Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products.

In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.     

人巨细胞病毒UL97基因耐药重组株的构建与鉴定

目的 构建含UL97基因耐药相关突变的重组人巨细胞病毒(rHCMV),并通过耐药表型和基因型加以鉴定.方法 采用重叠延伸剪接PCR在UL97基因中引入Pine I位点和耐药相关位点做定点突变,将所获突变基因片段按比例与人巨细胞病毒(HCMV)AD169标准株DNA混合,经脂质体介导共转染成纤维细胞MRC-5.利用间接免疫荧光检测HCMV PP65抗原,证实MRC-5已被rHCMV感染,观察同源重组病毒形成的细胞病变达100%后收获该重组病毒.用不同浓度更昔洛韦进行噬斑筛选纯化目的 病毒,并通过噬斑减少试验和耐药基因(UL97和UL54)序列分析对重组病毒进行鉴定.结果 成功构建目的 突变UL97基因片段,同病毒基因组DNA共转染7 d后可见明显细胞病变,PP65抗原检测证实为rHCMV感染灶.经过克隆筛选得到的重组病毒株UL97基因型分析结果与预期一致,UL54基因测序未发现突变.阳性克隆重组病毒对更昔洛韦敏感性显示半数抑制浓度(IC50)为15 μmol/L,是标准株的12倍,具耐药表型.结论 成功将HCMV基因组DNA与耐药突变目的 基因片段共转染MRC-5细胞,通过更昔洛韦筛选和噬斑纯化获得目的 重组病毒株.该方法的建立为在用药过程中不断出现的新的HCMV耐药突变株的鉴定分析提供了有效的技术平台.     

Association of DNA-dependent and -independent Ribonucleoside Triphosphatase Activities with dnaB Gene Product of Escherichia coli

Preparations of E. coli dnaB gene product contain ribonucleoside triphosphatase activity that is stimulated 10-fold by DNA. The products of the triphosphatase activity are nucleoside diphosphates and P(i). The dnaB complementing activity in the varphiX174 DNA-dependent system and these triphosphatase activities copurify over the last 20-fold of an extensive (about 40,000-fold) purification procedure. Acrylamide gel electrophoresis of the purified material shows a single band of protein coincident with eluted dnaB complementing and DNA-dependent and -independent nucleoside triphosphatase activities.     

T4 DNA-delay proteins, required for specific DNA replication, form a complex that has ATP-dependent DNA topoisomerase activity.

Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60. By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified. The purified fraction also contains complementing activities for T4 genes 52 and 60. On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein. This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction. The analogs adenylyl imidodiphosphate and adenyl [beta, gamma-methylene]diphosphonate cannot be used to replace ATP. The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase. The possible relationship among the three polypeptides and their biological activities is discussed.     

Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein.

Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, oriS and oriL. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on oriS, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described previously by Elias et al. [Elias, P., O'Donnell, M. E., Mocarski, E. S. & Lehman, I. R. (1986) Proc. Natl. Acad. Sci. USA 83, 6322-6326] is the product of the UL9 gene.     

Nucleoprotein complex formed between herpes simplex virus UL9 protein and the origin of DNA replication: inter- and intramolecular interactions.

The UL9 gene of herpes simplex virus type 1 encodes an origin-binding protein. UL9 protein purified from baculovirus vector-infected insect cells forms a stable complex with DNA containing the herpes simplex virus origin of DNA replication, oriS. Contained within oriS are two UL9 protein-binding sites, I and II, bracketing an (A + T)-rich region. UL9 protein, visualized by electron microscopy, binds selectively at the site of the origin and covers approximately 120 base pairs. Upon formation of the nucleoprotein complex, the apparent contour length of the DNA is shortened, suggesting that this amount of DNA is wrapped or condensed by the protein. A nucleoprotein complex of similar size and structure forms on an inactive origin deleted for binding site II. Multiple intermolecular interactions occur. In particular, UL9 nucleoprotein complexes interact in trans with other UL9 nucleoprotein complexes such that dimer DNA molecules are formed with a junction at the position of protein binding. The DNA molecules in these intermolecular complexes are aligned predominantly in a parallel orientation.     

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to oris, an origin of viral DNA replication

We have identified cellular proteins that interact with the herpes simplex virus type 1 (HSV-1) origin-binding protein (UL9 protein) by screening a HeLa cell complementary DNA library by using the yeast two-hybrid system. Approximately 7 x 10(5) colonies were screened. Five of the 48 positive clones contained cDNAs that encoded the p150(Glued) component of the dynactin complex, three contained cDNAs for the neural F Box 42-kDa protein (NFB42), which is highly enriched in neural tissue, and three contained hTid-1, a human homologue of the bacterial DnaJ protein. We have focused in this report on the interaction of the viral UL9 protein with the cellular hTid-1. In vitro immunoprecipitation experiments confirmed that hTid-1 interacts with the UL9 protein. Electrophoretic mobility-shift assays indicated that the hTid-1 enhances the binding of UL9 protein to an HSV-1 origin, ori(s), and facilitates formation of the multimer from the dimeric UL9 protein. hTid-1 had no effect on the DNA-dependent ATPase or helicase activities associated with the UL9 protein. These findings implicate hTid-1 in HSV-1 DNA replication, and suggest that this cellular protein may provide a chaperone function analogous to the DnaJ protein in Escherichia coli DNA replication.     

人巨细胞病毒gp52-pp65-pp150的融合表达及应用

目的克隆表达人巨细胞病毒(human cytomegalovirus,HCMV)特异性强的抗原决定簇基因,制备并纯化重组蛋白,并通过组装成捕获法ELISA试剂盒对其抗原性进行评价。方法提取人巨细胞病毒AD169株的DNA,用PCR扩增HCMV的pp150(UL32)、gp52(UL44)、pp65(UL83)基因片段,将3个基因片段串联克隆至原核表达载体pGEX-5x-3进行融合表达和纯化,采用SDS-PAGE电泳法和免疫印迹法(Western blot)对融合基因的克隆及重组蛋白的表达进行鉴定,并组装成捕获法ELISA试剂盒对临床标本进行检测,评价试剂盒的各项性能指标。结果经序列测定各基因片段序列正确,成功构建了HCMV的高效表达融合基因的重组载体gp52-pp65-pp150,表达的融合蛋白经Western blot分析,分子量在50 ku,具有良好的抗原性。将该蛋白组装成捕获法ELISA试剂盒,经232份血清标本的检测,与进口试剂盒相比,本试剂盒的灵敏度为93.91%;特异度为97.43%;粗一致性为96.1%;约登指数为0.901;批内变异系数为12.4%;稳定性良好,37℃保存试剂与4℃保存试剂进行...     

Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA.

phiX174 DNA-dependent DNA synthesis is catalyzed in vitro by the combination of at least 11 purified protein fractions: dnaB, dnaC(D), and dnaG gene products, DNA polymerase III, DNA elongation factors I and II, DNA binding protein, and replication factors W, X, Y, and Z. The reaction requires ATP, 4 dNTPs, and Mg+2 and is specific for phiX174 (or phiXahb) DNA. Purified replication factor Y contains phiX174 (or phiXahb) DNA-dependent ATPase (or dATPase) activity. The ATPase activity is poorly stimulated by other single-stranded DNA, by double-stranded DNA, or by RNA. The products of the phiX174 DNA-dependent ATPase activity of factor Y are Pi and ADP (or dADP). The association of phiX174 DNA-dependent ATPase activity with factor Y was shown in the following ways: (a) the two activities copurified with a constant ratio; (b) they comigrated on native polyacrylamide gel electrophoresis; (c) both activities were heat-inactivated at the same rate; and (d) both showed identical patterns of N-ethylmaleimide sensitivity.     

Differentiation of human cytomegalovirus genotypes in immunocompromised patients on the basis of UL4 gene polymorphisms

The variety of clinical manifestations of human cytomegalovirus infection probably results from both viral and host factors. Because genetic markers that span the viral genome are needed to identify such viral factors, polymorphisms at the UL4 gene locus were analyzed. DNA sequence analyses revealed 4 UL4-based genotypes, 2 of which were closely related but distinguishable by an uncommon polymorphism that results in overexpression of gpUL4. Similarities in the spectra of polymorphisms detected in various sets of samples reveal that all UL4 types infect a diversity of organs in different patient groups and in different geographic locales. Simultaneous infection by >1 UL4 type is common in AIDS patients. Data from sequencing analyses and from a rapid and simple UL4 typing assay did not detect linkage between UL4 and glycoprotein B types, which suggests that UL4 genotyping should be useful for studies that attempt to identify cytomegalovirus genes involved in disease pathogenesis.     

Comparison of cytomegalovirus (CMV) UL97 gene sequences in the blood and vitreous of patients with acquired immunodeficiency syndrome and CMV retinitis

Cytomegalovirus (CMV) resistance to ganciclovir occurs via mutations in the UL97 gene. CMV DNA, from vitreous and blood specimens and from culture isolates from 87 patients with acquired immunodeficiency syndrome and CMV retinitis who received a ganciclovir implant, was sequenced to identify the relationship between the UL97 DNA sequences in the eye and peripheral blood. There was 93.5% agreement between the UL97 gene sequences from paired vitreous specimens and blood specimens. Sequence analysis of vitreous specimens showed that 15% (13/87) of the patients had either a ganciclovir resistance-conferring mutation or a polymorphism in the CMV UL97 gene. Eleven of the 13 mutations or polymorphisms in the vitreous also were identified in blood. Although the number of mutations limits definitive interpretation, these data suggest that blood specimens may reflect the events occurring in the eyes of patients with CMV retinitis.     

人巨细胞病毒UL147基因在婴儿巨细胞病毒肝炎临床株中的多态性

目的:探讨人巨细胞病毒(human cytomegalov-irus,HCMV)UL147基因在婴儿巨细胞病毒肝炎临床株中的多态性及其与致病性之间的关系.方法:对25株经荧光定量PCR方法检测HCMVDNA为阳性的临床株的尿液上清液进行HCMVUL147全序列PCR扩增,并对PCR扩增产物进行序列测定及分析.结果:25株临床株扩增阳性产物进行HCMVUL147全序列PCR扩增,结果有19株PCR扩增阳性;与HCMVToled株进行序列比较分析,所有UL147开放读码框架(open-reading-fraem,ORF)长度在477-480bp,序列呈现较高多态性,UL147基因变异率为2.7%-10.9%;氨基酸变异集中在30-40氨基酸位点,变异率为4.3%-8.75%;所有研究标本中没有序列与Toledo完全一致.序列差异多位于序列的5’端,巨细胞病毒肝炎患儿和无症状患儿之间的UL147基因DNA序列是一致的.NCBIPROS数据库预测UL147编码蛋白功能区显示几乎所有的UL147序列中都存在1个CKP和1个PKC位点.结论:巨细胞病毒肝炎患儿临床株中的HCMVUL147基因序列呈现高度多态性.UL147基因多态性与其对肝脏损害的程度无关.     

UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells

The human cytomegalovirus UL82 gene encodes a protein (pp71) that is localized in the tegument domain of the virus particle. The UL82 gene product is delivered to the nucleus at the time of infection, and it is believed to function in gene activation. We have constructed a human cytomegalovirus mutant, ADsubUL82, that lacks a substantial portion of the UL82 coding region. It was propagated on human diploid fibroblasts expressing the UL82 gene product, and it was possible to produce a mutant virus lacking the UL82 protein by passaging virus stocks for one cycle of growth on normal, noncomplementing fibroblasts. The UL82-deficient mutant displays a multiplicity-dependent growth defect in normal human fibroblasts. The growth of ADsubUL82 is severely restricted at low input multiplicities (0.01-0.1 plaque-forming units per cell), producing a yield that is reduced by a factor of about 10(5) in comparison to wild-type virus. At higher input multiplicities (10 plaque-forming units per cell), ADsubUL82 grew nearly as well as the wild-type virus. By using a human cytomegalovirus gene array, we demonstrated that UL82 functions to facilitate virus mRNA accumulation very early during the human cytomegalovirus replication cycle. The growth phenotype associated with the UL82 mutant seems to result from its inability to efficiently activate human cytomegalovirus immediate early genes.     

Conversion of ?X174 and fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia coli

varphiX174 and M13 (fd) single-stranded circular DNAs are converted to their replicative forms by extracts of E. coli pol A1 cells. We find that the varphiX174 DNA-dependent reaction requires Mg(++), ATP, and all four deoxynucleoside triphosphates, but not CTP, UTP, or GTP. This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, but not that of dnaF (ribonucleotide reductase). The in vitro conversion of fd single-stranded DNA to the replicative form requires all four ribonucleoside triphosphates, Mg(++), and all four deoxynucleoside triphosphates. The reaction involves the product of gene dnaE but not those of genes dnaC, dnaD, dnaF, or dnaG. The reaction with fd DNA is inhibited by rifampicin or antibody to RNA polymerase, while the reaction with varphiX174 DNA is not affected by either. With the varphiX174 DNA-dependent reaction, activities have been detected that specifically complement extracts of dnaA, dnaB, dnaC, dnaD, or dnaG mutants.     

A multisubunit complex containing the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products isolated from yeast.

A complex containing the products of theSWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 genes and four additionalpolypeptides has been purified from extracts of the yeast Saccharomycescerevisiae. Physical association of these proteins was demonstrated bycopurification and coimmunoprecipitation. A potent DNA-dependent ATPasecopurified with the complex, and this activity was evidently associated withSWI2/SNF2.