Complete nucleotide sequence of the nucleoprotein gene of influenza B virus.

A DNA copy of influenza B/Singapore/222/79 viral RNA segment 5, containing the gene coding for the nucleoprotein (NP), has been cloned in Escherichia coli plasmid pBR322, and its nucleotide sequence has been determined. The influenza B NP gene contains 1,839 nucleotides and codes for a protein of 560 amino acids with a molecular weight of 61,593. Comparison of the influenza B NP amino acid sequence with that of influenza A NP (A/PR/8/34) reveals 37% direct homology in the aligned regions, indicating a common ancestor. However, influenza B NP has an additional 50 amino acids at its N-terminal end. As is the case with influenza A NP, influenza B NP is a basic protein, with its charged residues relatively evenly distributed rather than clustered. The structural homology suggests functional similarity between the NP of influenza A and B viruses.     

Total nucleotide sequence of a nearly full-size DNA copy of satellite tobacco necrosis virus RNA

pSTNV-1 is a chimera plasmid that contains a nearly full-size double-stranded DNA copy of the satellite tobacco necrosis virus RNA genome (see preceding paper by van Emmelo et al., 1980) and we report here the complete nucleotide sequence of this STNV² DNA insert. The results show that except for 23 nucleotide pairs corresponding to the 5′ end of STNV RNA, a full-size STNV DNA copy is present in pSTNV-1. The total nucleotide sequence of the STNV genome contains 1239 residues. The amino acid sequence of the coat protein can be deduced from the 5′ half of the DNA message strand and shows a rather hydrophobic carboxyl-terminal region and a basic amino-terminal region. The 3′ untranslated part of the viral RNA is 622 nucleotides long. A secondary structure model for the 5′ end showing an interaction with a segment in the 3′ half is proposed. The 3′ end region can be folded into a transfer RNA cloverleaf-like structure with an anticodon for AUG.     

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.     

Nucleotide sequence of human influenza A/PR/8/34 segment 2.

The nucleotide sequence of RNA segment 2 of human influenza strain A/PR/8/34 has been determined. Segment 2 in 2341 nucleotides long and encodes a protein of 757 amino acids (86,500 daltons molecular weight) which is involved in RNA synthesis. Although segment 2 is identical in size to segment 1, which encodes a protein of related function, neither the nucleotide sequences of these two RNA segments nor the amino acid sequences of the encoded proteins appear to be homologous. The sequence of segment 2 completes the sequence of the virus (total 13,588 nucleotides).     

Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.     

Influenza C virus RNA 7 codes for a nonstructural protein.

The complete nucleotide sequence of RNA segment 7 of influenza C/California/78 virus was determined by using cloned cDNA derived from viral RNA. The gene is 934 nucleotides long and possesses a long open reading frame which can code for a protein of 286 amino acids. Hybrid arrest translation experiments with the cloned cDNA fragment and poly(A)-containing RNA isolated from virus-infected cells showed that a 28,500-molecular-weight protein is coded for by RNA 7. Comparison of the proteins induced in the cell-free system and in virus-infected cells with those found in purified virus suggests that the 28,500-molecular-weight protein is a nonstructural protein.     

Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic.     

Sequence of the nucleocapsid gene from murine coronavirus MHV-A59.

The nucleotide sequence of the RNA encoding the nucleocapsid protein of coronavirus MHV-A59 has been determined. Copy DNA was prepared from mRNA isolated from virally infected cells, fragmented and cloned in the phage vector M13 mp8 for direct sequence determination. A sequence of 1817 nucleotides, adjacent to the viral poly-A tail, was obtained. It contains a single long open reading frame encoding a protein of mol. wt. 49660, which is enriched in basic residues.     

Sub-etioplast localization of the enzyme NADPH: protochlorophyllide oxidoreductase

The complete nucleotide sequence of the influenza A/PR/8/34 nucleoprotein gene was determined after cloning for dsDNA copy in pBR322. The nucleoprotein gene is 1517 nucleotides long of which 1446 nucleotides code for 482 amino acids. The calculated amino acid composition is in good agreement with those published for influenza A nucleoprotein genes. The amino acid sequence of the nucleoprotein contains clusters of basic amino acids and proline, a property shared with other nucleic-acid-associated proteins like Semliki forest virus nucleocapsid protein, VP1 protein of polyoma virus and Simian virus 40, and the core antigen of hepatitis B virus. The described nucleoprotein structure brings the number of sequenced genes of influenza A/PR/8/34 to five out of eight genes.     

Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

The nucleocapsid (NC) protein of human immunodeficiency virus HIV-1 (NCp7) is responsible for packaging the viral RNA by recognizing a packaging site (PSI) on the viral RNA genome. NCp7 is a molecule of 55 amino acids containing two zinc fingers, with only the first one being highly conserved among retroviruses. The first zinc finger is flanked by two basic amino acid clusters. Here we demonstrate that chemically synthesized NCp7 specifically binds to viral RNA containing the PSI using competitive filter binding assays. Deletion of the PSI from the RNA abrogates this effect. The 35 N-terminal amino acids of NCp7, comprising the first zinc finger, are sufficient for specific RNA binding. Chemically synthesized mutants of the first zinc finger demonstrate that the amino acid residues C-C-C/H-C/H are required for specific RNA binding and zinc coordination. Amino acid residues F16 and T24, but not K20, E21 and G22, located within this zinc finger, are essential for specific RNA binding as well. The second zinc finger cannot replace the first one. Furthermore, mutations in the basic amino acid residues flanking the first zinc finger demonstrate that R3, 7, 10, 29 and 32 but not K11, 14, 33 and 34 are also essential for specific binding. Specific binding to viral RNA is also observed with recombinant NCp15 and Pr55Gag. The results demonstrate for the first time specific interaction of a retroviral NC protein with its PSI RNA in vitro.     

中国西藏小反刍兽疫病毒China/Tib/Gei/07-30磷蛋白基因的分子特征及其编码蛋白特性分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行磷蛋白基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出病毒磷蛋白基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。小反刍兽疫病毒China/Tib/Gej/07-30磷蛋白基因由1 655个核苷酸组成,编码2个相互交叠的开放阅读框(ORF)。第一个ORF长度为1 530个核苷酸,编码的P蛋白长度为509个氨基酸。第二个ORF长度为534个核苷酸,编码的C蛋白长度为177个氨基酸。第一个ORF通过基因编辑在751位插入1个G核苷酸,转录生成第二个mRNA,长度为897个核苷酸,编码的V蛋白长度为298个氨基酸。小反刍兽疫病毒China/Tib/Gej/07-30的P蛋白与其他分离株氨基酸序列同源性为86.1%～97.3%,C蛋白氨基酸序列相似性为84.3%～94.9%,V蛋白为82.9%～96.3%。China/Tib/Gej/07-30的P蛋白第315～387位氨基酸是一段高度保守的七肽重复序列。     

Molecular characterization of messenger RNAs for 'pathogenesis related' proteins la, lb and lc, induced by TMV infection of tobacco

A cDNA library was made to poly(A)-containing RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco plants and clones corresponding to mRNAs for the `pathogenesis-related' (PR) proteins 1a, 1b and 1c were identified. One clone was found to contain a complete copy of PR-1b mRNA. The structural organization of this RNA is: a leader sequence of 29 nucleotides, an open reading frame of 504 nucleotides encoding a 30 amino acid long signal peptide and a 138 amino acid long mature protein, and a 3'-non-coding region of 235 nucleotides. Two other clones were found to contain partial copies of PR-1a and PR-1c mRNAs. The data indicate an ~90% homology between the amino acid sequences of PR-1a, -1b and -1c. Using one of the clones as probe it was shown that in the TMV-inoculated lower leaves and the non-inoculated upper leaves of a tobacco plant, the PR-1 mRNAs become detectable from 2 and 8 days after inoculation, respectively.     

A cDNA clone for the precursor of rat mitochondrial ornithine transcarbamylase: comparison of rat and human leader sequences and conservation of catalytic sites.

We have cloned a DNA complementary to the messenger RNA encoding the precursor of ornithine transcarbamylase from rat liver. This complementary DNA contains the entire protein coding region of 1062 nucleotides and 86 nucleotides of 5'- and 298 nucleotides of 3'-untranslated sequences. The predicted amino acid sequence has been confirmed by extensive protein sequence data. The mature rat enzyme contains the same number of amino acid residues (322) as the human enzyme and their amino acid sequences are 93% homologous. The rat and human amino-terminal leader sequences of 32 amino acids, on the other hand, are only 69% homologous. The rat leader contains no acidic and seven basic residues compared to four basic residues found in the human leader. There is complete sequence homology (residues 58-62) among the ornithine and aspartate transcarbamylases from E. coli and the rat and human ornithine transcarbamylases at the carbamyl phosphate binding site. Finally, a cysteine containing hexapeptide (residues 268-273), the putative ornithine binding site in Streptococcus faecalis, Streptococcus faecium, and bovine transcarbamylases, is completely conserved among the two E. coli and the two mammalian transcarbamylases.     

Structure of a bacterial photosynthetic membrane: integrity of reaction centers following proteolysis and detergent solubilization

cDNAs were molecularly cloned for proteins specifically expressed in embryo as well as in a chemically induced rat pancreatic B cell tumor in which virally related oncogenes such as v-myc, v-src, v-yes, v-mos and v-kis were previously demonstrated not to be expressed. A plasmid cDNA library consisting of 48,000 independent colonies was constructed from poly(A) containing cytoplasmic RNA isolated from 12 day rat embryo. The library was screened by hybridization with 32p-labelled cDNA synthesized from poly(A) containing RNA of rat pancreatic B cell tumor or normal islet B cells. Two clones were obtained which showed a clearly positive reaction only with tumor probe. Nucleotide sequence of one of them harboring insert of 615 nucleotides was determined and its amino acid sequence of 119 residues was deduced, which showed that the protein encoded by this mRNA is highly basic, basic residues/acidic residues being 1.63.     

Identification of basic amino acids at the N-terminal end of the core protein that are crucial for hepatitis C virus infectivity

A major function of the hepatitis C virus (HCV) core protein is the interaction with genomic RNA to form the nucleocapsid, an essential component of the virus particle. Analyses to identify basic amino acid residues of HCV core protein, important for capsid assembly, were initially performed with a cell-free system, which did not indicate the importance of these residues for HCV infectivity. The development of a cell culture system for HCV (HCVcc) allows a more precise analysis of these core protein amino acids during the HCV life cycle. In the present study, we used a mutational analysis in the context of the HCVcc system to determine the role of the basic amino acid residues of the core protein in HCV infectivity. We focused our analysis on basic residues located in two clusters (cluster 1, amino acids [aa]6 to 23; cluster 2, aa 39 to 62) within the N-terminal 62 amino acids of the HCV core protein. Our data indicate that basic residues of the first cluster have little impact on replication and are dispensable for infectivity. Furthermore, only four basic amino acids residues of the second cluster (R50, K51, R59, and R62) were essential for the production of infectious viral particles. Mutation of these residues did not interfere with core protein subcellular localization, core protein-RNA interaction, or core protein oligomerization. Moreover, these mutations had no effect on core protein envelopment by intracellular membranes. Together, these data indicate that R50, K51, R59, and R62 residues play a major role in the formation of infectious viral particles at a post-nucleocapsid assembly step.     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.     

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 haemagglutinin gene from a human influenza virus of the Hong Kong subtype.

The complete nucleotide sequence has been determined for a cloned double-stranded DNA copy of the haemagglutinin gene from the human influenza strain A/NT/60/68/29C, a laboratory-isolated variant of A/NT/60/68, an early strain of the Hong Kong subtype. The gene is 1765 nucleotides long and contains information sufficient to code for a protein of 566 amino acids, which includes a hydrophobic leader peptide (16 residues), HA1 (328), HA2 (221) and an arginine residue which joins the HA subunits. Comparison of the predicted amino acid sequence for 29C haemagglutinin with protein sequence data available for HA from other influenza strains shows that no potential coding information is lost by processing of the mRNA. A comparison of the amino acid sequences predicted from the gene sequences for 29C and fowl plague virus haemagglutinins, (1) indicates the extent to which changes can occur in the primary sequence of different regions of the protein, while maintaining essential structure and function.