeLa细胞株,流式细胞术检测MHC Ⅱ类抗原表达,RT-PCR检测CⅡTA mRNA水平.结果表明,Rz464与CⅡTA靶序列体外切割产物电泳见预期切割条带.pRz464+HeLa细胞与对照组比较,HLA-DR、DP、DQ抗原诱导型表达分别降低了79.21%、90.31%及48.30%;同时CⅡTA的诱导型mRNA含量明显减少.Rz464通过切割CⅡTA mRNA,进而阻止了后者调控的MHC Ⅱ类分子的表达.     

人巨细胞病毒UL138～142基因在临床低传代分离株中的多态性研究

目的　研究人巨细胞病毒 (humancytomegalovirus ,HCMV)UL138～UL14 2基因在临床低传代分离株中的多态性及其与HCMV先天感染致病性之间的关系。方法　对经荧光定量PCR方法检测HCMV DNA为阳性的临床分离株进行UL138～UL14 2基因全序列PCR扩增 ,对扩增阳性的标本进行全序列的测序及结果分析。结果　HCMV临床分离株的UL138、UL14 2ORF(openreadingframe)高度保守 ;UL139ORF呈现高度多态性 ,可被明确划分为 3个基因型 ,且核苷酸及氨基酸的变异主要集中在序列的 5′端 ;所有临床分离株的UL14 0ORF在Toledo株第 174位核苷酸处插入 1个胞嘧啶核苷酸 ,其ORF较Toledo株增加了 2 31个核苷酸 ;所有临床分离株的UL14 1ORF在Toledo株第 2 2 7位核苷酸处缺失了 1个胸腺嘧啶核苷酸 ,故形成UL14 1a及UL14 1b 2个新的ORF。HCMV临床分离株的UL14 0蛋白较Toledo株新增了ScAMP磷酸化和酪蛋白激酶Ⅱ磷酸化位点 ,其它基因编码蛋白的重要功能区域相对保守。结论　HCMV临床分离株UL139ORF的 5′端呈现高度多态性 ,而且被明确地分成 3个基因型 ,故其可能在HCMV先天感染的致病性差异方面起一定作用 ;尚未发现UL138～UL14 2中某个特定基因与HCMV先天感染致病性有本质联系     

Ganciclovir resistance mutations in UL97 and UL54 genes of Human cytomegalovirus isolates resistant to ganciclovir

Human cytomegalovirus (HCMV) resistance to ganciclovir results from mutations in viral phosphotransferase (UL97) and/or DNA polymerase (UL54) genes. The HCMV isolates from the blood of immunocompromised patients with persisting presence of the pp65 antigen in the blood in spite of ganciclovir therapy were tested for ganciclovir susceptibility by an immediate-early antigen plaque reduction assay, and the UL54 and UL97 genes were sequenced. Nine isolates from eight patients (six patients with acquired immune deficiency syndrome (AIDS), one liver transplant recipient and one renal transplant recipient) showed phenotypic resistance to ganciclovir. All these ganciclovir-resistant HCMV isolates harbored one or more of the following UL97 mutations: M460V, A594V, A594T, L595S, C603W, and M615V. Two isolates harbored the P522S mutation in the UL54 gene. The M615V mutation in the UL97 gene has not been reported earlier and its role in ganciclovir resistance remains to be elucidated. In ganciclovir-resistant HCMV isolates the UL54 gene was less frequently mutated than the UL97 gene. The P522S mutation was relatively frequent in UL54-mutated HCMV isolates.     

Inhibition of UL54 and UL97 genes of human cytomegalovirus by RNA interference

Short interfering RNAs (siRNAs), namely siUL54-1 and siU54-2 targeting UL54 (DNA polymerase) gene, and siUL97-1 and siUL97-2 targeting UL97 (phosphotransferase) gene, were used to inhibit respective genes of Human cytomegalovirus (HCMV) and consequently the virus infection process in human foreskin fibroblast (HFF) cultures. The virus infection was monitored by cell morphology (CPE), levels of UL83 and IE86 mRNAs, and virus antigen. The results showed that siUL97-2 remarkably inhibited viral CPE while other siRNAs were less inhibitory. The siRNAs reduced the levels of UL83 mRNA but not that of IE86 mRNA; again, siUL97-2 was most inhibitory. Particularly, siUL97-2 reduced the UL83 mRNA level 14, 19, 203, and 37 times at 24, 48, 72, and 96 hrs post infection (p.i.), respectively. When tested for the effect on viral antigen by immunofluorescent assay (IFA), UL97-2 exerted a marked inhibition. These results demonstrate the effectiveness of siRNAs against experimental HCMV infection and indicate their therapeutic potential.     

Rapid detection by reverse hybridization of mutations in the UL97 gene of human cytomegalovirus conferring resistance to ganciclovir.

BACKGROUND OF STUDY: Diseases due to human cytomegalovirus (HCMV) infection constitute a major threat in marrow and solid organ transplant recipients. Ganciclovir (GCV) is widely used in prophylaxis and pre-emptive therapy of active HCMV infection. Resistance to ganciclovir (GCV) may arise at variable frequency under GCV therapy and is conferred by mutations (i) in the UL97 gene (codons 460, 520, and 591-607) encoding a phosphotransferase which is essential for monophosphorylation of GCV and, to a lesser extent, (ii) in the UL54 gene coding for the DNA polymerase of HCMV. OBJECTIVE: The purpose was to develop a rapid assay to screen for emerging GCV resistance mutations in the UL97 gene of HCMV whereby avoiding virus isolation and nucleotide sequencing procedures. STUDY DESIGN: A nested PCR (nPCR) amplifying UL97 codons 450-672 was developed. Nested amplicons were subsequently sequenced directly. Oligonucleotides for use in a reverse hybridization assay were designed to detect relevant non-synonymous mutations at codons UL97 460, 520, 603 and 607. Strain AD169 served as a wild-type control. RESULTS: UL97-specific nPCR amplicons were obtained from 18 EDTA blood samples of ten transplant recipients receiving GCV for more than 30 days. In three consecutive samples from a single patient a GCV resistance mutation at codon 603 (C-->W) was detected. In addition, two out of four cell culture-adapted HCMV isolates known to exhibit GCV resistance in vitro revealed mutations at codons 460 (M-->V) and 607 (C-->Y), respectively. By reverse hybridization a discrimination of single nucleotide changes at codons 460, 520, 603 and 607 was possible whereby matching exactly the results of the nucleotide sequence analysis for all 23 amplicons examined. CONCLUSIONS: Reverse hybridization appeared to be a rapid and convenient alternative to nucleotide sequencing when screening the UL97 gene of HCMV for selected markers of GCV resistance.     

稳定表达人巨细胞病毒蛋白pUL23细胞系建立及其功能研究

目的:建立稳定表达人巨细胞病毒UL23基因的HELF细胞系,研究病毒蛋白在宿主细胞内行为,为进一步研究人巨细胞病毒蛋白pUL23的功能提供依据。方法:通过PCR技术从人巨细胞病毒基因组中扩增出UL23基因,通过分子克隆技术构建重组逆转录病毒表达载体pLEGFP-N1-FLAG-UL23。将该载体导入Am-phoPackTM-293细胞,收获重组逆转录病毒,然后感染HELF细胞,HELF经持续G418抗性筛选后获得稳定表达UL23基因的细胞系。采用激光共聚焦显微镜观察病毒蛋白在细胞内的定位。结果:RT-PCR、Western blotting结果证实病毒基因UL23能够整合到宿主细胞基因组中,并能在宿主细胞中稳定表达病毒蛋白。共聚焦显微镜观察到病毒蛋白pUL23定位于细胞质,处于细胞核周边。结论:利用逆转录病毒载体介导的基因转移技术,成功构建了稳定表达UL23基因的转基因细胞系。该病毒蛋白在宿主细胞质中的定位,提示病毒蛋白发挥功能的空间位于细胞核周边,有利地推进了人巨细胞病毒蛋白pUL23功能研究的进程。     

Characterization of Rhesus Cytomegalovirus Genes Associated with Anti-viral Susceptibility

Studies were initiated to determine whether rhesus cytomegalovirus (RhCMV)-infected macaques could serve as an animal model for evaluating anti-CMV compounds, as macaques have a naturally occurring CMV that is similar to human CMV (HCMV). Utilizing plaque reduction assays, RhCMV was tested for anti-viral susceptibility. By these assays, RhCMV displayed anti-viral susceptibility to ganciclovir at a 50% effective dose (ED₅₀) of 0.8 μM, acyclovir at an ED₅₀of 15 μM, and foscarnet at an ED₅₀of 250 μM. By Southern blot analysis with HCMV UL97 (phosphotransferase) and DNA polymerase (pol) genes as probes, we isolated viral DNA fragments that strongly hybridized. DNA sequence analysis of these DNA fragments revealed two open reading frames with homology to HCMV UL97 and DNA polymerase. Steady-state RNA analysis revealed that the RhCMV UL97 homologue andpolgenes are transcribed as early late and early genes, respectively. Comparison against HCMV showed the RhCMV UL97 homologue exhibits 54.4% amino acid (aa) sequence identity to HCMV UL97 and the RhCMV DNA polymerase 59.2% aa sequence identity to HCMV DNA polymerase. Results from anti-viral assays and molecular characterization of these two viral genes suggest that RhCMV-infected rhesus macaques should serve as an excellent animal model for evaluating future anti-CMV compounds.     

Molecular Epidemiology of Cytomegalovirus UL97 and UL54 variants in Taiwan

BackgroundThe antiviral resistance of cytomegalovirus (CMV) infections is associated with mutations in the CMV UL54 and UL97 gene regions and is a serious problem in immunocompromised patients. However, the molecular epidemiology of UL54 and UL97 in Taiwan is unclear.MethodsWe conducted a retrospective study of patients with CMV infections between January and December 2016 in two tertiary hospitals, one regional hospital in Taiwan. CMV DNAemia was confirmed by elevated CMV DNA titers. Then the regions of the UL54 and UL97 mutations were amplified by PCR and sequenced.ResultsOf 729 patients with CMV syndrome, 112 CMV DNAemia patients were enrolled. Twelve novel variants in UL54 (P342S, S384F, K434R, S673F, T754M, R778H, C814S, M827I, G878E, S880L, E888K, and S976N) and one novel variant in UL97 (M615T) were discovered. UL97 antiviral resistance mutations (L595S, M460I, and M460V) were found in four patients (3.6%). In the drug resistance strains, the mutation events occurred after 83–150 days of therapy, and drug resistance was also observed in these patients. The following high frequency variants were observed: D605E in UL97 and A885T, N898D, V355A, N685S, and A688V in UL54.ConclusionThe results demonstrate that the positive rate of CMV DNAemia was 15.3% (112/729) among the patients with clinical CMV infection symptoms. The proportion of antiviral resistance CMV strains within CMV DNAemia patients was 3.6%. With the information of polymorphism incidence in the UL54 and UL97 patients from our study, determination of the genetic profile of UL54 and UL97 among immunocompromised populations with refractory CMV infection is recommended.     

Detection of ganciclovir resistance after valacyclovir-prophylaxis in renal transplant recipients with active cytomegalovirus infection

Whether valaciclovir (VCV) prophylaxis could be responsible for ganciclovir (GCV)-resistance of Human cytomegalovirus (HCMV) in transplantation has never been documented. A multicentric retrospective pilot study was undertaken to detect GCV-resistance through mutations within the UL97 gene in renal transplant recipients who experienced active HCMV infection and received valacyclovir prophylaxis. Twenty-three patients who experienced HCMV antigenaemia or DNAemia during or at the end of prophylaxis were included. UL97 genotyping was carried out on peripheral blood samples, using a nested in-house PCR, which amplified the full-length UL97 gene. One patient has a resistance-related mutation (M460I); the major risk factor for emergence of resistance in this patient was the presence of early and persistent antigenaemia. GCV-resistance during VCV-prophylaxis was rare after renal transplantation. However, special attention must be paid to patients developing early active HCMV infection under prophylaxis.     

Multiple ganciclovir-resistant strains in a newborn with symptomatic congenital human cytomegalovirus infection

A case of human cytomegalovirus (HCMV) drug-resistance in a congenitally infected newborn is described. Unusual aspects of this case include: (i) the detection of an extremely complex virus population, composed of a mixture of wild-type (wt) and multiple mutant ganciclovir (GCV) and valganciclovir (val-GCV) resistant strains carrying a variety of known mutations in UL97; (ii) the identification of novel UL97 mutations and (iii) the first time detection of combined UL97 drug resistance mutations in the same viral strain. In detail, four known UL97 single-nucleotide mutations (A594T/V, M460V/I, C592G), a new amino-acid substitution (C607S), and a new deletion (597-600) in one of the three UL97 hot spots for GCV/val-GCV resistance (codons 460, 520 and 590-607) were detected. In addition, the combination of M460V+A594V and M460V+C592G was observed for the first time. The emergence of HCMV drug-resistance in symptomatic congenital infections chronically treated with GCV or val-GCV should be taken into account. The immaturity of the neonatal immune system may contribute to selection of complex virus populations in these patients.     

Contrasting effects on ganciclovir susceptibility and replicative capacity of two mutations at codon 466 of the human cytomegalovirus UL97 gene

BackgroundHuman cytomegalovirus (HCMV) infections cause significant morbidity in immunocompromised hosts. Resistance to ganciclovir is predominantly associated with alterations in the HCMV UL97 kinase and, more occasionally, with mutations in the HCMV DNA polymerase gene.ObjectivesThe aim of this study was to investigate the impact of two different mutations found at the same UL97 codon on drug susceptibility and viral replicative capacity. Mutation V466G was observed in a solid organ transplant recipient whereas mutation V466M was observed in a patient with AIDS.Study designTwo HCMV UL97 mutations, V466M and V466G, were transferred to recombinant viruses using a bacterial artificial chromosome system. Susceptibility testing of the recombinant wild-type and mutant viruses was performed using a standard plaque reduction assay. Replication kinetics of recombinant viruses was investigated using a yield assay.ResultsMutant V466G was resistant to ganciclovir and had significant replicative defect whereas mutant V466M was drug susceptible and had unaltered replication kinetics. Furthermore, mutant V466G formed small viral plaques with intracellular inclusions.ConclusionsTo our knowledge, this is the first report of such contrasting phenotypes for drug susceptibility and replicative capacity for HCMV mutations found at the same codon of the UL97 gene.