对HCMV UL54 mRNA片段特异性切割的M1GS构建

人巨细胞病毒是一种DNA病毒,在人群中一般呈亚临床感染和潜伏感染。为研究病毒基因沉默工具和抗病毒制剂,以人巨细胞病毒UL54基因mRNA序列设计互补的外部引导序列,共价结合到大肠杆菌来源RNaseP催化核心M1RNA上,从而构建成M1GS-T6核酶。通过对DNA聚合酶UL54基因亚克隆片段转录产物体外切割研究,证实该核酶具备对UL54mRNA片段的特异切割能力。     

RNase P核酶对人巨细胞病毒UL54基因mRNA体外切割作用

外部引导序列(EGSs)是mRNA靶序列互补并引导RNase P切割的小RNA片段.我们设计与人巨细胞病毒HCMV(Human Cytomegalovirus) UL54基因mRNA序列互补的EGSs,将其与大肠杆菌来源RNase P催化核心M1 RNA构建成M1GS核酶.通过对UL54基因亚克隆片转录产物体外切割研究,证实该核酶具备对UL54 mRNA片段的特异切割能力,可以发展成为一种抗病毒试剂.     

RNase P核酶对人巨细胞病毒UL54基因mRNA体外切割作用

外部引导序列(EGSs)是mRNA靶序列互补并引导RNaseP切割的小RNA片段。我们设计与人巨细胞病毒HCMV(Human Cytomegalovirus)UL54基因mRNA序列互补的EGSs,将其与大肠杆菌来源RNaseP催化核心M1RNA构建成M1GS核酶。通过对UL54基因亚克降片转录产物体外切割研究,证实该核酶具备对UL54 mRNA片段的特异切割能力,可以发展成为一种抗病毒试剂。     

核酶对人巨细胞病毒mRNA片段的体外切割

引导序列GSs(GuideSequences)是能与mRNA互补,引导核酶RNaseP催化核心M1RNA对互补区域特异切割的小片段游离RNA。针对人巨细胞病毒HCMV(humancytomegalovirus)DNA聚合酶mRNA序列设计GS,共价结合到大肠杆菌来源M1RNA中,构建成M1GST7核酶。通过对巨细胞病毒DNA聚合酶亚克隆片段转录产物体外切割实验,表明该核酶具备对DNA聚合酶mRNA片段的特异切割能力 。     

GS介导RNase P对人巨细胞病毒ul54基因mRNA的切割作用

引导序列(Guide Sequences,GSs)是与mRNA靶序列互补并引导RNase P切割的小RNA片段。设计与人巨细胞病毒HCMV(Human Cytomegalovirus,HCMV)ul54基因D片段mRNA序列互补的GS,将其共价结合到大肠杆菌来源RNase P催化核心M1 RNA,构建成T7-M1GS核酶。通过对ul54基因D片段转录产物体外切割实验和将T7-M1GS构建在含有U6启动子的逆转录病毒载体,与构建在真核载体pEGFP-N1的ul54基因D片段共转染人宫颈癌细胞系HeLa的体内切割实验,证实该核酶具备对ul54基因D片段mRNA的特异切割能力,为利用核酶治疗HCMV感染提供实验基础。     

GS介导RNaseP对人巨细胞病毒μ154基因mRNA的切割作用

引导序列（Guide Sequences,GSs）是与mRNA靶序列互补并引导RNase P切割的小RNA片段。设计与人巨细胞病毒HCMV（Human Cytomegalovirus,HCMv）μ/54基因D片段mRNA序列互补的GS,将其共价结合到大肠杆菌来源RNase P催化核心M1 RNA,构建成T7-M1GS核酶。通过对μ/54基因D片段转录产物体外切割实验和将T7-M1GS构建在含有U6启动子的逆转录病毒载体,与构建在真核载体pEGFP-N1的μ/54基因D片段共转染人宫颈癌细胞系HeLa的体内切割实验,证实该核酶具备对μ/54基因D片段mRNA的特异切割能力,为利用核酶治疗HCMV感染提供实验基础。     

DNA-EGS1386胞内诱导核酶P抑制人巨细胞病毒UL49基因的表达

外部引导序列(EGSs)是一类与mRNA靶序列互补并能引导核酶P切割靶mRNA的小分子RNA。本实验构建稳定表达UL49基因的HeLa细胞系,设计合成了针对于人巨细胞病毒(HCMV)UL49基因的12ntDNA性质的EGS1386,通过转染稳定表达UL49基因的细胞系,荧光定量PCR和Western blotting检测细胞内目的基因UL49的表达情况。结果显示在DNA-EGS1386作用下UL49基因的表达量降低了50%,表明DNA-EGS1386可以有效引导人的核酶P切割目标mRNA。因此,DNA-EGS可以发展成为一种新的基因沉默技术和潜在的抗病毒试剂。     

HCMV UL97 mRNA序列特异性M1GS的构建及其体外切割活性研究

HCMV UL97基因编码一种蛋白激酶,该酶参与调控病毒DNA的复制和衣壳的形成,且序列异常保守,可作为抗HCMV治疗的重要靶位。基于HCMV UL97 mRNA T3位点附近的序列,设计一段与该位点互补的引导序列（Guide Sequence,GS）,并将其与大肠杆菌核酶P催化亚基（M1 RNA）的3’末端共价连接,构建了一种序列特异性的M1GS（M1-T3）。体外实验证实,所构建的M1-T3可与UL97 mRNA的T3位点特异性结合并产生有效的切割作用。进一步研究M1-T3的结构与其对底物片段靶向切割活性的关系,结果发现在M1 RNA与GS之间增加一段88核苷酸桥连序列的M1-T3（即M1-T3’）,其靶向切割活性大大增强。此外,去除M1-T3 3’末端的CCA序列,其靶向切割活性将基本丧失。上述结果表明,这段桥连序列和3’末端的CCA序列是M1-T3重要的结构元件。这不仅有助于阐明M1GS与其底物的相互作用机制,同时也为进一步评价M1-T3在体内对UL97基因表达及病毒复制的抑制活性奠定了基础。     

改进型外部引导序列抑制人巨细胞病毒UL49 基因的表达

外部引导序列（EGS）技术（The EGS-based technology）是一种新型的基因沉默技术,能诱导内源性的核酶 P（RNase P）对靶mRNA进行有效切割. 以人类巨细胞病毒（HCMV）UL49基因mRNA片段为靶序列,基于前人实验基础上设计出更为精简与高效的改进型EGS (miniEGS),DNA片段长度仅为12 bp. 构建稳定表达HCMV UL49的细胞系,通过应用荧光定量PCR及Western 印迹分别鉴定miniEGS对内源性UL49的抑制效率.结果显示,miniEGS能在HeLa细胞中能达到很高的转染效率（97.9%）,并且在转染稳定表达UL49的HeLa细胞系后,发现UL49基因的mRNA与蛋白表达水平都出现明显下降（50%）.研究表明, 改进型的EGS序列不仅能有效抑制目的基因的表达,同时因其序列设计的精简性与高效性,可更好地应用到以后的抗病毒研究中.     

引导RNaseP对HCMVUL54mRNA片段体外切割的EGS构建

HCMV是一种广泛存在的疱疹病毒,在免疫抑制和免疫功能低下人群中,HCMV感染可引起严重疾病。RNaseP是细胞内催化tRNA5’末端成熟的酶,当EGSs与靶mRNA互补结合并形成类似tNRA的复合物时,大肠杆菌RNaseP催化亚基M1RNA可具备对靶mRNA特异的催化切割活性。为研究抗病毒制剂,针对HCMVDNA多聚酶UL54mRNA设计并构建特异性的EGS—C6,通过对觇舅基因亚克隆片段转录产物体外切割研究,证实该EGS具备引导M1RNA对UL54mRNA特异切割的能力,可发展成一种新型抗病毒制剂。     

In vitro construction of effective M1GS ribozymes targeting HCMV UL54 RNA segments

Seven sequence-specific ribozymes (M1GS RNAs) derived in vitro from the catalytic RNA subunit of Escherichia coli RNase P and targeting the mRNAs transcribed by the UL54 gene encoding the DNA polymerase of human cytomegalovirus were screened from 11 ribozymes that were designed based on four rules: (1) the NCCA-3′ terminal must be unpaired with the substrate; (2) the guide sequence (GS) must be at least 12 nt in length; (3) the eighth nucleotide must be U, counting from the site-1; and (4) around the cleavage site, the sites -1/ 1/ 2 must be U/G/C or C/G/C. Further investigation of the factors affecting the cleavage effect and the optimal ratio for M1GS/substrate was carried out. It was determined that the optimal ratio for M1GS/substrate was 2:1 and too much M1GS led to substrate degrading. As indicated above, several M1GS that cleaved HCMV UL54 RNA segments in vitro were successfully designed and constructed.Our studies support the use of ribozyme M1GS as antisense molecules to silence HCMV mRNA in vitro, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.     

Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection.

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J.A. Kerry, M.A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.     

Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.     

Characterization of Drug Resistance-Associated Mutations in the Human Cytomegalovirus DNA Polymerase Gene by Using Recombinant Mutant Viruses Generated from Overlapping DNA Fragments

A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts, and their drug susceptibility profiles were determined. Amino acid substitutions located in UL54 conserved region IV (N408D, F412C, and F412V), region V (A987G), and δ-region C (L501I, K513E, P522S, and L545S) conferred various levels of resistance to cidofovir and ganciclovir. Mutations in region II (T700A and V715M) and region VI (V781I) were associated with resistance to foscarnet and adefovir. The region II mutations also conferred moderate resistance to lobucavir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region III (L802M, K805Q, and T821I) were associated with various drug susceptibility profiles. Mutations located outside the known UL54 conserved regions (S676G and V759M) did not confer any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations with respect to the final drug resistance phenotype was demonstrated. Finally, the influence of selected UL54 mutations on the susceptibility of viral DNA replication to antiviral drugs was characterized by using a transient-transfection-plus-infection assay. Results of this work exemplify specific roles of the UL54 conserved regions in the development of HCMV drug resistance and may help guide optimization of HCMV therapy.