Nucleotide sequence of the gene encoding the matrix protein of mumps virus (Miyahara strain)

The nucleotide sequence of the gene encoding the matrix (M) protein of mumps virus (MuV), Miyahara strain, has been determined from several overlapping cDNA clones. The M protein mRNA is 1248 nucleotides in length, exclusive of the poly(A) tail, and codes for a protein of 375 amino acids (Mr41,556). Comparison of the deduced amino acid sequence of the M protein of the Miyahara strain with that of the SBL-1 strain revealed that the M proteins of both strains are highly conserved. A significantly lower rate of nucleotide differences conducive to amino acid differences in the M gene compared with other genes appeared to indicate the importance of the conserved primary structure of the M protein for its function.Requests for reprints should be addressed to Kiyoshi Tanabayashi, Department of Measles Virus, National Institute of Health, 4-7-1 Gakuen, Musashimurayama, Tokyo 190-12, Japan.     

Japanese encephalitis virus nonstructural protein NS3 has RNA binding and ATPase activities

Sequence data suggest that Japanese encephalitis virus (JEV) protein NS3 is a multifunctional protein with sequence motifs characteristic of a protease and a helicase. To examine the functions of JEV-NS3, a fusion protein of NS3 inEscherichia coli was generated. Analysis by Western blot using monospecific rabbit antisera generated against the fusion protein (anti-MBJEN3) showed that NS3 was localized in the membrane fraction of JEV-infected cells and the particulate fraction of bacteria extracts. The addition of anti-MBJEN3 sera reduced JEV-specific RNA synthesis activity in a in vitro system. In addition, NS3 was shown to exhibit RNA binding and ATPase activities, suggesting this protein has an important role in viral RNA replication in virus-infected cells.     

Nucleotide sequence of the coat protein coding region of tulip breaking virus

cDNA of tulip breaking virus-tulip (TBV-tulip) RNA was synthesized and cloned inE. coli. One clone that contains a 4.5 kb insert was identified by restriction enzyme analysis, dot immunobinding assay (DIBA), and partial sequencing. Then 1479 nucleotides of the 3-terminus of the clone were sequenced and revealed that the sequence contains one open reading frame (ORF), followed by an untranslated region of 255 nucleotides and a poly(A) tract. The deduced amino acid sequence was found to include the C terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative NIa protease cleavage site at the N terminus of the coat protein.The nucleotide sequence data reported in this paper have been submitted to GenBank nucleotide sequence database and have been assigned the accession number X63630.     

Characterization of Asn130-to-Ala mutant of dengue type 1 virus NS1 protein

The nonstructural protein 1 (NS1) of flavivirus has two N-glycosylation sites that are thought to be important for viral replication. Effects of NS1 glycosylation site mutations on viral replication have been reported in several flaviviruses, but the results have differed. In this report, we examined the role of glycosylation site of NS1 on the replication of dengue type 1 virus (DENV-1). DENV-1 production was not detectable when full-length DENV-1 RNA, which has an N-glycosylation site Asn130-to-Ala (Asn130Ala) mutation in NS1, was transfected into mammalian and mosquito cells. However, replication and secretion of recombinant DENV-1 with the NS1 Asn130Ala mutation were recovered by exogenously expressed wild-type DENV-1 NS1. A growth kinetics experiment showed that propagation of wild-type DENV-1 was prevented by NS1 Asn130Ala mutant expression in trans. Our results suggest that Asn130 of the DENV-1 NS1 is important for viral replication in both mammalian and mosquito cells.     

Nucleotide sequence of cDNA to the rinderpest virus mRNA encoding the nucleocapsid protein

The full-length cDNA corresponding to the mRNA encoding the nucleocapsid protein (NP) of rinderpest virus (RV) was cloned and its complete nucleotide sequence was determined. The gene of RV-NP was composed of 1683 nucleotides and contained a single large open reading frame, which is capable of encoding a protein of 525 amino acids with a molecular weight of 58,241 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of measles virus (MV) and canine distemper virus (CDV). The nucleotide sequence of the coding region of RV-NP (53–1630) revealed a homology of 68.1% and 63.0% with MV and CDV-NP, respectively. Relatively moderate homologies of 68.7% (MV) and 64.3% (CDV) were found at nucleotides 53–592. The highest homology of 75.3–74.3% was equally present between RV and both MV and CDV in the middle region at nucleotides 593–1312. The homologies of the predicted amino acids in this region were 88.3% (MV) and 86.3% (CDV). Relatively low (MV) or little (CDV) homology was detected in the last 318 nucleotides toward the 3 terminus (1313–1630). The predicted secondary structures of amino acids at the C terminus differed between the three viruses.     

Nucleotide sequence of the avian influenza virus A/fowl plague/Rostock/34 segment 1 encoding the PB2 polypeptide

The nucleotide sequence of segment 1 of fowl plague virus (A/FPV/Rostock/34) was determined from a DNA copy inserted at the Pst I site of pBR322. Segment 1 is 2341 nucleotides long and encodes a highly basic protein of 759 amino acid residues (PB2). This protein forms part of the viral polymerase complex and is involved in the recognition of cap structures and endonucleolytic cleavage of eukaryotic mRNAs. Comparisons made between segment 1 of fowl plague virus and three human strains of influenza virus show approximately 85% nucleotide sequence homology and 94% homology between amino acid sequences.     

抗Ⅳ型登革病毒NS1单克隆抗体的制备及特性鉴定

目的:研制Ⅳ型登革病毒(DENV-4)非结构蛋白1(NS1)特异性的单克隆抗体(mAbs),并鉴定其特异性。方法:在表达具有良好抗原性的重组DENV4-NS1蛋白的基础上,用重组DENV4-NS1蛋白和灭活的DENV-4免疫BALB/c小鼠,取免疫小鼠的脾细胞与小鼠骨髓瘤细胞进行融合,用间接ELISA法筛选阳性的杂交瘤细胞,并结合免疫荧光测定法(IFA)和Western blot分析对mAb的特异性进行鉴定;通过竞争抑制试验对mAb识别的抗原位点进行分析。结果:获得15株特异性针对DENV4-NS1的mAbs,与其他3型DENV的NS1不产生交叉反应。mAb的Ig亚类测定均为IgG1。竞争抑制试验显示,15株mAbs存在2个不同识别抗原位点。结论:成功地获得特异性针对DENV4-NS1蛋白的mAbs,为进一步研究DENV4-NS1蛋白的结构与功能及研制DENV-4的临床诊断试剂奠定了基础。     

Vaccinia virus recombinant expressing gene of tick-borne encephalitis virus non-structural NS1 protein elicits protective activity in mice

A vaccinia virus recombinant containing the non-structural tick-borne encephalitis virus (TBEV) NS1 gene was developed. The recombinant expressed native dimeric form of the NS1 protein in infected cells and protected mice against lethal infection with TBEV.     

Expression of bluetongue virus serotype 17 NS1 protein from a cloned gene

A full-length copy of the coding region of segment 6 from bluetongue virus (BTV) serotype 17 was constructed from five overlapping cDNA clones. The gene coding for the NS1 protein was cloned into an expression plasmid under the control of a bacteriophage T7 promoter and expressed both in vitro and in Escherichia coli BL21(DE3) cells which contain a T7 RNA polymerase gene in their chromosome. Expression in both systems resulted in the synthesis of a protein comigrating with NS1 and a minor polypeptide comigrating with another viral-induced protein, NS1a, sometimes seen in BTV-infected cells. The proteins induced in E. coli were synthesized to high levels as insoluble products.     

Ⅰ型登革病毒NS1抗原捕获ELISA的建立和初步临床诊断应用

目的以登革病毒特异性非结构蛋白1（NS1）单克隆抗体为基础建立Ⅰ型登革病毒（DEN1）抗原检测的酶联免疫吸附（ELISA）法，并探索从病人早期血清样品中检测DEN1-NS1的可行性。方法利用已制备的抗DEN1-NS1单克隆抗体（单抗），进行多种抗体组合配对优化模式的分析，建立双抗体夹心抗原捕获ELISA，以469份健康人血清样品确定cut off值，检测DEN1感染患者急性期血清样品。结果对多种抗体组合反复筛选，最终确立了最佳的包被单抗和酶标测定单抗，建立了抗体夹心捕获DEN1-NS1抗原的酶联免疫测定方法，能特异检测DEN1，与其他血清型登革病毒不发生交叉反应。检测16例临床确诊DEN1感染病人急性期血清样品，15例呈特异的抗原反应阳性。结论成功建立了DEN1-NS1抗原捕获ELISA并应用于临床血清样品的检测，为登革热的早期实验室诊断提供技术方法。     

Nucleotide and deduced amino acid sequence of the nonstructural phosphoprotein, NS2, of bluetongue virus serotype 17: comparison to two isolates of serotype 10

The nucleotide sequence of bluetongue virus (BTV) serotype 17 segment 8 from North America (NA) coding for the nonstructural phosphoprotein, NS2, was determined. This segment contains 1125 base pairs and codes for a protein of 40,581 daltons containing 354 amino acids with a net charge of -8.5 at pH 7.0. The carboxyl terminal portion of the protein is very hydrophilic and has a high degree of potential alpha-helix. Serine is the major, if not the exclusive, phosphorylated amino acid residue and ten of the twenty serine residues present in NS2 are found in consensus phosphorylation sites. Comparison of the nucleotide sequence of BTV-17NA segment 8 with the sequence of BTV-10NA and BTV-10 South Africa (SA) revealed a greater degree of homology between different serotypes within the same geographical area, i.e., 17NA and 10NA, than between isolates of the same serotype located in different areas, i.e., 10NA and 10SA. The same homology relationship as above was found at the amino acid level.     

Nucleotide sequence of cDNA encoding the coat protein of beet yellows virus

A cDNA clone of beet yellows viral RNA expressed the viral coat protein gene inE. coli. The sequence of the 2724 nucleotide insert revealed three open reading frames, the 3 of which was shown to be the coat protein cistron. This cistron is expressed inE. coli, in spite of there being no obvious ribosome binding site upstream.     

Rescue of influenza virus expressing GFP from the NS1 reading frame

In this study, several influenza NS1 mutants were examined for their growth ability in interferon (IFN)-deficient Vero cells treated with human interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed similar growth properties as the wild-type virus, whereas viruses carrying an impaired RNA binding domain were dramatically attenuated. Relying on the ability of the first half of the NS1 protein to antagonize the IFN action, we established a rescue system for the NS gene based on the transfection of one plasmid expressing recombinant NS vRNA and subsequent coinfection with an IFN sensitive helper virus followed by adding of human IFN-alpha as a selection drug. Using this method, a recombinant influenza A virus expressing green fluorescence protein (GFP) from the NS1 reading frame was rescued. To ensure the posttranslational cleavage of GFP from the N-terminal 125 amino acids (aa) of NS1 protein, a peptide sequence comprising a caspase recognition site (CRS) was inserted upstream the GFP protein. Although a rather long sequence of 275 aa was inserted into the NS1 reading frame, the rescued recombinant vector appeared to be genetically stable while passaging in Vero cells and was able to replicate in PKR knockout mice.     

5株呼吸道合胞病毒地方株F蛋白基因序列分析

目的呼吸道合胞病毒（ＲＳＶ）Ｆ蛋白是ＲＳＶ感染免疫中最重要的病毒蛋白，为了解我国ＲＳＶ地方株Ｆ蛋白的基因状况和变异特征，随机选取北京、广州、长春和河北四个地区具有不同流行特征的ＲＳＶ地方株（Ａ亚型）５株，进行ＲＳＶＦ蛋白全基因的核苷酸序列分析。方法以提取的病毒ｍＲＮＡ为模板进行ＲＴ－ＰＣＲ扩增、目的基因的克隆及序列测定，对地方株及原型株的序列进行比较分析。结果地方株Ｆ蛋白基因与原型株Ａ２株有很高的同源性，核苷酸全序列的同源性为９５．１％～９６．１％，氨基酸同源性为９６．７％～９７．４％。核苷酸有义突变率为２２．６％～２５．９％。３非编码区的核苷酸序列比蛋白编码区变异显著。河北地方株（Ｅ７３株）在３非编码区有６个核苷酸的插入。Ｆ２亚单位的氨基酸变异高于Ｆ１亚单位。在北京地方株（ＺＨＳ１３株）Ｆ１亚单位内，由具有中和能力单克隆抗体所识别的抗原表位区中存在一个氨基酸的变异。结论我国ＲＳＶ地方株与原型株之间的Ｆ蛋白基因尽管存在一定的变异，但仍有很高的同源性。地方株间Ｆ蛋白的核苷酸、氨基酸变异的位置及形式很相似，提示我国ＲＳＶ的不同流行特征可能并非由于Ｆ蛋白的基因变异所致。     

Nucleotide sequence of the entire protein coding region of canine distemper virus polymerase-associated (P) protein mRNA

The entire coding region of the polymerase-associated (P) protein gene of canine distemper virus has been sequenced. A single cDNA clone which represents 98% of the mRNA encoding this protein was used to determine the nucleotide sequence. The sequence predicts a major protein of 507 amino acids and a molecular weight of 54 936. There is also a second, overlapping, open reading frame with a start signal 21 bases downstream of the first AUG which could code for a protein of 174 amino acids with a predicted molecular weight of 20 292. This arrangement of the genome for the P protein of canine distemper virus is exactly analogous to that published recently for the P gene of measles virus (Bellini, W.J. et al., 1985, J. Virol. 53, 908-919). When the sequences are aligned at the first AUG, considerable homology is seen at both the nucleotide and protein sequence level.     

Nucleotide sequence of the capsid protein gene of potato virus Y (PVY)

The nucleotide sequence of the 3 terminal region of potato virus Y (PVY) was determined. Starting with a poly(A) tail of 18 residues a non-coding region of 335 nucleotides precedes the region encoding for the virus coat protein (cp) 801 nucleotides long ending with a TGA. This region was located by comparing the predicted amino acid sequence with the one determined for the PVY capsid protein by Shukla et al. (1). Both sequences contained 267 amino acids sharing about 94% homology. They differ, however, at several positions presumably due to base transitions within their respective nucleotide sequences. Restriction endonuclease sites in and around the cp coding region were identified.     

Conserved nucleotide sequence of rabies virus cDNA encoding the nucleoprotein

The cDNAs of rabies virus (the CVS strain) encoding the structural proteins (G, N, NS, and M) were cloned. Of these clones, the nucleotide sequence of the cDNA encoding the nucleoprotein was determined to compare with those of other strains of rabies virus. The comparison confirmed that the nucleotide sequences and deduced amino acid sequences are highly conserved among strains including an avirulent strain.     

Comparison of the reovirus serotype 1, 2, and 3 S3 genome segments encoding the nonstructural protein sigma NS

The sequences of the S3 genome segments of reovirus serotype 1 and 2 prototype strains are presented and these sequences are compared with the sequence of the serotype 3 S3 genome segment. The S3 genome segment encodes the nonstructural protein sigma NS which possesses affinity for ssRNA and appears to function in reovirus morphogenesis. The three S3 genome segments are closely related: all are 1198 nucleotides long and possess a single long open reading frame 366 codons long. They exhibit a serotype 1:3 relatedness pattern: there are only 13% mismatches between the S3 genome segments of serotypes 1 and 3, but 27 and 26% mismatches, respectively, between those of serotype 1 and 2 and serotype 3 and 2. The amino acid mismatches for the three sigma NS proteins are much lower (2.7, 13.9, and 13.7%, respectively), because the majority of nucleotide mismatches are in third base codon positions. The three sigma NS proteins possess a conserved secondary structure that is rich in alpha-helix content; in fact, the predicted alpha-helix content of these nonstructural proteins (about 50%) is much higher than that of the three other sigma size class proteins (about 20%), all of which are structural proteins. We also sequenced the S3 genome segment of a ts mutant of serotype 3 generated by treatment with nitrous acid and found a single nucleotide change that specifies an amino acid change that introduces a five-residue-long beta-sheet prone configuration into a long (80 amino acids) highly conserved alpha-helix in the C-terminal half of sigma NS. This change could account for this mutant's ts character. Finally, the three sigma NS proteins have diverged in only about 10% of positions, whereas the three sigma 1 proteins have diverged in about 70%. The rapid evolutionary divergence of the latter may be a result of several factors, including (i) the fact that sigma 1, but not sigma NS, is under immunologic selective pressure; (ii) the fact that the functions of sigma 1 (antigenicity and cell attachment) probably reside in two rather small domains that are not restricted spatially with respect to each other; and (iii) the fact that the functions of sigma NS, namely RNA binding and protein binding (during morphogenesis), require a highly specific overall protein configuration that may permit little variation.     

CDK/ERK-mediated phosphorylation of the human influenza A virus NS1 protein at threonine-215

Posttranslational modification of viral proteins by cellular enzymes is a feature of many virus replication strategies. Here, we report that during infection the multifunctional human influenza A virus NS1 protein is phosphorylated at threonine-215. Substitution of alanine for threonine at this position reduced early viral propagation, an effect apparently unrelated to NS1 antagonizing host interferon responses or activating phosphoinositide 3-kinase signaling. In vitro, a subset of cellular proline-directed kinases, including cyclin dependent kinases (CDKs) and extracellular signal-regulated kinases (ERKs), potently phosphorylated NS1 protein at threonine-215. Our data suggest that CDK/ERK-mediated phosphorylation of NS1 at threonine-215 is important for efficient virus replication.