==================================BSR56================================== 56. Display the 15 base pairs upstream of the coding region for structural proteins. 1 UI - 87092279 AU - Conboy J ; Kan YW ; Shohet SB ; Mohandas N TI - Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton. AB - Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of non-erythroid tissues. In mammalian erythrocytes, it plays a key role in regulating membrane physical properties of mechanical stability and deformability by stabilizing spectrin-actin interaction. We report here the molecular cloning and characterization of human erythrocyte protein 4.1 cDNA and the complete amino acid sequence of the protein derived from the nucleotide sequence. Probes prepared from the cloned erythrocyte protein 4.1 cDNA hybridized with distinct mRNA species from a wide variety of non-erythroid tissues, including brain, liver, placenta, pancreas, and intestine, implying substantial homology between erythroid and non-erythroid protein 4.1. The availability of cloned erythrocyte protein 4.1 cDNA should facilitate the study of the functional characteristics of this protein in erythroid as well as non-erythroid cells. MH - Amino Acid Sequence ; Base Sequence ; Blood Proteins/*GENETICS ; Cloning, Molecular ; DNA/GENETICS ; Erythrocyte Membrane/*ULTRASTRUCTURE ; Human ; Membrane Proteins/*GENETICS ; Protein Conformation ; Reticulocytes/ PHYSIOLOGY ; RNA, Messenger/METABOLISM ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Tissue Distribution SO - Proc Natl Acad Sci USA 1986 Dec;83(24):9512-6 2 UI - 87085488 AU - Spriggs MK ; Collins PL TI - Sequence analysis of the P and C protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins. AB - The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared. MH - Amino Acid Sequence ; Base Sequence ; Capsid/ANALYSIS/*GENETICS ; Comparative Study ; Distemper Virus, Canine/ANALYSIS/GENETICS ; DNA ; *Genes, Viral ; Human ; Measles Virus/ANALYSIS/GENETICS ; Nucleic Acid Hybridization ; Para-Influenza Virus Type 1/ANALYSIS/GENETICS ; Para-Influenza Virus Type 3/ANALYSIS/*GENETICS ; Para-Influenza Viruses/ *GENETICS ; Paramyxoviridae/*ANALYSIS/GENETICS ; Peptide Mapping ; Respiratory Syncytial Viruses/ANALYSIS/GENETICS ; RNA, Messenger/GENETICS ; RNA, Viral/*GENETICS ; Viral Core Proteins/ANALYSIS/*GENETICS ; Viral Proteins/ANALYSIS/*GENETICS SO - J Gen Virol 1986 Dec;67 ( Pt 12):2705-19 3 UI - 87075646 AU - H:jrup P ; Andersen SO ; Roepstorff P TI - Primary structure of a structural protein from the cuticle of the migratory locust, Locusta migratoria. AB - The complete amino acid sequence of a structural protein isolated from pharate cuticle of the locust Locusta migratoria was determined. The protein has an unusual amino acid composition: 42% of the residues are alanine and only 14 of the 20 common amino acid residues are present. The primary structure consists of regions enriched in particular amino acid residues. The N-terminal region and a region close to the C-terminus are enriched in glycine. The rest of the protein is dominated by alanine, except for two short regions enriched in hydrophilic residues. Almost all the proline residues are situated in the alanine-rich regions in a conserved sequence 'A-A-P-A/V'. An internal duplication has taken place covering most of the protein except for the glycine-rich regions. Owing to the unusual features of the protein a combination of automated Edman degradations and plasma-desorption m.s. was used to determine the complete sequence. The protein does not show sequence homology to other proteins, but proteins divided into regions enriched in the same kind of amino acid residues have been isolated from other insect structures. MH - Amino Acid Sequence ; Animal ; Chromatography, High Pressure Liquid ; Chymotrypsin ; Grasshoppers/GENETICS/*METABOLISM ; Peptide Mapping ; *Proteins/GENETICS ; Sequence Homology, Nucleic Acid ; Spectrum Analysis, Mass ; Support, Non-U.S. Gov't ; Trypsin SO - Biochem J 1986 Jun 15;236(3):713-20 4 UI - 87071658 AU - Deubel V ; Kinney RM ; Trent DW TI - Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype. AB - The nucleotide sequence of the 5'-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5'-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5'-C-prM(M)-E-3' and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses. MH - Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Codon ; Comparative Study ; Dengue Virus/CLASSIFICATION/*GENETICS ; DNA/GENETICS ; Flaviviruses/GENETICS ; Protein Conformation ; Solubility ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Viral Proteins/*GENETICS SO - Virology 1986 Dec;155(2):365-77 5 UI - 87053878 AU - Portela A ; Melero JA ; de la Luna S ; Ort:in J TI - Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes. AB - Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes. MH - Animal ; Base Sequence ; Cell Line ; *Cell Transformation, Viral ; Cercopithecus aethiops ; Cloning, Molecular ; DNA Restriction Enzymes ; Gene Amplification ; *Genes, Viral ; Orthomyxoviridae/*GENETICS ; Plasmids ; Support, Non-U.S. Gov't ; Vero Cells ; Viral Proteins/ *GENETICS SO - EMBO J 1986 Sep;5(9):2387-92 6 UI - 87048799 AU - Munemitsu SM ; Atwater JA ; Samuel CE TI - Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide sigma 1a and the nonstructural polypeptide sigma 1bNS. AB - Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames. The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS. Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment. MH - Base Sequence ; Capsid/*GENETICS ; Cloning, Molecular ; DNA ; Escherichia Coli/GENETICS ; Genes, Structural ; Reoviridae/*GENETICS ; RNA/ANALYSIS ; RNA, Double-Stranded/ANALYSIS ; RNA, Messenger/ANALYSIS ; RNA, Viral/ *ANALYSIS ; Support, U.S. Gov't, P.H.S. ; Viral Proteins/*GENETICS SO - Biochem Biophys Res Commun 1986 Oct 30;140(2):508-14 7 UI - 87044106 AU - Zhao B ; Mackow E ; Buckler-White A ; Markoff L ; Chanock RM ; Lai CJ ; Makino Y TI - Cloning full-length dengue type 4 viral DNA sequences: analysis of genes coding for structural proteins. AB - DNA sequences (approximately 11,000 nucleotides) representing the full-length genome of the dengue virus type 4 were cloned. The sequence of the first 2,429 nucleotides at the 5' terminus which includes the coding region for the structural proteins is presented. The virion structural proteins are encoded in one long open reading frame specifying a polyprotein precursor which is apparently proteolytically cleaved by a mechanism resembling that proposed for expression of structural proteins of other flaviviruses such as yellow fever (YF) and West Nile (WN) viruses. The N terminus for each of the dengue virus structural proteins was tentatively assigned by homology alignment to the corresponding sequence of YF or WN virus. Comparison of sequence homology of structural proteins suggests that dengue virus is more closely related to WN virus than to YF virus or Murray Valley encephalitis virus. Finally, analysis of the extreme 5'- and 3'-terminal nucleotides of the dengue virus genome revealed sequences that may be involved in transcription, replication, and packaging of viral RNA. MH - Amino Acid Sequence ; Base Sequence ; Chromosome Mapping ; Cloning, Molecular ; Comparative Study ; Dengue Virus/*GENETICS ; DNA, Viral/ GENETICS ; Flaviviruses/GENETICS ; Nucleic Acid Conformation ; Protein Conformation ; RNA, Viral/*GENETICS ; Solubility ; Viral Proteins/ *GENETICS SO - Virology 1986 Nov;155(1):77-88 8 UI - 87044104 AU - Galinski MS ; Mink MA ; Lambert DM ; Wechsler SL ; Pons MW TI - Molecular cloning and sequence analysis of the human parainfluenza 3 virus mRNA encoding the P and C proteins. AB - The sequence of the mRNA encoding the phosphoprotein (P protein) of the human parainfluenza virus 3 (PF3) was determined by molecular cloning. In other Parmyxoviridae the P protein mRNA is functionally bicistronic and encodes an additional smaller nonstructural protein termed C. In this report three open reading frames (ORF) are described. These consist of a single long ORF encoding the P protein, and two shorter ORFs encoding the structural Vp18 protein (analogous to the Sendai C protein) and a putative polypeptide termed D protein. The encoded phosphoprotein consists of 603 amino acids and has a predicted molecular weight of 67,683. The C protein consists of 199 amino acids and has a predicted molecular weight of 23,288. The D protein consists of 140 amino acids and has a predicted molecular weight of 16,270. Although the D protein has not yet been demonstrated in vivo its synthesis could be demonstrated in vitro using a rabbit reticulocyte lysate system. Thus it appears that unlike the other paramyxoviruses, the PF3 P protein mRNA may be functionally tricistronic. MH - Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA/GENETICS ; Evolution ; Genes, Viral ; Molecular Weight ; Para-Influenza Virus Type 1/ GENETICS ; Para-Influenza Virus Type 3/*GENETICS ; Para-Influenza Viruses/ *GENETICS ; Phosphoproteins/*GENETICS ; Protein Conformation ; RNA, Messenger/GENETICS ; Solubility ; Translation, Genetic ; Viral Proteins/ *GENETICS SO - Virology 1986 Nov;155(1):46-60 9 UI - 87036916 AU - Cotmore SF ; McKie VC ; Anderson LJ ; Astell CR ; Tattersall P TI - Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments. AB - Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights of 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Restriction endonuclease fragments of this cloned B19 genome were treated with BAL 31 and shotgun cloned into the open reading frame expression vector pJS413. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus. MH - Adolescence ; Anemia, Sickle Cell/MICROBIOLOGY ; Base Sequence ; Capsid/ *ANALYSIS/BLOOD/GENETICS ; Cloning, Molecular ; DNA, Viral/GENETICS ; Female ; Fetal Diseases/MICROBIOLOGY ; Genes, Viral ; Human ; Liver/ MICROBIOLOGY ; Male ; Molecular Weight ; Parvoviridae/*ANALYSIS/GENETICS ; Parvovirus Infections/*MICROBIOLOGY ; Pregnancy ; Support, U.S. Gov't, P.H.S. ; Viral Proteins/*ANALYSIS/BLOOD/GENETICS SO - J Virol 1986 Nov;60(2):548-57 10 UI - 86306669 AU - Johnson BJ ; Kinney RM ; Kost CL ; Trent DW TI - Molecular determinants of alphavirus neurovirulence: nucleotide and deduced protein sequence changes during attenuation of Venezuelan equine encephalitis virus. AB - The nucleotide and deduced amino acid sequences of the structural proteins of the TC-83 vaccine strain of Venezuelan equine encephalitis (VEE) virus have been determined from a cDNA clone containing the 26S mRNA coding region. A cDNA clone encoding the equivalent region of the virulent parent VEE virus [Trinidad donkey strain (TRD)] has been sequenced previously. Comparison of the sequences of the TC-83 and TRD cDNA clones revealed 13 nucleotide differences. Neither the organization of the structural proteins (5'-capsid-E3-E2-6K-E1-3') nor the length (3762 nucleotides) of the open reading frame coding for the viral polyprotein precursor was altered during attenuation. Of the 13 nucleotide differences between the cDNA clones of TC-83 and TRD, nine occurred in the dominant population of the respective genomic RNAs from plaque-purified viruses. Six of the nine mutations were clustered in the E2 surface glycoprotein gene. All five of the nucleotide changes which produced non-conservative amino acid substitutions in the encoded proteins were located in the E2 gene. Two mutations occurred in the E1 glycoprotein gene; one was silent and the other did not alter the chemical character of the E1 protein. One nucleotide difference was found in the non-coding region immediately preceding the 5'-end of the 26S mRNA. The E2 and non-coding region mutations are candidates for the molecular determinants of VEE virus neurovirulence. MH - Amino Acid Sequence ; Animal ; Base Sequence ; Brain/*MICROBIOLOGY ; DNA ; Encephalitis Virus, Venezuelan Equine/GENETICS/*PATHOGENICITY ; Genes, Viral ; Glycoproteins/*GENETICS ; Mutation ; RNA, Viral/GENETICS ; Viral Proteins/*GENETICS ; Virulence SO - J Gen Virol 1986 Sep;67 ( Pt 9):1951-60 11 UI - 86263392 AU - Kinney RM ; Johnson BJ ; Brown VL ; Trent DW TI - Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins. AB - A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses. MH - Amino Acid Sequence ; Amino Acids/ANALYSIS ; Animal ; Anthropoidea ; Base Sequence ; Capsid ; Cell Line ; Cloning, Molecular ; DNA/ANALYSIS ; Encephalitis Virus, Venezuelan Equine/*GENETICS ; Genes, Structural ; RNA, Messenger/*ANALYSIS ; Translation, Genetic ; Viral Proteins/ *ANALYSIS SO - Virology 1986 Jul 30;152(2):400-13 12 UI - 86220784 AU - Hudson PJ ; McKern NM ; Fahey KJ ; Azad AA TI - Predicted sequence of the host-protective immunogen of infectious bursal disease virus. AB - The genome of Australian strain 002-73 of infectious bursal disease virus (IBDV) has been cloned as cDNA fragments into an expression library based on pUR plasmid vectors. Recombinant colonies were selected with a monoclonal antibody specific for the 32-kDa host-protective immunogen of IBDV and fully characterized by nucleotide sequence analysis. The amino acid sequence of several tryptic peptides derived from the native 32-kDa structural protein has confirmed the coding region assignment and nucleotide sequence data. We believe this to be the first published sequence of a birnavirus-encoded protein and these data may provide the basis for an effective subunit vaccine against IBDV. MH - Amino Acid Sequence ; Base Sequence ; DNA/GENETICS ; DNA, Recombinant ; Genes, Viral ; *Infectious Bursal Agent/GENETICS ; *Reoviridae/GENETICS ; RNA, Viral/GENETICS ; Viral Proteins/GENETICS/*IMMUNOLOGY ; Viral Vaccines SO - FEBS Lett 1986 May 26;201(1):143-6 13 UI - 86281885 AU - Soussi T TI - DNA-binding properties of the major structural protein of simian virus 40. AB - We investigated whether the VP1 protein of simian virus 40 binds to DNA. In vitro DNA-binding experiments clearly indicate that VP1 bound strongly to double-stranded and single-stranded DNA, with a higher affinity for the latter; additional experiments show that VP1 did not bind to a specific sequence of simian virus 40 DNA. MH - Base Sequence ; DNA/METABOLISM ; DNA-Binding Proteins/*METABOLISM ; DNA, Single-Stranded/*METABOLISM ; DNA, Viral/*METABOLISM ; Support, Non-U.S. Gov't ; SV40 Virus/*METABOLISM ; Viral Proteins/*METABOLISM SO - J Virol 1986 Sep;59(3):740-2 14 UI - 86264103 AU - Uckert W ; Hertling I ; Kraft R ; Bossmann H ; R:ossler H ; Meyer U TI - Structural components of bovine leukemia virus: further biochemical and immunological characterization of major structural proteins and glycoproteins. AB - Proteins from bovine leukemia virus (BLV) were investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence and absence of 14C amino acids. Virus grown either in fetal lamb kidney (FLK) or bat lung (BL) cells revealed seven major proteins designated p10, p12, p15(1), p15(2), p24, gp30, and gp64. By tryptic peptide analysis, N-terminal amino acid analysis and radioimmunoassay it could be shown that p10, a basic protein with hydrophobic properties, is a cleavage product of p15(2), the acidic and slightly hydrophobic phosphoprotein of BLV. The structural protein p12 was shown to be a phosphorylated protein identifying it as a second major viral protein to contain phosphorous. Investigation of [3H]glucosamine incorporation, N-terminal amino acid analysis and hydrophobic properties by chloroform-methanol extraction confirmed properties of gp30 and gp64 predicted by nucleotide sequence data. The two p15 proteins have been characterized in more detail. MH - Amino Acid Sequence ; Antigens, Viral/IMMUNOLOGY ; Base Sequence ; Bovine Leukemia Virus/*ANALYSIS/IMMUNOLOGY ; Cross Reactions ; Electrophoresis, Polyacrylamide Gel ; Glycoproteins/*ANALYSIS/IMMUNOLOGY ; Peptides/ ANALYSIS ; Phosphoproteins/ANALYSIS ; Radioimmunoassay ; Retroviridae/ *ANALYSIS ; Viral Proteins/*ANALYSIS/IMMUNOLOGY SO - Virus Res 1986 Jun;4(4):343-56 15 UI - 86220766 AU - Pletnev AG ; Yamshchikov VF ; Blinov VM TI - Tick-borne encephalitis virus genome. The nucleotide sequence coding for virion structural proteins. AB - RNA of a flavivirus, tick-borne encephalitis virus (TBEV; strain Sofjin), was subjected to reverse transcription and the DNA copy was transformed into double-stranded DNA by the action of E. coli DNA-polymerase I (Klenow fragment). This DNA was annealed with plasmid pBR322. The recombinant plasmids were cloned in E. coli K802. The nucleotide sequence of the inserts of the clones, coding for region structural proteins C, M, E and nonstructural protein NS1, was determined by the Maxam-Gilbert method. The genes of structural proteins form a compact cluster. Homology has been studied of the TBEV sequences found with the structures of proteins and RNAs of other flaviviruses, yellow fever virus and West Nile virus, and a high degree of homology was found. MH - Amino Acid Sequence ; Base Sequence ; Encephalitis Viruses, Tick-Borne/ *GENETICS ; *Genes, Viral ; Viral Proteins/*GENETICS SO - FEBS Lett 1986 May 12;200(2):317-21 16 UI - 86124703 AU - Castle E ; Leidner U ; Nowak T ; Wengler G ; Wengler G TI - Primary structure of the West Nile flavivirus genome region coding for all nonstructural proteins. AB - The genome RNA of the flavivirus West Nile (WN) virus has been transcribed into cDNA, the cDNA has been cloned, and the nucleotide sequences coding for the structural proteins have been determined (Castle et al., 1985; Wengler et al., 1985). We have now determined the nucleotide sequence coding for all viral nonstructural proteins which comprises 7929 nucleotides. Together with our earlier sequence analyses these data show that a long open reading frame (ORF) containing 10,290 nucleotides is present on the genome of WN virus. The two largest nonstructural proteins which can be detected in flavivirus-infected cells are the proteins NV5 and NV4 which have an apparent molecular mass of 97,000 and 74,000 Da, respectively. Both proteins were isolated by preparative polyacrylamide gel electrophoresis, and partial amino acid sequences of peptides derived from these proteins were determined. These analyses allow us to localize the nucleotide regions which code for these proteins and show that the region coding for the NV5 protein is located at the 3'-terminus of the long ORF. Together with our earlier analyses these data show that the protein sequences of virus-specific proteins are present on the viral polyprotein translated from the long ORF in the order V2-NV2-V3-(nonstructural proteins of up to 75,000 Da)-NV4-(nonstructural proteins of up to 45,000 Da)-NV5. Our data indicate that virus-specific structural and nonstructural proteins which are synthesized from a single long ORF accumulate in large amounts in infected cells. A possible role of the presence of these molecules, which are associated to cellular membranes, in the accumulation of membrane vesicles which characteristically occurs in flavivirus-infected cells is discussed. MH - Amino Acid Sequence ; Animal ; Base Sequence ; Cell Line ; Cloning, Molecular ; Codon ; DNA ; Electrophoresis, Polyacrylamide Gel ; Genes, Viral ; Hamsters ; Molecular Weight ; RNA, Viral/*GENETICS ; Support, Non-U.S. Gov't ; Translation, Genetic ; Viral Proteins/*GENETICS/ ISOLATION & PURIFICATION ; West Nile Virus/*GENETICS SO - Virology 1986 Feb;149(1):10-26