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Replication-associated activities of purified human papillomavirus type 11 E1 helicase

PROTEIN EXPRESSION AND PURIFICATION 2000年第2期18卷 148-159页

作者： Rocque, WJ Porter, DJT Barnes, JA Dixon, EP Lobe, DC Su, JL Willard, DH Gaillard, R Condreay, JP Clay, WC Hoffman, CR Overton, LK Pahel, G Kost, TA Phelps, WC Glaxo Wellcome Inc Dept Mol Sci Res Triangle Pk NC 27709 USA Glaxo Wellcome Inc Dept Mol Biochem Res Triangle Pk NC 27709 USA Glaxo Wellcome Inc Dept Virol Res Triangle Pk NC 27709 USA

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme. (C) 2000 Academic Press.

关键词：昆虫/细胞学昆虫/病毒学核苷三磷酸酶乳头状瘤病毒科/化学点突变蛋白质结构二级病毒蛋白质类/化学病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯病毒蛋白质类/代谢女(雌)性人类小鼠

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Helix-destabilizing properties of the baculovirus single-stranded DNA-binding protein (LEF-3)

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VIROLOGY 2000年第1期270卷 180-189页

作者： Mikhailov, VS NK Koltsov Dev Biol Inst Moscow 117808 Russia Inst Phys & Chem Res Lab Mol Entomol & Baculovirol Wako Saitama Japan

The helix-destabilizing properties of a single-stranded DNA-binding protein, LEF-3, of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Partial duplexes of DNA containing single-stranded (ss) tails of different sizes and orientations were used as substrates for assay of the unwinding ability of LEF-3. Upon noncooperative binding to ssDNA, LEF-3 was capable of unwinding the duplexes with 5' ss tails. However, it did not cause melting of the duplexes containing 3' ss tails, even at oversaturation of ssDNA adjacent to the duplexes. Upon cooperative binding to long ss tails, LEF-3 also produced the polar melting effect;it unwound the duplexes with long 5' ss tails, but not those with long 3' ss tails. These data suggest that LEF-3 has a preferential direction for entry into duplex DNA, namely 5' to 3' with respect to the bound DNA strand. In agreement with its polarity, LEF-3 efficiently melted the primer-template complexes which serve as substrates for DNA polymerases. However, the formation of a complex with viral DNA polymerase before addition of LEF-3 protected the primer-templates from the destabilization effect of LEF-3. Although the destabilization effect of LEF-3 was highly sensitive to monovalent and divalent salts, the protein was capable of melting DNA duplexes in a polar manner at physiological conditions, i.e., 30 degrees C in 0.15 M NaCl. Therefore, the polar destabilization effect of LEF-3 seems to be physiologically important and may be connected, in particular, with the polar action of viral helicase holoenzyme during baculovirus replication. (C) 2000 Academic Press.

关键词：腺苷三磷酸/药理学杆状病毒科/酶学杆状病毒科/遗传学碱基序列蚕属/病毒学 DNA/生物合成 DNA/化学 DNA/遗传学 DNA/代谢 DNA螺旋酶类/分离和提纯 DNA螺旋酶类/代谢 DNA复制 DNA 单链/化学 DNA 单链/遗传学 DNA 单链/代谢 DNA结合蛋白质类/分离和提纯 DNA结合蛋白质类/代谢指导DNA的DNA聚合酶/分离和提纯指导DNA的DNA聚合酶/代谢氯化镁/药理学核酸构象/药物作用核酸变性作用/药物作用蛋白质结合氯化钠/药理学底物特异性模板遗传病毒蛋白质类/分离和提纯病毒蛋白质类/代谢动物

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Recombinant expression and modification analysis of protein agno-1b encoded by avian polyomavirus BFDV

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ARCHIVES OF VIROLOGY 2000年第6期145卷 1211-1223页

作者： Liu, Q Hintz, M Li, J Linder, M Geyer, R Hobom, G Univ Giessen Inst Mikrobiol & Mol Biol D-35392 Giessen Germany Univ Giessen Klinikum Inst Biochem D-6300 Giessen Germany

Among two pairs of agnoproteins encoded in upstream positions in the late mRNAs of avian polyomavirus BFDV, either agno-1a or its splice derivative agno-1b are required for viral propagation. Out of the two proteins both of which consist of multiple electrophoretic subspecies, the smaller and less complex agno-1b has been cDNA-cloned into an influenza-virus /RNA-polymerase I expression system for production of higher amounts of this protein in infected chicken embryo fibroblasts. Fractional modification of agno-1b by phosphorylation at residues serine 51, serine 53, and threonine 73 is demonstrated through dephosphorylation by alkaline phosphatase, mass spectrometry of individual protein species isolated by strong anion exchange chromatography, and single or multiple alanine substitutions of serine or threonine residues in site-directed mutagenesis.

关键词：氨基酸序列印迹法蛋白质内肽酶类/代谢分子序列数据诱变定点磷酰化多瘤病毒属/代谢蛋白质加工转译后重组遗传丝氨酸/化学光谱法质量基质辅助激光解吸电离苏氨酸/化学病毒蛋白质类/化学病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯病毒蛋白质类/代谢动物鸡胚

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Crystallization and preliminary X-ray analysis of the matrix protein from Ebola virus

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2000年第6期56卷 758-760页

作者： Dessen, A Forest, E Volchkov, V Dolnik, O Klenk, HD Weissenhorn, W European Mol Biol Lab F-38000 Grenoble France Inst Biol Struct Jean Pierre Ebel F-38027 Grenoble France Inst Virol D-35037 Marburg Germany

The matrix protein from Ebola virus is a membrane-associated molecule that plays a role in viral budding. Despite its functional similarity to other viral matrix proteins, it displays no sequence similarity and hence may have a distinct fold. X-ray diffraction quality crystals of the Ebola VP40 matrix protein were grown by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 64.4, b = 91.1, c = 47.9 Angstrom, beta = 96.3 degrees. A data set to 1.9 Angstrom resolution has been collected using synchrotron radiation. The unit cell contains one molecule of molecular weight 35 kDa per asymmetric unit, with a corresponding volume solvent content of 35%.

关键词：结晶结晶学 X线埃博拉病毒/化学因子Ⅹa/遗传学遗传载体肽碎片/生物合成肽碎片/遗传学重组融合蛋白质类/生物合成重组融合蛋白质类/遗传学病毒基质蛋白质类/生物合成病毒基质蛋白质类/化学病毒基质蛋白质类/遗传学病毒基质蛋白质类/分离和提纯病毒蛋白质类/生物合成病毒蛋白质类/化学病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯

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Synthesis, subcellular localization and VP16 interaction of the herpes simplex virus type 2 UL46 gene product

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ARCHIVES OF VIROLOGY 2000年第10期145卷 2149-2162页

作者： Kato, K Daikoku, T Goshima, F Kume, H Yamaki, K Nishiyama, Y Nagoya Univ Sch Med Dis Mechanism & Control Res Inst Nagoya Aichi 4668550 Japan Nagoya Univ Sch Med Dept Internal Med 2 Nagoya Aichi 466 Japan

We developed a rabbit polyclonal antiserum reactive against a recombinant 6x His-UL46 fusion protein expressed in Escherichia coli, and using this antiserum identified the UL46 gene product of herpes simplex virus type 2 (HSV-2) to be phosphoproteins with apparent molecular masses of 82-, 84-, and 86-kDa in infected Vero cells. The UL46 protein was produced in the late phase of infection in a manner highly dependent on viral DNA synthesis, and was mainly distributed at the edge of the nucleus in the cytoplasm. Although its kinetics of production and its progress of distribution were different from those of the major tegument protein VP16 (the UL48 gene product or alpha -trans-inducing factor (alpha TIF)), most of the UL46 protein colocalized with VP16 in the late phase of infection, and copurified with it in column chromatography. Moreover, our data showed that the HSV-2 UL46 protein, when coexpressed with VP16, enhanced alpha4 promotor-regulated gene expression in a transient luciferase reporter assay, while the expression of the UL46 protein alone suppressed it.

关键词：疱疹病毒2型人/致病力疱疹病毒2型人/生理学萤光素酶类/遗传学萤光素酶类/代谢显微镜检查共焦沉淀素试验 Vero细胞病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯病毒蛋白质类/代谢人类兔

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Human cytomegalovirus - no longer just a DNA virus

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NATURE MEDICINE 2000年第8期6卷 863-864页

作者： Huang, ES Johnson, RA Univ N Carolina Dept Med Chapel Hill NC 27599 USA Univ N Carolina Lineberger Comprehens Canc Ctr Chapel Hill NC 27599 USA

Human cytomegalovirus (HCMV) is classified as a DNA herpesvirus. A recent study showed that the HCMV virion contains not only DNA, but also four species of mRNA, indicating that this virus is more complex than previou... 详细信息

关键词：巨细胞病毒/遗传学巨细胞病毒/生长和发育巨细胞病毒/生理学 DNA 病毒/分离和提纯基因病毒 RNA 信使/分离和提纯 RNA 病毒/分离和提纯病毒蛋白质类/分离和提纯人类

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Differential functional behavior of viral φ29, Nf and GA-1 SSB proteins

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NUCLEIC ACIDS RESEARCH 2000年第10期28卷 2034-2042页

作者： Gascón, I Lázaro, JM Salas, M Univ Autonoma Madrid Ctr Biol Mol Severo Ochoa CSIC UAM E-28049 Madrid Spain

DNA replication of phi 29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage phi 29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of phi 29 and the related phages Nf and GA-l,their ability to eliminate non-productive binding of phi 29 DNA polymerase to ssDNA and their stimulatory effect on replication by phi 29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during phi 29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs, First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by phi 29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the phi 29 and Nf SSBs do not show any effect, On the other hand, the phi 29 SSB is the only one of the three SSBs able to increase the replication rate of phi 29 DNA polymerase in primed M13 ssDNA replication. From the fact that the phi 29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of phi 29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB-ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on phi 29-like DNA replication has been proposed.

关键词：氨基酸序列芽孢杆菌噬菌体/遗传学芽孢杆菌噬菌体/代谢枯草芽孢杆菌/病毒学 DNA复制 DNA 单链/化学 DNA 单链/遗传学 DNA 单链/代谢 DNA结合蛋白质类/化学 DNA结合蛋白质类/分离和提纯 DNA结合蛋白质类/代谢指导DNA的DNA聚合酶/代谢动力学分子序列数据核酸构象序列比对序列同源性氨基酸病毒蛋白质类/化学病毒蛋白质类/分离和提纯病毒蛋白质类/代谢

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Viral coinfection in salmonids:: Infectious pancreatic necrosis virus interferes with infectious hematopoietic necrosis virus

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ARCHIVES OF VIROLOGY 1999年第4期144卷 657-673页

作者： Alonso, M Rodríguez, S Pérez-Prieto, SI CSIC Ctr Invest Biol Dept Mol Microbiol E-28006 Madrid Spain

Coinfection of farm-reared salmonids involving two viruses has been described, but there is no report on the interactions between viruses. Here we examine whether infectious pancreatic necrosis virus (IPNV) strain Sp interferes with the growth of infectious hematopoietic necrosis virus (IHNV) strain S46, a coinfected isolate from rainbow trout. When BF-2 cell culture was inoculated with S46 the infective titer of the IHNV fraction decreased by 3 log(10) units compared to the growth curve of IHNV in the single infection. RT-PCR assay confirmed this reduction, which after successive passages of the co-infected sample led to a decrease in IHNV mRNA and the absence of the specific PCR product for IHNV. Flow cytometry showed that only 13% of the cells inoculated with S46 strain were infected with IHNV at 48-72 h post infection, in contrast to the 50-80% of cells that were positive for IPNV. Exposure of cells to IHNV for 24 h before infection with IPNV did not affect the infective titers of either virus or the PCR results obtained in simultaneous coinfections. Moreover IHNV was not inhibited when the IPNV inoculum was reduced. So, a multiplicity of infection dependence was demonstrated for IPNV-IHNV interference;the RT-PCR assay described here was found to be a suitable technique for identifying and studying dual viral infections.

关键词：鲈形目/病毒学原病毒/生理学 RNA 病毒/遗传学逆转录聚合酶链反应弹状病毒科/遗传学弹状病毒科/分离和提纯弹状病毒科/生理学鲑科病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯病毒结构蛋白质类/遗传学病毒复制

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Identification of MaTu-MX agent as a new strain of lymphocytic choriomeningitis virus (LCMV) and serological indication of horizontal spread of LCMV in human population

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VIROLOGY 1999年第1期257卷 73-83页

作者： Reiserová, L Kaluzová, M Kaluz, S Willis, AC Závada, J Závodská, E Závadová, Z Ciampor, F Pastorek, J Pastoreková, S Slovak Acad Sci Inst Virol Bratislava 84246 Slovakia Univ Oxford Dept Biochem MRC Immunochem Unit Oxford OX1 3QU England Acad Sci Czech Republ Inst Mol Genet Prague Czech Republic

In this study we elucidated the molecular character of MaTu-MX, previously described as an unusual transmissible agent. Amino acid sequencing of peptides generated from a 58-kDa MX-related protein purified from MaTu human carcinoma cells allowed us to identify it as a nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). Northern blot analysis detected LCMV-specific RNAs in MaTu cells. Comparative immunoprecipitations showed cross-reactivity between NP of LCMV strain WE end MX NP. Using RT-PCR, we have cloned MX NP cDNA. According to sequence comparison, MX LCMV is as closely related to both LCMV strains WE and Armstrong as these strains are to one another. Based on this finding we propose that MX is a new strain of LCMV. We also showed that the stability of MX NP in MaTu cells is very high and that the virus is transmissible by cell-to-cell contact or by cell-free extract to human HeLa and monkey Vero cells, but not to human AGS, canine MDCK, mouse NIH 3T3, and hamster CHO cells. Finally, employing MX LCMV NP in immunoprecipitation and solid-phase radioimmunoassay, we found 37.5% prevalence of anti-LCMV antibodies in human sera, suggesting possible horizontal spread of the virus in the human population. (C) 1999 Academic Press.

关键词： 3T3细胞氨基酸序列抗体病毒/血液乳腺肿瘤/病毒学 CHO细胞共同培养技术仓鼠亚科 DNA 互补/化学疾病传播传染性 HeLa细胞淋巴细胞性脉络丛脑膜炎/免疫学淋巴细胞性脉络丛脑膜炎/传播淋巴细胞性脉络丛脑膜炎病毒/遗传学淋巴细胞性脉络丛脑膜炎病毒/免疫学淋巴细胞性脉络丛脑膜炎病毒/分离和提纯分子序列数据核蛋白类/化学核蛋白类/遗传学核蛋白类/分离和提纯 RNA 病毒/分析肿瘤细胞培养的/病毒学病毒蛋白质类/化学病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯青少年成年人老年人动物狗人类小鼠中年人

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Histidine codons appended to the gene encoding the RP022 subunit of vaccinia virus RNA polymerase facilitate the isolation and purification of functional enzyme and associated proteins from virus-infected cells

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VIROLOGY 1999年第2期258卷 469-479页

作者： Katsafanas, GC Moss, B NIAID Viral Dis Lab NIH Bethesda MD 20892 USA

Vaccinia virus encodes a eukaryotic-like RNA polymerase composed of two large and six small subunit protein species. A replication-competent virus with 10 histidine codons added to the single endogenous J4R open reading frame was constructed. The altered migration of the 22-kDa subunit of RNA polymerase on SDS-polyacrylamide gel electrophoresis confirmed that J4R encoded the RPO22 subunit and that the mutant virus was genetically stable. The histidine-tagged RNA polymerase bound quantitatively to metal-affinity resins and was eluted in an active form upon addition of imidazole. Glycerol gradient sedimentation of the eluted fraction indicated that most of the RPO22 in infected cells is associated with RNA polymerase. Using stringent washing conditions, metal-affinity chromatography resulted in a several hundred-fold increase in RNA-polymerase-specific activity, and substantially pure enzyme was obtained with an additional conventional chromatography step. When mild conditions were used for washing the metal-affinity resin, the vaccinia virus-encoded capping enzyme, early transcription factor, and nucleoside triphosphate phosphohydrolase I specifically co-eluted with the tagged RNA polymerase, consistent with their physical association. The ability to selectively bind RNA polymerase to an affinity column provided a simple and rapid method of concentrating and purifying active enzyme and protein complexes.

关键词：细胞系长尾猴黑色谱法亲和密码子指导DNA的RNA聚合酶类/遗传学指导DNA的RNA聚合酶类/分离和提纯指导DNA的RNA聚合酶类/代谢基因病毒 HeLa细胞组氨酸金属重组遗传痘苗病毒/酶学痘苗病毒/遗传学病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯病毒蛋白质类/代谢动物人类

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