Evolutionary relatedness of the predicted gene product of RNA segment 2 of the tick-borne Dhori virus and the PB1 polymerase gene of influenza viruses

The complete nucleotide sequence of the second largest RNA segment of Dhori/India/1313/61 virus was determined and the deduced amino acid sequence was compared with the polymerase (P) proteins of influenza A, B, and C viruses. RNA segment 2 (2224 nucleotides) of Dhori virus contains a single long open reading frame that can encode a 716-amino acid polypeptide (81.3 kDa). The predicted polypeptide shares between 27 and 31% sequence identities with the PB1 polypeptides of influenza A, B, and C viruses. Among the regions most highly conserved are the sequences around the Asp-Asp motif common to many RNA polymerases. In spite of the high level of sequence identity between the Dhori RNA segment 2 gene product and the influenza A, B, and C virus PB1 proteins the amino acid composition of the Dhori protein indicates an acidic charge feature at pH 7.0 in contrast to the basic nature of the PB1 proteins of the influenza viruses. We suggest that the Dhori PB1-like protein be designated the P alpha protein of this virus.     

Influenza B virus PB1 protein; nucleotide sequence of the genome RNA segment predicts a high degree of structural homology with the corresponding influenza A virus polymerase protein

The complete nucleotide sequence of a cloned cDNA copy of the genome RNA segment encoding the influenza B/Lee/40 virus PB1 polymerase protein has been determined. The genome RNA segment is 2368 nucleotides in length and is capable of encoding a polymerase (PB1) protein of 752 amino acids with a calculated mol mass of 84,407 Da. As expected, the protein is highly basic with a net charge of +20 at pH 7.0. Sequence comparison between the influenza A and B virus PB1 proteins reveals that they share 61% amino acid homology. An internal hydrophobic domain and 90% of the proline residues found within the influenza A virus PB1 protein are conserved in the influenza B virus molecule. The influenza A and B virus PB1 proteins share the highest homology yet seen between proteins encoded by these disparate viruses. This remarkable conservation of primary structure argues for severe functional constraint on the evolution of this influenza virus polymerase protein.     

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain H?lln?s B1 coding for the viral RNA polymerase

The L RNA segment of the nephropathia epidemica virus (NEV) strain H?lln?s B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3' and 5' termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30-40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza type A. These results indicate that the L RNA segment of the NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.     

Comparison of the three large polymerase proteins of influenza A, B, and C viruses

The three large RNA segments of influenza C virus C/JJ/50 were cloned and sequenced, and the deduced amino acid sequences were compared with those of the polymerase (P) proteins of influenza A and B viruses. The coding strategy of the C virus RNA segments is the same as that for the large A and B virus segments as one long open reading frame is present in each segment. RNA segment 1 of influenza C virus encodes the equivalent of the PB2 protein; it has an approximate 25% sequence identity with the corresponding (cap binding) influenza A and B virus PB2 proteins. The PB1 protein of influenza C virus, coded for by segment 2, has an approximate 40% sequence identity with the corresponding proteins of influenza A and B viruses including the Asp-Asp sequence motif found in many RNA polymerase molecules. The PB1 polymerase is thus the most highly conserved protein among the influenza A, B, and C viruses. Although the protein coded for by RNA 3 of influenza C virus shows an approximate 25% sequence identity with the acid polymerase (PA) proteins of the A and B viruses, its sequence does not display any acid charge features at neutral pH. This protein is thus referred to as the P3 (rather than the PA) protein of influenza C virus.     

Rescue of synthetic RNAs into thogoto and influenza A virus particles using core proteins purified from Thogoto virus

The ribonucleoprotein (RNP) complexes of Thogoto virus (THOV), a tick-borne orthomyxovirus, have been purified from detergent-lysed virions. The purified RNPs were then disrupted by centrifugation through a CsCl-glycerol gradient to obtain fractions highly enriched in nucleoprotein (NP) and virtually devoid of viral genomic RNA. When these NP-enriched fractions were incubated with a synthetic THOV-like RNA, and the mixtures were transfected into THOV-infected cells, the synthetic RNA was expressed and packaged into THOV particles. Similarly, hybrid mixtures containing purified THOV NP and influenza A virus synthetic RNAs (either a model CAT RNA or a gene encoding the viral neuraminidase), were prepared and transfected into influenza A virus-infected cells. The synthetic CAT RNA, was shown to be expressed and packaged into virus particles, and the neuraminidase gene was rescued into influenza virions. These data are discussed in terms of the similarities observed between THOV and influenza A virus and the potential application of the THOV purified proteins for rescuing synthetic genes into infectious viruses.     

Nucleotide sequence and coding capacity of the large (L) genomic RNA segment of Seoul 80-39 virus, a member of the hantavirus genus.

The nucleotide sequence of the large (L) genomic RNA segment of Seoul 80-39 virus was determined from overlapping cDNA clones. The virion L RNA segment is 6530 nucleotides long. The 3' and 5' terminal sequences are inversely complementary for 15 bases. The viral complementary-sense RNA contains a single open reading frame from an AUG codon at nucleotide position 37-39 to a UAA stop codon at nucleotide position 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 kDa) which likely corresponds to the L protein detected in purified viral particles (Elliott et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). Comparison of the L protein of the Seoul 80-39 virus with the polymerase proteins encoded by other negative-stranded RNA viruses revealed 44% similarity only with the part of the Bunyamwera virus L protein (Elliott, 1989) and a very weak homology with the PB1 protein of influenza virus.     

流感病毒RNA聚合酶蛋白PB1在小鼠中可诱导亚型间交叉免疫保护

目的探索流感病毒RNA聚合酶PB2和PB1亚基作为实验性流感疫苗候选抗原的可能性.方法以复制型质粒（pSCA）为载体分别构建表达甲1型和甲3型流感PB2和PB1的复制型DNA疫苗,免疫小鼠后分别用甲1型流感病毒（A/PR/8/34）进行鼻腔攻击,观察针对不同亚型流感病毒的复制型DNA疫苗的免疫保护效果.结果本实验所构建的复制型DNA疫苗在真核细胞中均可表达外源基因;本实验采用的复制型质粒载体（pSCA）与传统质粒载体（pcDNA3）在诱导小鼠产生抗体方面无差异,并且都诱导了偏向TH1类的免疫反应;表达甲1型和甲3型流感PB1基因的复制型DNA疫苗均可保护小鼠抵御甲1型流感病毒（A/PR/8/34）的攻击.结论表达甲型流感病毒PB1的复制型DNA疫苗能保护小鼠抵御同型和异型流感病毒的攻击,本实验为流感疫苗研究提供新的候选抗原.     

Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus

Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.     

RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses

The sequence of the RNA genome of bovine ephemeral fever virus (BEFV) was determined from the start of the L (polymerase) gene to the end of the untranslated 5′ trailer sequence, completing the sequence of the 14 900 nucleotide (nt) genome. The 6470 nt L gene encodes a single long ORF of 2144 amino acids with a deduced molecular weight of 249 766 Da. The 70 nt BEFV 5′ trailer region displays partial terminal complementarity with the 3′ leader sequence and contains a 26 nt direct repeat of the U-rich domain of the 3′ leader region. The 47 nt 5′ trailer region of Adelaide River virus (ARV) displays terminal sequence similarity to the BEFV trailer and partial terminal complementarity with the ARV 3′ leader sequence, but does not contain the direct repeat sequence. The BEFV L protein contains all characteristic sequence motifs of amino acid blocks I–VI, conserved among RNA polymerase proteins of single-stranded (−) RNA viruses, separated by regions of lower homology. Phylogenetic analysis using the complete BEFV L protein sequence indicated a closer relationship to vesicular stomatitis virus than to rabies virus. Sequence comparison of two conserved central domains encompassing blocks II and III and block VI of the BEFV and ARV L proteins indicated they are closely related. An extended phylogenetic analysis using the block III sequence, confirmed the relationship of these ephemeroviruses to vesiculo- and lyssaviruses and to other single-stranded (−) RNA viruses.     

Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae

The complete nucleotide sequence of the large (L) genome segment of Bunyamwera virus has been determined from overlapping cDNA clones. The segment is 6875 nucleotides long and has a base composition of 29.8% A, 17.9% C, 15.4% G, and 36.9% U. Eighteen of the terminal 19 nucleotides at the 3' and 5' ends are complementary. In the viral-complementary (+ sense) RNA there is a single long open reading frame (ORF) from AUG at bases 51-53 to a UAG stop codon at bases 6765-6767; this ORF encodes a polypeptide of 2238 amino acids (MW 259,000), corresponding to the L protein which has been mapped to the L RNA segment by analysis of reassortants of Bunyamwera, Batai, and Maguari viruses. The amino-terminal 46 amino acids of the L protein show strong homology (63% identity) with the amino-termini of ORFs predicted from limited sequence analysis of the L segments of La Crosse and snowshoe hare bunyaviruses. Comparison with the polymerase proteins encoded by other negative-strand viruses showed weak homology with part of the influenza virus PB1 protein, but no homology was detected with the other influenza virus polymerase proteins nor with the L proteins of arenaviruses, paramyxoviruses, and rhabdoviruses. At the 5' end of genomic (- sense) RNA there is an AUG-initiated ORF potentially encoding a protein of 14,700; the significance of this ORF is unknown at present.     

Nucleotide sequences of the PA and PB1 genes of B/Ann Arbor/1/66 virus: comparison with genes of B/Lee/40 and type A influenza viruses

The complete sequences of the PA and PB1 genome RNA segments of B/Ann Arbor/1/66 virus have been determined. The PA vRNA is 2308 bases long. Its complementary RNA has a single open reading frame of 2187 bases, capable of encoding a PA protein of 726 amino acids with a molecular weight of 83,175 Da. The predicted PA polypeptide has an overall net charge of -7.5 at pH 7.0. The PB1 vRNA is 2369 bases long. Its complementary RNA has a single open reading frame of 2277 bases, capable of encoding a PB1 protein of 752 amino acids with a molecular weight of 84,332 Da. The predicted PB1 polypeptide has an overall net charge of +18.5 at pH 7.0. Sequence homology comparisons of the PA and PB1 polypeptides from B/Ann Arbor/1/66 virus to the PA and PB1 polypeptides of type A influenza virus reveal respective homologies of approximately 38 and 60%. This high cross-type homology (61%) was previously reported for the PB1 protein of B/Lee/40 virus (Kemdirim et al., 1986). The cross-type homology for the PA protein is similar to that of other non-polymerase proteins, but is substantially lower than that seen for the PB1 protein. Thus, the high cross-type homology that exists for the PB1 gene does not appear to be a characteristic of all polymerase genes.     

汉坦病毒A9株全长L片段cDNA克隆和核苷酸序列测定

目的克隆我国分离的汉坦病毒A9株L片段全长cDNA,并测定其核苷酸序列.方法用逆转录-聚合酶链反应(RT-PCR)技术分段扩增汉坦病毒A9株全部L片段,用T-A克隆方法进行PCR产物克隆,测定PCR产物的核苷酸序列.通过亚克隆将分段的L片段连接成全长cDNA克隆.结果A9株的基因组L片段长度为6533个核苷酸,腺嘌呤核苷酸和尿嘧啶核苷酸丰富(%A+U=62.47).包含有一个单一的开放读码框架(ORF),编码一个标准的质量为2.46×105的蛋白,含有2151个氨基酸.A9株与76-118、C1-1和C1-2株的同源性最高,达到83.8%.与TULA病毒的关系较远,其核酸序列的同源性为65.8%.将推导的A9株编码的氨基酸序列与其他21种负链RNA病毒的依赖RNA的RNA聚合酶的氨基酸序列以及汉坦病毒几个代表株的L片段氨基酸序列进行比较,显示A9编码的RNA聚合酶也有6个比较保守的区域以及几个极端保守的氨基酸残基.结论汉坦病毒A9株L片段具有和其他汉坦病毒RNA聚合酶相似的核苷酸一级结构,通过对推导的氨基酸分析,该片段具有一些在RNA病毒聚合酶中都存在的保守区域.     

Molecular cloning and sequencing of influenza virus A/Victoria/3/75 polymerase genes: sequence evolution and prediction of possible functional domains

The influenza virus A/Victoria/3/75 (H3N2) polymerase genes encoding PB1, PB2 and PA have been cloned by cDNA synthesis and insertion into bacterial vectors. The complete sequence for each polymerase gene has been obtained from random M13 subclones and compared to other influenza virus polymerase genes. A total of 45, 74 and 78 nucleotide changes were fixed in the period 1968-1975, corresponding to 10, 12 and 9 amino acid changes, for PB1, PB2 and PA genes, respectively. The amino acid sequence of PB1 polypeptide contains motifs found in a series of positive- and negative-RNA virus polymerase genes and that of PA polypeptide share invariant residues common to DNA and presumptive RNA helicases.     

Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2)

Nucleotide sequences have been obtained for RNA segments encoding the PB2, PB1, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.     

Sequence analysis of coat protein gene of Wuhan nodavirus isolated from insect

Wuhan nodavirus (WhNV) particles are isometric, non-enveloped, and about 29 nm in diameter. In the previous study, we determine its physiochemical characterization and the nucleotide sequence of the larger genomic segment, RNA1 and identify it a nodavirus. WhNV RNA1 is 3,149 nt in length, encoding protein A, catalytic subunit of RNA-dependent RNA polymerase (RdRp). In this report, we complete the sequence determination of the smaller genomic segment, RNA2 of WhNV. WhNV RNA2 is determined to be 1,562 nt long, containing a 430-amino-acid open reading frame (ORF) encoding the coat protein of WhNV with a calculated molecular mass of 47,856 Da. The homology of the coat protein of WhNV and the homologous proteins of other nodaviruses either alphanodaviruses or betanodaviruses is very low. WhNV coat protein shares the highest identity (24%) with that of Lates calcarifer encephalitis virus (LCEV), a betanodavirus, and shares less than 16% identical amino acids with each of the alphanodaviruses. Furthermore, the prediction of WhNV capsid structure by 3D-PSSM shows that the capsid structure of WhNV resembles that of tomato bushy stunt virus (TBSV), a tombusvirus, which contains two domains, rather than the expected single-domain capsid protein of insect nodaviruses. The phylogenetic analysis indicates that WhNV is the most distantly related of both the alphanodaviruses and betanodaviruses, which provides significant new data for understanding the evolution of the nodavirus family.