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An internalization-competent influenza hemagglutinin mutant causes the redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in clathrin free clusters

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BIOCHEMISTRY 1999年第46期38卷 15166-15173页

作者： Brown, CM Roth, MG Henis, YI Petersen, NO Univ Western Ontario Dept Chem London ON N6A 5B7 Canada Univ Texas SW Med Ctr Dept Biochem Dallas TX 75235 USA Tel Aviv Univ George S Wise Fac Life Sci Dept Neurobiochem IL-69978 Tel Aviv Israel

Image correlation spectroscopy and cross correlation spectroscopy were used to demonstrate that similar to 25% of the internalization-competent influenza virus hemagglutinin mutant, HA+8, is colocalized with clathrin and AP-2 at the plasma membrane of intact cells, while wild-type HA (which is excluded from coated pits) does not colocalize with either protein. Clathrin and AP-2 clusters were saturated when HA+8 was overexpressed, and this was accompanied by a redistribution of AP-2 into existing coated pits. However, de novo coated pit formation was not observed. In nontreated cells, the number of clusters of clathrin or AP-2 colocalized with HA+8 was always comparable. Hypertonic treatment which disperses the clathrin lattices resulted in more clusters containing AP-2 and HA+8 than clathrin and HA+8, Less colocalization of HA+8 with clathrin was also observed after cytosol acidification, which causes the formation of deeply invaginated pits, where the HA+8 may be inaccessible to extracellular labeling by antibodies, and blocks coated vesicle budding. However, cytosol acidification elevated the number of clusters containing both HA+8 and AP-2, suggesting an increase in their level of association outside of the deep invaginations. Our results imply that AP-2 and HA+8 can colocalize in clusters devoid of clathrin, at least in cells treated to alter the clathrin lattice structure. Although we cannot ascertain whether this also occurs in untreated cells, we propose that AP-2 binding to membrane proteins carrying internalization signals can occur prior to the binding of AP-2 to clathrin. While such complexes can in principle serve to recruit clathrin for the formation of new coated pits, the higher affinity of the internalization signals for clathrin-associated AP-2 [Rapoport, I., et al, (1997) EMBO J, 16, 2240-2250] makes it more likely that once the AP-2-membrane protein complexes form, they are quickly recruited into existing coated pits.

关键词：氢离子浓度高渗溶液/代谢图像处理计算机辅助流感病毒a型/遗传学流感病毒A型/代谢大分子物质显微镜检查共焦/方法转录因子AP-2 转录因子/代谢转染/遗传学

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Selective perturbation of early endosome and/or trans-Golgi network pH but not lysosome pH by dose-dependent expression of influenza M2 protein

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第14期274卷 9854-9860页

作者： Henkel, JR Popovich, JL Gibson, GA Watkins, SC Weisz, OA Univ Pittsburgh Renal Electrolyte Div Lab Epithelial Cell Biol Pittsburgh PA 15213 USA Univ Pittsburgh Dept Cell Biol & Physiol Pittsburgh PA 15213 USA

Many sorting stations along the biosynthetic and endocytic pathways are acidified, suggesting a role for pH regulation in protein traffic, However, the function of acidification in individual compartments has been difficult to examine because global pH perturbants affect all acidified organelles in the cell and also have numerous side effects, To circumvent this problem, we have developed a method to selectively perturb the pH of a subset of acidified compartments, We infected HeLa cells with a recombinant adenovirus encoding influenza virus M2 protein (an acid-activated ion channel that dissipates proton gradients across membranes) and measured the effects on various steps in protein transport, Al low multiplicity of infection (m.o.i.), delivery of influenza hemagglutinin from the trans-Golgi network to the cell surface was blocked, but there was almost no effect on the rate of recycling of internalized transferrin, At higher m.o.i., transferrin recycling was inhibited, suggesting increased accumulation of M2 in endosomes, Interestingly, even at the higher m.o.i., M2 expression had no effect on lysosome morphology or on EGF degradation, suggesting that lysosomal pH was not compromised by M2 expression. However, delivery of newly synthesized cathepsin D to lysosomes was slowed in cells expressing active M2, suggesting that acidification of the TGN and endosomes is important for efficient delivery of lysosomal hydrolases, Fluorescence labeling using a pH-sensitive dye confirmed the reversible effect of M2 on the pH of a subset of acidified compartments in the cell, The ability to dissect the role of acidification in individual steps of a complex pathway should be useful for numerous other studies on protein processing and transport.

关键词：腺病毒科生物转运组织蛋白酶D/代谢细胞区室化细胞极性剂量效应关系药物内颗粒/代谢表皮生长因子/代谢遗传载体高尔基体/代谢 HeLa细胞氢离子浓度流感病毒a型/遗传学流感病毒A型/代谢离子通道/遗传学离子通道/代谢溶酶体/代谢病毒基质蛋白质类/生物合成人类

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Epitope mapping of the influenza A virus RNA polymerase PA using monoclonal antibodies

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ARCHIVES OF VIROLOGY 2000年第5期145卷 957-964页

作者： Hatta, M Asano, Y Masunaga, K Ito, T Okazaki, K Toyoda, T Kawaoka, E Ishihama, A Kida, H Hokkaido Univ Grad Sch Vet Med Dept Dis Control Sapporo Hokkaido 0600818 Japan Natl Inst Genet Dept Mol Genet Shizuoka Japan Rat Drug Design Labs Fukushima Japan Kurume Univ Sch Med Dept Virol Fukuoka Japan Kurume Univ Sch Med Dept Pediat Fukuoka Japan Tottori Univ Fac Agr Dept Vet Publ Hlth Tottori 680 Japan Univ Wisconsin Sch Vet Med Dept Pathobiol Sci Madison WI 53706 USA

To obtain reagents to functionally map the PA protein, we produced monoclonal antibodies specific to this protein. Twenty-two monoclonal antibodies reacting with PA protein in ELISA were divided into 10 groups on the basis of competitive binding patterns to this protein. Of these, seventeen monoclonal antibodies bound to PA polypeptide spanning amino acids 101-400 and three bound to that of amino acids 518-600, while the other two did not react with any PA polypeptides tested with the exception of the intact PA. Among these monoclonal antibodies, only five reacted with PA in A/PR/8/34 virus-infected cells in indirect immunofluorescence assay. Thus, we obtained monoclonal antibodies that recognize at least 10 distinct regions of the PA molecule. These monoclonal antibodies should be useful in dissecting functions of the PA protein.

关键词：抗体单克隆结合竞争性细胞系酶联免疫吸附测定表位作图/方法流感病毒A型/酶学流感病毒a型/遗传学流感病毒A型/免疫学 RNA复制酶/遗传学 RNA复制酶/免疫学动物鸡胚人类小鼠

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Transient bicistronic vRNA segments for indirect selection of recombinant influenza viruses

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VIROLOGY 1999年第1期262卷 93-103页

作者： Flick, R Hobom, G Inst Mikrobiol & Mol Biol D-35392 Giessen Germany

The 5'- and 3'-terminal regions of influenza vRNA molecules are known to constitute the promoter structure upon association with viral RNA polymerase in an activated complementary conformation. An inherent requirement for their location at the very ends of the vRNA molecules always has been implied because of that natural structure, but this study demonstrates that one or both of the promoter sequences may be relocated into vRNA-internal positions and still retain their polymerase-binding function. External extensions of vRNA molecules employed include either single-stranded RNA sequences less than or equal to 750 nucleotides in length or complementary, and hence potentially double-stranded sequences, or promoter duplications. 5' RACE analyses of internally promoted cRNA and mRNA molecules prove initiation to occur at exactly the 3' standard template position (1) over bar, as defined by the regular promoter structure. Thereby any template extensions are lost from the resulting RNA molecules and progeny virions. These observations have been used to construct bicistronic vRNAs with an additional 3'-promoter sequence located between the two reading frames. During propagation, these spontaneously give rise to monocistronic vRNAs upon internal initiation reactions. Accordingly designed bicistronic vRNAs can be employed for indirectly selecting any foreign gene encoded in the resulting monocistronic VRNA for incorporation into recombinant influenza viruses, (C) 1999 Academic Press.

关键词： 3'非翻译区/遗传学 5'非翻译区/遗传学基因扩增流感病毒a型/遗传学流感病毒A型/分离和提纯核酸构象启动区(遗传学)/遗传学 RNA/遗传学 RNA剪接/遗传学 RNA 互补/遗传学 RNA 信使/遗传学 RNA 病毒/遗传学序列分析 RNA 病毒复制/遗传学动物狗

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Strain-specific differences in the effect of influenza A virus neuraminidase on vector-expressed hemagglutinin

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ARCHIVES OF VIROLOGY 1999年第4期144卷 781-786页

作者： Kaverin, NV Klenk, HD Univ Marburg Inst Virol Marburg Germany

In order to evaluate the efficiency of the removal of sialic acid residues from the influenza virus hemagglutinin by the viral neuraminidase in the course of the virus replication cycle, CV-1 cells expressing the hemagglutinin of H7 subtype from an SV40-based vector were superinfected with influenza virus strain A/Duck/Ukraine/63 (H3N8) or A/USSR/90/77 (H1N1). Vector-expressed hemagglutinin was immunoprecipitated from cell lysates and analyzed by polyacrylamide gel electrophoresis. The data indicate that the removal of sialic acid residues from the vector-expressed H7 hemagglutinin by N1 neuraminidase of A/USSR/90/77 virus in the course of the virus replication cycle is incomplete. The results are discussed in connection with previously published data showing that the low activity of NA in wild-type influenza virus results in incomplete removal of sialic acid residues from virion components.

关键词：细胞系长尾猴黑鸭遗传载体 HN蛋白质/生物合成 HN蛋白质/遗传学流感病毒A型/酶学流感病毒a型/遗传学流感病毒A型/生理学神经氨酸酶/代谢猴病毒40 种特异性病毒复制动物

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Prevalence of swine influenza virus subtypes on swine farms in the United States

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ARCHIVES OF VIROLOGY 2002年第6期147卷 1209-1220页

作者： Choi, YK Goyal, SM Joo, HS Univ Minnesota Dept Clin & Populat Sci St Paul MN 55108 USA

Serologic and virologic prevalence of infection with different swine influenza virus (SIV) subtypes was investigated using swine sera, nasal swabs and lung samples that had been submitted for a diagnosis to the Minnesota Veterinary Diagnostic Laboratory. A total of 111,418 pig sera were tested for SIV antibody between 1998 and 2000, and 25,348 sera (22.8%) were found to be positive by the hemagglutination inhibition (HI) test. Of the positive samples, 16,807 (66.7%) and 8,541 (33.7%) had antibody to H1 and H3 subtypes, respectively. Between January 1998 and May of 2001, a total of 3,561 nasal swabs or lung samples were examined for the presence of SIV, and SIV was isolated from 1,124 samples (31.7%). Of these isolates, 869 (77.3%) and 255 (22.7%) were subtyped as H1 and H3, respectively, by the HI method. For further characterization, 120 SIV isolates each from 1998 to 2001 were randomly selected from a culture collection and their hemagglutinin (HA) and neuraminidase genes examined by reverse transcription-PCR and sequencing. Of the 480 isolates, 322 (67.1%), 22 (4.6%) and 129 (26.9%) were subtyped as H1N1, H1N2 and H3N2, respectively. The remaining 7 samples (1.5%) were found to contain both H1N1 and H3N2 viruses. The SIV H1N2 subtype was isolated from 1, 8, and 13 samples in 1999, 2000, and 2001, respectively. The 22 H1N2 isolates originated from 9 different states of the United States. Genetic screening of the HA genes of 12 selected H1N2 isolates showed that 8 of them had a close phylogenetic relationship with the Indiana isolate of H1N2 (A/Swine/Indiana/9K035/99), while 4 isolates were closely related to classical SIV H1N1.

关键词：氨基酸序列畜牧业抗体病毒/血液血凝集抑制试验血凝素糖蛋白类流感病毒/遗传学流感病毒A型/分类流感病毒a型/遗传学流感病毒A型/分离和提纯肺/病毒学分子序列数据鼻/病毒学正黏病毒科感染/流行病学正黏病毒科感染/兽医学正黏病毒科感染/病毒学种系发生患病率逆转录聚合酶链反应序列比对序列分析 DNA 猪疾病/流行病学猪疾病/病毒学美国/流行病学动物

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Characterization of the 1918 "Spanish" influenza virus neuraminidase gene

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第12期97卷 6785-6790页

作者： Reid, AH Fanning, TG Janczewski, TA Taubenberger, JK Armed Forces Inst Pathol Dept Cellular Pathol Div Mol Pathol Washington DC 20306 USA

The "Spanish" influenza pandemic of 1918 was characterized by exceptionally high mortality, especially among young adults. The surface proteins of influenza viruses, hemagglutinin and neuraminidase, play important roles in virulence, host specificity, and the human immune response. The complete coding sequence of hemagglutinin was reported last year. This laboratory has now determined the complete coding sequence of the neuraminidase gene of the 1918 virus. Influenza RNA fragments were isolated from lung tissue of three victims of the 1918 flu;complete sequence was generated from A/Brevig Mission/1/18, with confirmatory sequencing carried out on A/South Carolina/1/18 and A/New York/1/18. The 1918 neuraminidase gene sequence was compared with other N1 subtype neuraminidase genes, including 9 N1 strains newly sequenced for this study. The 1918 neuraminidase shares many sequence and structural characteristics with avian strains, including the conserved active site, wild-type stalk length, glycosylation sites, and antigenic sites. Phylogenetically, the 1918 neuraminidase gene appears to be intermediate between mammals and birds, suggesting that it was introduced into mammals just before the 1918 pandemic.

关键词：氨基酸序列碱基序列催化域糖基化流感病毒A型/分类流感病毒A型/酶学流感病毒a型/遗传学分子序列数据神经氨酸酶/化学神经氨酸酶/遗传学种系发生毒力成年人女(雌)性人类男(雄)性

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The development of live attenuated cold-adapted influenza virus vaccine for humans

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REVIEWS IN MEDICAL VIROLOGY 1999年第4期9卷 237-244页

作者： Maassab, HF Bryant, ML Univ Michigan Sch Publ Hlth Dept Epidemiol Ann Arbor MI 48109 USA Aviron Mountain View CA 94043 USA

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation;ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attentuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups. Copyright (C) 1999 John Wiley & Sons, Ltd.

关键词：适应生理学冷流感病毒a型/遗传学流感病毒A型/生长和发育流感病毒A型/免疫学流感病毒B型/遗传学流感病毒B型/生长和发育流感病毒B型/免疫学流感疫苗/免疫学流感人/预防和控制疫苗减毒/免疫学人类

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Mapping of a protective helper T cell epitope of human influenza A virus hemagglutinin

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第1期270卷 190-198页

作者： Gogolák, P Simon, A Horváth, A Réthi, B Simon, I Berkics, K Rajnavölgyi, É Tóth, GK Univ Szeged Dept Med Chem H-6720 Szeged Hungary Eotvos Lorand Univ Dept Immunol God Hungary Hungarian Acad Sci Inst Enzymol Budapest Hungary

The synthetic peptide comprising the 317-341 region of human influenza A virus (H1N1 subtype) hemagglutinin elicits peptide-specific antibody and helper T cell responses and confers protection against lethal virus infection. Molecular mapping of the 317-329 region, which encompasses the epitope recognized by peptide-specific T cells, revealed that the minimal size required for T cell activation was the 317-326 segment. The most likely peptide alignment, which placed 320Leu to pocket 1 of the I-E(d) peptide binding groove, was predicted by molecular mechanics calculations performed with the parental and with the Ala-substituted analogs. In Line with the prediction data, the results of the peptide binding assay, where the relative binding efficiency to I-E(d) molecules expressed on the surface of antigen-presenting cells was monitored, identified the 320-326 core sequence interacting with the major histocompatibility class II peptide binding groove. Functional analysis of Ala-substituted variants by functional assays and by calculating the surface-accessible areas of the single peptidic amino acids in the I-E(d)-peptide complexes demonstrated that 324Pro is a primary contact residue for the T cell receptor. Our results show that this type of analysis offers a suitable tool for molecular mapping of helper T cell epitopes and thus provides valuable data for subunit vaccine design. (C) 2000 Academic Press.

关键词：丙氨酸/遗传学计算机模拟表位作图表位血凝素糖蛋白类流感病毒/遗传学血凝素糖蛋白类流感病毒/免疫学组织相容性抗原Ⅱ类/化学组织相容性抗原Ⅱ类/免疫学流感病毒a型/遗传学流感病毒A型/免疫学白细胞介素2/生物合成淋巴细胞活化诱变定点肽碎片/遗传学肽碎片/免疫学蛋白质结合受体抗原 T细胞 T淋巴细胞辅助诱导/免疫学

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Generation of influenza A viruses entirely from cloned cDNAs

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第16期96卷 9345-9350页

作者： Neumann, G Watanabe, T Ito, H Watanabe, S Goto, H Gao, P Hughes, M Perez, DR Donis, R Hoffmann, E Hobom, G Kawaoka, Y Univ Wisconsin Sch Vet Med Dept Pathobiol Sci Madison WI 53706 USA Hokkaido Univ Sch Vet Med Dept Dis Control Microbiol Lab Sapporo Hokkaido 0600818 Japan Univ Nebraska Dept Vet & Biomed Sci Lincoln NE 68583 USA Inst Mikro & Mol Biol D-35392 Giessen Germany

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA. of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 x 10(3) plaque-forming units (pfu) of virus per mi of supernatant at is hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x 10(4)-5 x 10(7) pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

关键词：细胞系 DNA 互补 DNA 病毒/遗传学 HN蛋白质/遗传学流感病毒a型/遗传学流感病毒A型/生理学肾质粒 RNA聚合酶Ⅰ/代谢 RNA 病毒/遗传学逆转录聚合酶链反应转染病毒结构蛋白质类/遗传学病毒复制动物鸡胚狗人类小鼠

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