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Selective nuclear export of viral mRNAs in influenza-virus-infected cells

TRENDS IN MICROBIOLOGY 2000年第8期8卷 376-383页

作者： Chen, ZY Krug, RM Univ Texas Inst Cellular & Mol Biol Sect Mol Genet & Microbiol Austin TX 78712 USA

The NS1A protein of influenza A virus specifically inhibits the cellular machinery that processes the 3' ends of cellular pre-mRNAs by targeting two of the essential proteins of this machinery. Because the virus does not use this cellular machinery to synthesize the 3' poly(A) ends of viral mRNA, the nuclear export of cellular but not viral mRNAs is selectively inhibited.

关键词：细胞核/代谢细胞核/病毒学流感病毒A型/代谢核蛋白质类/代谢 Poly(A)结合蛋白质类 RNA加工转录后 RNA 信使/代谢 RNA 病毒/代谢 RNA结合蛋白质类/代谢病毒非结构蛋白质类/代谢 mRNA剪切和多聚腺苷酸化因子类动物人类

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Characterization of a monoclonal antibody directed to the surface of MA104 cells that blocks the infectivity of rotaviruses

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VIROLOGY 2000年第1期273卷 160-168页

作者： López, S Espinosa, R Isa, P Merchant, MT Zárate, S Méndez, E Arias, CF Univ Nacl Autonoma Mexico Inst Biotechnol Dept Genet & Fisiol Mol Cuernavaca 62250 Morelos Mexico Univ Nacl Autonoma Mexico Fac Med Dept Microbiol & Parasitol Cuernavaca 62250 Morelos Mexico

Rhesus rotavirus (RRV) binds to sialic acid residues on the surface of target cells, and treatment of these cells with neuraminidase greatly reduces virus binding with the consequent reduction of infectivity. Variants that can efficiently infect neuraminidase-treated cells have been isolated, indicating that attachment to sialic acid is not an essential step for animal rotaviruses to infect cells. To identify and characterize the neuraminidase-resistant receptor for rotaviruses, we have isolated a hybridoma that secrets a monoclonal antibody (MAb) (2D9) that specifically blocks the infectivity of wild-type (wt) RRV and of its sialic acid-independent variant nar3, in untreated as well as in neuraminidase-treated cells. The infectivity of a human rotavirus was also inhibited, although to a lesser extent. MAb 2D9 blocks the binding of the variant to MA104 cells, while not affecting the binding of wt RRV;in addition, this MAb blocked the attachment of a recombinant glutathione S-transferase (GST)-VP5 fusion protein, but did not affect the binding of GST-VP8. Altogether these results suggest that MAb 2D9 is directed to the neuraminidase-resistane receptor. This receptor seems to mediate the direct attachment of the variant to the cell, through VP5, while the receptor is used by wt RRV for a secondary interaction, after its initial binding to sialic acid, through VP8. MAb 209 interacts specifically with the cell surface by indirect immunofluorescence, immunoelectron microscopy, and FAGS. By a solid-phase immunoisolation technique, MAb 2D9 was found to react with three proteins of ca. 47 55, and 220 kDa, which might form a complex. (C) 2000 Academic Press.

关键词：抗体特异性生物素化作用细胞系表位/免疫学表位/代谢流式细胞术荧光抗体技术间接杂交瘤/免疫学恒河猴/病毒学显微镜检查免疫电子分子量突变/遗传学 N-乙酰神经氨酸/代谢神经氨酸酶/代谢 RNA结合蛋白质类/代谢受体病毒/拮抗剂和抑制剂受体病毒/免疫学受体病毒/代谢重组融合蛋白质类/拮抗剂和抑制剂重组融合蛋白质类/代谢轮状病毒属/化学轮状病毒属/药物作用轮状病毒属/遗传学轮状病毒属/代谢变异(遗传学)/遗传学病毒非结构蛋白质类/拮抗剂和抑制剂病毒非结构蛋白质类/代谢人类小鼠

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Cleavage of hepatitis C virus nonstructural protein 5A by a caspase-like protease(s) in mammalian cells

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VIROLOGY 2000年第2期270卷 476-487页

作者： Satoh, S Hirota, M Noguchi, T Hijikata, M Handa, H Shimotohno, K Tokyo Inst Technol Fac Biosci & Biotechnol Midori Ku Yokohama Kanagawa 2268501 Japan Kyoto Univ Inst Virus Res Sakyo Ku Kyoto 6068507 Japan

Nonstructural 5A protein (NS5A) of hepatitis C virus (HCV) is localized in the cytoplasm although it has a functional nuclear localization signal. To clarify the determinant of NS5A cytoplasmic localization, various N- or C-terminal deleted NS5A mutants were generated and their subcellular localization was analyzed in cell lines after transient expression. N-terminal deleted forms of NS5A were localized in the nucleus, and the sequence of the N-terminal 27 amino acids of NS5A had sufficient function to cause retention of a normally nuclear protein in the cytoplasm. These observations indicated that cytoplasmic localization of NS5A is determined primarily by the N-terminal region of the molecule. In addition, we found proteolytic processing of NS5A in transiently expressing cells. In these cells, cleavage occurred at a few sites located in the N- and C-terminal regions of NS5A. This cleavage in cells was enhanced by apoptotic stimuli and was Inhibited by the caspase inhibitor Z-VAD-FMK, suggesting that a caspase-like protease(s) contributes to the cleavages of NS5A Based on the results of mutational analysis of NS5A, we predicted one cleaved form, which had lost both the N- and the C-terminal portions of NS5A, to be composed of amino acid residues 155 to 389. Peptide containing the same amino acid sequence as this cleaved product was localized in the nucleus. Furthermore, we found that a fusion protein consisting of Gal4 DNA-binding domain fused with this cleaved form showed transcriptional activity only when the cu-catalytic subunit of protein kinase A (PKA) was coproduced, suggesting that the transcriptional activity of this product was regulated by PKA. These results suggested that the cleavage product of NS5A by a caspase-like protease(s) plays a role in transcriptional regulation of the host cell gene(s) in HCV-infected cells. (C) 2000 Academic Press.

关键词：氨基酸序列半胱氨酸天冬氨酸蛋白酶/代谢肝炎病毒属/代谢肝炎丙型/代谢肝炎丙型/病毒学分子序列数据突变 RNA复制酶/代谢序列比对信号传导肿瘤细胞培养的病毒融合蛋白质类/遗传学病毒融合蛋白质类/代谢病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢人类

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Yellow fever virus NS2B-NS3 protease: Charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function

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VIROLOGY 2000年第2期275卷 335-347页

作者： Droll, DA Murthy, HMK Chambers, TJ St Louis Univ Sch Med Dept Mol Microbiol & Immunol St Louis MO 63104 USA Univ Alabama Birmingham Ctr Macromol Crystallog Birmingham AL 35294 USA

Charged-to-alanine substitutions and deletions within the yellow fever virus NS2B-NS3(181) protease were analyzed for effects on protease function. During cell-free translation of NS2B-3(181) polyproteins, mutations at three charge clusters markedly impaired cis cleavage activity: a single N-terminal cluster in the conserved domain of NS2B (residues ELKK52-55) and two in NS3 (ED21-221 and residue H-47). These mutations inhibited other protease-dependent cleavages of a transiently expressed nonstructural polyprotein, although differential effects occurred. NS2B and NS3(181) proteins harboring these mutations were impaired in their ability to associate for trans cleavage activity. N-terminal deletions in NS3 also implicated residues ED21-22 in the association with NS2B. Deletions within NS2B revealed that the conserved domain alone provided minimal cofactor activity, with optimal function requiring both flanking hydrophobic regions. NS2B-3(181)- and NS3(181)-green fluorescent protein fusion proteins were used to determine the intracellular distribution of the protease complex. The former localized in membrane-based vesicular structures, whereas the latter localized poorly. The data suggest that NS2B-NS3 complex formation requires charge interactions involving the N-terminus of the conserved domain of NS2B and 22 N-terminal residues of NS3. A role for the putative transmembrane regions of NS2B in targeting of NS3 to intracellular membranes is also suggested. (C) 2000 Academic Press.

关键词：氨基酸序列氨基酸取代电泳聚丙烯酰氨凝胶内肽酶类/遗传学内肽酶类/代谢荧光抗体技术绿色荧光蛋白质类发光蛋白质类/遗传学发光蛋白质类/代谢分子序列数据诱变定点 RNA解螺旋酶类重组融合蛋白质类/遗传学重组融合蛋白质类/代谢序列缺失丝氨酸内肽酶类病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢黄热病毒/酶学黄热病毒/代谢

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Intracellular hepatitis c virus RNA-dependent RNA polymerase activity

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ARCHIVES OF VIROLOGY 2001年第8期146卷 1553-1570页

作者： Goobar-Larsson, L Wiklund, L Schwartz, S Univ Uppsala BMC Dept Med Biochem & Microbiol S-75123 Uppsala Sweden

Studies of intracellular hepatitis C virus (HCV) RNA-dependent RNA polymerase activity (RdRp activity) have been limited by the poor replicative capacity of HCV in cell culture. We have developed a method that allows for the measurement of HCV specific RdRp activity in eukaryotic cells. This method is based on the transient expression of the HCV polymerise and its templates under the control of the T7 promoter in the presence of an infection with recombinant vaccinia virus (vTF7-3) expressing the bacteriophage T7 DNA-dependent RNA polymerase. Both negative-strand and positive-strand RNA synthesis were characterised, and the role of the other HCV non-structural proteins for polymerase activity was assessed. With this assay we were able to show that: a) Intracellular HCV RdRp activity is not restricted to, but is higher for templates containing HCV specific sequences, b) The HCV polymerise is active within the polyprotein precursor, c) Cleavage of NS5b from the polyprotein precursor does not determine template specificity, and d) HCV RdRp activity is higher in the presence of the other HCV non-structural proteins and lower within a protease-deficient polyprotein precursor. This method allows the measurement of intracellular HCV polymerase activity and may be used to test substances against the HCV polymerise in search of potential drugs for anti-HCV therapy.

关键词： HeLa细胞肝炎病毒属/酶学肝炎病毒属/遗传学质粒/遗传学 RNA复制酶/遗传学 RNA复制酶/代谢 RNA 病毒/生物合成重组蛋白质类/代谢模板遗传转染病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢人类

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Hepatitis C virus NS5A protein is phosphorylated by casein kinase II

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第3期257卷 777-781页

作者： Kim, J Lee, D Choe, J Korea Adv Inst Sci & Technol Dept Biol Sci Taejon 305701 South Korea

Hepatitis C virus (HCV) has a positive-strand RNA genome that encodes a polyprotein, which is posttranslationally processed by cellular and viral proteinases into three structural and six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists as two phosphorylated forms of 56 kDa and 58 kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from rat-1 cells by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein. This protein kinase was associated through the N-terminus of NS5A and was not regulated by cell cycle. The GST-NS5A was also phosphorylated in vitro by the purified casein kinase II (CKII), a member of the CMCG kinase family. Since CKII and the NS5A-associated protein kinase have the same molecular size and property by In-gel kinase assay and an inhibitor treatment test, we conclude that HCV NS5A protein is phosphorylated by CKII. (C) 1999 Academic Press.

关键词：重组融合蛋白质类/遗传学重组融合蛋白质类/代谢序列缺失时间因素病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢大鼠

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Hepatitis CNS3 protease: Restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第1期267卷 278-282页

作者： Butkiewicz, NJ Yao, NH Wright-Minogue, J Zhang, RM Ramanathan, L Lau, JYN Hong, Z Dasmahapatra, B Schering Plough Corp Res Inst Dept Antiviral Therapy Kenilworth NJ 07033 USA Schering Plough Corp Res Inst Dept Struct Chem Kenilworth NJ 07033 USA Schering Plough Corp Res Inst Dept Tumor Biol Kenilworth NJ 07033 USA

The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide sig -nificantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease. (C) 2000 Academic Press.

关键词：氨基酸序列氨基酸取代生物素化作用克隆分子肝炎病毒属/酶学模型分子分子序列数据诱变定点肽碎片/化学合成肽碎片/化学肽碎片/代谢蛋白质生物合成蛋白质构象重组蛋白质类/化学重组蛋白质类/代谢丝氨酸内肽酶类/化学丝氨酸内肽酶类/代谢病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢

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The movement protein NSm of tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第5期97卷 2373-2378页

作者： Soellick, TR Uhrig, JF Bucher, GL Kellmann, JW Schreier, PH Max Planck Inst Zuchtungsforsch D-58029 Cologne Germany Bayer AG PF F MWF BT D-51368 Leverkusen Germany Univ Rostock FB Biol Abt Biochim D-18051 Rostock Germany

The nonstructural NSm protein of tomato spotted wilt tospovirus (TSWV) represents a putative viral movement protein involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures. To study the molecular basis of NSm function, we expressed the protein in Escherichia coli and investigated protein-protein and protein-RNA interactions of NSm protein in vitro. NSm specifically interacts with TSWV N protein and binds single-stranded RNA in a sequence-nonspecific manner. Using NSm as a bait in a yeast two-hybrid screen, we identified two homologous NSm-binding proteins of the DnaJ family from Nicotiana tabacum and Arabidopsis thaliana.

关键词：氨基酸序列拟南芥属克隆分子大肠杆菌蛋白质类 HSP40热休克蛋白质类热休克蛋白质类/遗传学热休克蛋白质类/代谢分子序列数据核壳/遗传学核壳/代谢核壳蛋白质类植物蛋白质类/遗传学植物蛋白质类/代谢植物病毒运动蛋白质类植物有毒 RNA 病毒/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/代谢酵母菌酿酒烟草蕃茄斑萎病毒属/遗传学蕃茄斑萎病毒属/代谢病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢病毒蛋白质类/遗传学病毒蛋白质类/代谢

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CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mice

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第1期264卷 144-150页

作者： Ohshima, T Nakajima, T Oishi, T Imamoto, N Yoneda, Y Fukamizu, A Yagami, K Univ Tsukuba Inst Basic Med Tsukuba Ibaraki 3058575 Japan Univ Tsukuba Inst Appl Biochem Tsukuba Ibaraki Japan Univ Tsukuba Ctr Tsukuba Adv Res Alliance Tsukuba Ibaraki Japan Osaka Univ Sch Med Dept Anat & Cell Biol Osaka Japan

The nonstructural protein 2 (NS2) from parvovirus minute virus of mice (MVMp) is a 25-kDa polypeptide which localizes preferentially to the cytoplasm and associates with cellular proteins in cytoplasm. These lines of evidence suggest that NS2 is positively exported from the nucleus to cytoplasm and functions in cytoplasm. We report here that nuclear export of NS2 is inhibited by leptomycin B (LMB), a drug that specifically blocks nuclear export signal (NES)-chromosomal region maintenance 1 (CRM1) interactions. CRM1 binds specifically to the 81- to 106-amino-acid (aa) region of NS2, and the region of NS2 actually functions as a NES. Interestingly, this region appears to be distinct from a typical NES sequence, which consists of leucine-rich sequences. These results indicate that NS2 protein is continuously exported from the nucleus by a CRM1-dependent mechanism and suggest that CRM1 also exports to distinct type of NESs. (C) 1999 Academic Press.

关键词：氨基酸序列生物转运/药物作用载体蛋白质类/代谢细胞核/代谢细胞核/病毒学细胞质/药物作用细胞质/代谢脂肪酸类不饱和/药理学 HeLa细胞核胞浆转运蛋白类小鼠细小病毒/药物作用小鼠细小病毒/代谢分子序列数据受体胞质和核序列同源性氨基酸信号传导/药物作用病毒非结构蛋白质类/代谢动物人类小鼠

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