Nucleotide sequence of the influenza virus A/USSR/90/77 neuraminidase gene

The complete nucleotide sequence of the N1 neuraminidase gene of influenza virus A/USSR/90/77 was determined. Comparison of its predicted amino acid sequence with other N1 and N2 neuraminidases indicates that the N1 neuraminidases share most of the antigenic determinants mapped on the N2 neuraminidase but display at least one additional potentially antigenic region probably as a result of intersubtypic differences in glycosylation.     

Neuraminidase gene from the early Asian strain of human influenza virus, A/RI/5-/57 (H2N2)

The complete structure of the neuraminidase gene from the A/RI/5-/57 strain of influenza virus has been determined. It is 1467 nucleotides long and codes for a protein of 469 amino acid residues. Comparison with the gene sequence for the N1 strains A/WSN/33 and A/PR/8/34, the N2 strain A/Udorn/72 and the protein sequence for the N2 strain A/Tokyo/3/67 shows the amino acid sequence changes that have occurred during antigenic shift (60%) and drift (7-9%).     

Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33.

The complete nucleotide sequence of the neuraminidase (NA) gene of WSN/33 (H1N1) virus was determined. The entire sequence was derived from the insert of cDNA clones, except the last 20 nucleotides, which were determined by primer extension. The WSN NA gene contained 1,409 nucleotides beginning at the 5' end (sense strand), with an untranslated region of 19 nucleotides followed by 1,359 nucleotides coding for 453 amino acids and finally ending with a 31-nucleotide sequence of untranslated region at the 3' termini. The amino acid sequence of WSN NA, as deduced from the DNA sequence, showed the presence of a stretch of 29 amino acids (7 to 35) enriched in hydrophobic amino acids, which may anchor the protein into the viral or cellular membrane. When compared with the PR8 NA sequence, WSN NA appeared to possess a similar structure, including the identical location of all cysteine and proline residues. However, WSN NA contained only three of the five potential glycosylation sites present in PR8 NA. Additionally, WSN NA contained a substitution of a five-amino acid sequence for a six-amino acid sequence in PR8 NA. The possible significance of these sequence changes in the primary structure of WSN NA in the unique role of WSN NA as a virulence factor in mouse brain and MDBK cells is discussed.     

Evolution of alpha 2-macroglobulin. The demonstration in a variety of vertebrate species of a protein resembling human alpha 2-macroglobulin.

The amino acid sequence of the Pronase-released heads of neuraminidase subtype N2 from the A/Tokyo/3/67 strain of influenza virus was determined by a combination of peptide and nucleic acid sequence analysis. The results show that the Pronase-released heads contain 396 amino acid residues and extend from residue 74 in the original protein to the C-terminus at residue 469. The heads contain five potential glycosylation sites at asparagine residues 86, 146, 200, 234 and 402, but only the first four are glycosylated. The sequence homology with the corresponding region of the previously published sequence of the neuraminidase subtype N1 [Fields, Winter & Brownlee (1981) Nature (London) 290, 213-217] is 45%. Detailed evidence for the sequence data has been deposited as Supplementary Publication SUP 50116 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1981) 193, 5.     

Structure of an escape mutant of glycoprotein N2 neuraminidase of influenza virus A/Tokyo/3/67 at 3 A

The three-dimensional structure of the membrane glycoprotein neuraminidase of an escape mutant of the influenza virus strain A/Tokyo/3/67 has been determined to 3 A (1 A = 0.1 nm) resolution by X-ray diffraction. The mutant virus, selected by growing the virus in the presence of a monoclonal antibody to the neuraminidase, is shown to have undergone a single amino acid change of lysine to glutamic acid at residue 368. The three-dimensional structure of the neuraminidase is identical with that reported for A/Tokyo/3/67, except for a purely local adjustment of the structure at position 368.     

Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism

Different isolates of influenza virus show a high degree of amino acid sequence variation in their surface glycoproteins. Conserved residues located in the substrate-binding pocket of the influenza virus neuraminidase are therefore likely to be involved in substrate binding or enzyme catalysis. In order to study the structure and function of the active site of this protein, a full-length cDNA clone of the neuraminidase gene from influenza A/Tokyo/3/67 was subcloned into aN M13 vector and amino acid substitutions were made in selected residues by using the oligonucleotide mismatch technique. The mutant neuraminidase genes were expressed from a recombinant SV40 vector, and the proteins were analyzed for synthesis, transport to the cell surface, and proper three-dimensional folding by internal and surface immunofluorescence. The mutant neuraminidase proteins were then assayed to determine the effect of the amino acid substitution on enzyme activity. Twelve of the 14 mutant proteins were correctly folded and were transported to the cell surface in a manner identical with that of the wild type. Two of these have full enzyme activity, but seven mutants, despite correct three-dimensional structure, have completely lost neuraminidase activity. Two mutants were active at low pH. The properties of the mutant enzymes suggest a possible mechanism of neuraminidase action.     

Synthesis, cloning and determination of the primary structure of a full-size DNA copy of the neuraminidase gene from influenza virus type A subtype H1N1

Complete nucleotide sequence of the cloned full-length DNA copy of the influenza virus A (H1N1) neuraminidase gene has been determined. The predicted amino acid sequence is compared with sequences of neuraminidases from other influenza virus strains. A section of the neuraminidase is found to be homologous to the chicken lysozyme catalytic centre.     

Sequence determination of the Sendai virus HN gene and its comparison to the influenza virus glycoproteins

The nucleotide sequence of the Sendai virus (SV) HN (hemagglutinin-neuraminidase) gene was determined. The deduced primary structure of the protein showed only one hydrophobic domain likely to represent the transmembrane region, but at its N terminus. Since the SV F protein is anchored in the membrane at its C terminus, the two SV glycoproteins are thus membrane-anchored in opposite orientations, similar to the two influenza virus (FLU) glycoproteins. Amino acid sequence comparisons of the SV HN and the FLU HA and NA proteins revealed homologies between 100 amino acids of the hemagglutinin region of the FLU HA protein and the C terminus of the SV HN, and between 200 amino acids of the neuraminidase region of the FLU NA and the central region of SV HN. Alignment of the neuraminidase, hemagglutinin, and fusion regions shared by these glycoproteins suggest the structure of a possible ancestral gene.     

Using PC/GENE for protein and nucleic acid analysis

This paper describes a series of protein analyses using the molecular biology software package PC/GENE, which runs on an IBM or compatible microcomputer. A nucleic acid sequence was first edited and then translated into an amino acid sequence. The amino acid composition, isoelectric point, molecular weight, and other properties of the sequence were determined. Programs to predict secondary structure, alpha helix membrane associations, hydrophobic and hydrophilic regions, and surface and antigenic sites from the amino acid sequence were also used. A search was made in a data base for sequences containing a region similar to a region in the protein sequence. Sequence alignments and queries of data bases can also be performed.     

Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase.

As the initial step in generating a live attenuated influenza A vaccine, we attempted to substitute an unrelated amino acid sequence (FLAG) for a portion of the neuraminidase (NA) molecule in influenza virus A/WSN/33 (H1N1), using a recently developed technique (reverse genetics [W. Luytjes, M. Krystal, M. Enami, J. D. Parvin, and P. Palese, Cell 59:1107-1113, 1989]). This technique allowed us to rescue the NA molecules containing the FLAG sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) at the bottom portion of the boxlike head of the molecule immediately above the stalk region (amino acid residues 63 to 70 [WSN NA numbering]). An anti-FLAG monoclonal antibody immunoprecipitated the NA molecules with the FLAG sequence, demonstrating that the foreign epitope was exposed on the virion surface. The dose of FLAG-containing transfectant virus required to kill 50% of mice was 100-fold higher than the required dose of parent virus. The FLAG sequence was stably maintained in the NA molecule during passage of the virus in tissue culture and in mice. These findings demonstrate that live influenza A vaccine strains with stable attenuating mutations in the coding region of the viral genes can be generated by reverse genetics.     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒 ,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库 ,从中克隆了金属蛋白酶 解整合蛋白Ussurin ,并进行了序列分析。结果显示 ,Ussurin开框读码序列由 14 34bp组成 ,编码 4 78个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出 ,Ussurin最初的翻译产物是酶原前体 ;依次含有 18氨基酸组成的信号肽 ,171氨基酸组成的酶原区和由 2 89氨基酸组成的Ussurin(2 0 0氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和 73氨基酸组成的解整合蛋白结构域 )。Ussurin的金属蛋白酶结构域含有 3对二硫键 ;解整合蛋白结构域含有 6对二硫键和特征性RGD(精氨酸 甘氨酸 天冬氨酸 )结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶 解整合蛋白呈现高度同源性属于P Ⅱ。氨基酸序列blast比对发现 ,酶原区和解整链蛋白结构域呈现极高的同源性 ,而金属蛋白酶结构域却出现了极高的变异 ,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域     

Effects of single-point amino acid substitutions on the structure and function neuraminidase proteins in influenza A virus

In order to clarify the effect of amino acid substitutions on the structure and function of the neuraminidase (NA) protein of influenza A virus, we introduced single-point amino acid substitutions into the NA protein of the A/Tokyo/3/67 (H2N2) strain using PCR-based random mutation. The rate of tolerant random one amino acid substitutions in the NA protein was 47%. Rates of tolerant substitutions for the stalk and for the surface and inner portion of the head region of the NA protein were 79, 54, and 19%, respectively. Deleterious changes, such as those causing the NA protein to stop at the Golgi/endoplasmic reticulum, were scattered throughout the protein. On the other hand, the ratio of mutations with which the NA protein lost neuraminidase activity, but was transported to the cell surface, decreased in proportion to the distance from the structural center of enzyme active site. In order to investigate the effect of accumulated amino acid substitutions on the structural character of the N2NA protein during evolution, the same amino acid substitutions were introduced by site-directed mutagenesis at 23 homologous positions on N2 proteins of A/Tokyo/3/67, A/Bangkok/15/85 (H3N2), and A/Mie/1/2004 (H3N2). The results showed a shift, or discordance, in tolerance at some of the positions. An increase in discordance was correlated with the interval in years between virus strains, and the discordance rate was estimated to be 0.6-0.7% per year.     

A sequence homology between the pX genes of HTLV-I/II and the murine IL-3 gene

Searching the protein sequence database for amino acid sequences homologous to the x-lor sequence in the pX region of human T-cell leukemia virus types I and II (HTLV-I/II), we found that there is a region of 38 amino acids where the murine interleukin 3 (IL-3) sequence has a 40% homology with the x-lor sequence. A statistical analysis shows that this homology is highly significant with a probability of 1.57 X 10(-10). The biological implication of this homology is discussed.     

Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serological cross-reactivity and an amino acid sequence homology of approximately 50%. The three-dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X-ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demonstrates that absence of immunological cross-reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.     

A cDNA from human bone marrow encoding a protein exhibiting homology to the ATP1gamma1/PLM/MAT8 family of transmembrane proteins

A cDNA clone, IWU-1, was cloned from human bone marrow. Its putative open reading frame encoded a protein of 115 amino acids with a calculated molecular mass of 12.9 kDa. The deduced amino acid sequence exhibited high homology (>68%) to members of the ATP1gamma1/PLM/MAT8 family of single transmembrane proteins, primarily in the region containing the putative transmembrane domain. The sequence at the amino-terminal side exhibited high homology (>61%) to the cytoplasmic region of the angiotensin II type 1 receptors.     

Comparison of bovine serum transferrin A and D2. II. Glycopeptides

Glycopeptides are isolated from subtilisin and pronase digests of whole bovine serum transferrin A and D₂. The two variants yield glycopeptides with identical ami-no acid composition. Hence, there is probably no amino acid substitution in this region of the peptide chain. Amino acid sequence determination of one glycopeptide (subtilisin glycopeptide 8) gives the sequence: (CHO)Asn-Ser-Ser-Leu-Cys. This sequence is identical with that of residues 491–495 of the sequence for human serum transferrin (MacGillivray et al., 1982) except that in the bovine transferrin, Asp is replaced by Asn, enabling carbohydrate attachment. A second glycopeptide sequence Arg-(CHO)Asn-Ala-Thr-Tyr is observed, and the significance discussed in relation to carbohydrate moieties of serum glycoproteins.     

Mapping nucleolar localization sequences of 1A6/DRIM

Human 1A6/downregulated in metastasis (DRIM) is a nucleolar protein with multiple HEAT-repeat motifs (Huntington, elongation factor 3, a subunit of protein phosphatase 2A, target of rapamycin). The yeast homologue to 1A6/DRIM, Utp20, is part of the small subunit processome and functions in 18S RNA processing. In the present study, we utilized the green fluorescent protein as the fusion protein marker to investigate the sequence responsible for 1A6/DRIM accumulation in nucleolus. Deletion sequence analysis demonstrated that a single region located between amino acids 2744 and 2761 at the C-terminus of 1A6/DRIM is capable of nucleolar accumulation. Two basic amino acid clusters within this region are essential for nucleolar accumulation. The sequences required for nucleolar accumulation overlaps the putative nuclear localization signal of 1A6/DRIM.     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.     

Amino acid sequence and oligosaccharide distribution of the haemagglutinin from an early Hong Kong influenza virus variant A/Aichi/2/68 (X-31).

The amino acid sequence and oligosaccharide distribution for the haemagglutinin from the early Hong Kong influenza virus A/Aichi/2/68 (X-31) was investigated. The two polypeptide chains, HA1 and HA2, were fragmented by CNBr and enzymic digestion, and the amino acid sequence of each small peptide was deduced by comparing its chromatographic behaviour, electrophoretic mobility, amino acid composition and N-terminus with that of the corresponding peptide of the haemagglutinin of known structure from the influenza-virus variant A/Memphis/102/72. Those peptides in which changes were detected were sequenced fully. The complete amino acid sequence of the haemagglutinin HA1 chain (328 residues) and 188 of the 221 residues of the HA2 chain were established by this approach, and revealed only twelve differences between the amino acid sequences of variant-A/Aichi/68 and -A/Memphis/72 haemagglutinins. These occurred at positions 2, 3, 122, 144, 155, 158, 188, 207, 242 and 275 in the HA1 chain and 150 and 216 in the HA2 chain. The highly aggregated hydrophobic region (residues 180-121) near the C-terminal end of the HA2 chain was not resolved by peptide sequencing. The oligosaccharide distribution in variant-A/Aichi/68 haemagglutinin was identical with that found in that of A/Memphis/72, with sugar units attached at asparagine residues 8, 22 38, 81, 165 and 285 in the HA1 chain and 154 on the HA2 chain. The monosaccharide compositions of the individual carbohydrate units on variant-A/Aichi/68 haemagglutinin differed from those of the corresponding units in variant-A/Memphis/72 haemagglutinin, and evidence was found for heterogeneity in the oligosaccharide units attached at single glycosylation sites.