Molecular cloning and sequence analysis of the human parainfluenza 3 virus gene encoding the matrix protein

The sequence of the matrix (M) protein gene and contiguous intergenic regions of the human parainfluenza 3 virus (PF3) was determined by molecular cloning. The encoded M protein contains 354 amino acids and has a predicted mol wt of 39,506. The M protein amino acid sequence was compared to the homologous proteins from other members of the Paramyxoviridae family. The PF3 protein shared 61 % homology with the Sendai virus protein and approximately 35% homology with measles and canine distemper virus proteins. Little homology was observed with respiratory syncytial virus. The M protein appears to be the most highly conserved among the Paramyxoviridae proteins.     

Molecular cloning and sequence analysis of the rinderpest virus mRNA encoding the hemagglutinin protein

We cloned the full-length cDNAs corresponding to the mRNA for the hemagglutinin (H) protein of rinderpest virus (RV) and determined the nucleotide sequence of RV-H. The gene of RV-H was composed of 1952 nucleotides and contained a single large open reading frame, which was capable of encoding a protein of 609 amino acids with a molecular weight of 68,330 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of the measles virus (MV)-H. The 5' end of the message (nucleotides 1 to 485) was largely conserved, with a homology of 75.1% of the nucleotides and 78.0% of the predicted amino acids. In the middle portion (nucleotides 486-1310), where the potential glycosylation sites exist, 56.6% of the nucleotides and 49.5% of the amino acids were identical. In the 3' end of the message (nucleotides 1311-1850), 63.3% of the nucleotides and 58.1% of the amino acids were identical. Four potential glycosylation sites were found in RV-H protein and three of them were the same as those of MV-H protein. The positions of 13 cysteine residues of RV-H were absolutely identical to those of MV-H. The hydropathy profile of RV-H protein resembled that of MV-H. One major hydrophobic region long enough to be an anchor in the membrane was located near the N-terminus.     

Molecular cloning and sequence analysis of bovine respiratory syncytial virus mRNA encoding the major nucleocapsid protein

The nucleotide sequence of the gene encoding the major nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) has been determined. The N mRNA is 1196 nucleotides long with a single, large open reading frame. The derived polypeptide has 391 amino acids corresponding to a calculated molecular weight of 42,600 Da. This is in agreement with the molecular weight of 43,000 Da determined for the BRSV N protein by SDS-polyacrylamide gel electrophoresis (PAGE). Comparison of the nucleotide sequence of BRSV N gene with the sequence of the N gene of human respiratory syncytial virus (HRSV) revealed a homology of 80.7%. There is a 93.3% homology at the amino acid level between the N proteins of BRSV and HRSV. The 5'- and 3'-terminal untranslated sequences that are conserved among HRSV mRNAs were also identified in the N mRNA of BRSV. The results indicate that the N genes are highly conserved in the bovine and human strains of respiratory syncytial virus.     

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.     

Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium.

Cowdria ruminatium, the causative agent of heartwater disease, expresses an immunodominant and conserved 32-kilodalton protein (MAP1; formerly called Cr32), which is currently in use for serodiagnosis of the disease. The gene encoding this protein, designated map1, was detected, cloned, and characterized. The gene is conserved between four different stocks of C. ruminantium originating from Senegal, Sudan, South Africa, and Zimbabwe. Homology searches revealed MAP1 to be homologous to the Anaplasma marginale surface protein MSP4, a potential protective antigen. The MAP1 protein, expressed in Escherichia coli fused with glutathione S-transferase, is specifically recognized by sera from animals infected with seven different stocks of C. ruminantium.     

Molecular cloning and sequence analysis of the duck enteritis virus UL5 gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, the DEV UL5 gene was cloned and sequenced from a vaccine virus. According to the consensus sequence of herpesvirus UL5 and UL3 gene degenerate oligonucleotide primers were designed and were used in the polymerase chain reaction (PCR) to amplify DNA products with 4577 bp in size. DNA sequence analysis revealed a 2568 bp open reading frame (ORF) encoding a 855 amino acid polypeptide homologous to herpesvirus UL5 proteins. The DEV UL5 gene has a base composition of 769 adenine (29.95%), 556 cytosine (21.65%), 533 guanine (20.76%) and 710 thymine (27.65%). Sequence comparison revealed that the nucleotide sequence of the DEV UL5 gene was highly similar to other alphaherpesviruses. Phylogenetic tree analysis showed that the fifteen herpesviruses viruses analyzed fell into four large groups, and the duck enteritis virus itself branched and was most closely related to meleagrid herpesvirus 1, gallid herpesvirus 2 and gallid herpesvirus 3 subtrees.     

Molecular cloning and sequence analysis of the scpZ gene encoding the serine carboxypeptidase of Absidia zychae

Carboxypeptidase Z is a serine carboxypeptidase secreted by Absidia zychae NRIC 1199. The cDNA and genomic DNA carrying the scpZ gene encoding carboxypeptidase Z were cloned and sequenced. The nucleotide sequences of the cDNA (1.4 kb) and the genomic DNA (3.3 kb) were analyzed and the intervening sequences were located by a comparison of the two. It was found that the scpZ gene was interrupted by 11 short introns, 50–75 nucleotides in length. Genomic Southern analysis showed that there was only one scpZ gene in the genome of A. zychae. The gene encoded a putative pre-proenzyme composed of 409 amino-acid residues of the mature carboxypeptidase Z (Mr 45 421) and an additional N-terminal sequence of 51 amino-acid residues. The amino-acid sequence around the active serine residue of carboxypeptidase Z (-G-E-S-Y-G-G-) differed from the consensus (-G-E-S-Y-A-G-) which is conserved in most of the serine carboxypeptidases so far analyzed.     

Sequence analysis of the mumps virus mRNA encoding the P protein

The nucleotide sequence of the mumps virus phospho- or polymerase-associated (P) protein mRNA has been determined by sequencing a full-length cDNA clone and confirmed by partially sequencing the mRNA and the genome. The mRNA contains 1311 nucleotides excluding the poly(A) and encodes a protein of 390 amino acids with a calculated molecular weight of 41,574. Three small polypeptides were seen in in vitro translation of viral mRNA and hybrid-selected P mRNA, possibly representing internal initiation in the same reading frame of the P protein. A second overlapping reading frame is predicted from the sequence which has a capacity to code for two polypeptides of 56 and 34 amino acids, respectively. Whether these two polypeptides are expressed in infected cells is not known. Comparison of the P protein sequence with that of Sendai virus, measles virus, parainfluenza virus type 3, and canine distemper virus (CDV) showed no distinct homology but comparison with the P protein of Newcastle disease virus (NDV) showed 25.6% homology.     

Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein

The fusion (F) protein of mumps virus was purified by immunoaffinity chromatography using an anti-F monoclonal antibody. The F protein was reduced and alkylated, and the F1 and F2 chains were isolated by high-pressure size exclusion chromatography. Twenty-three amino acid residues from the amino terminus of each chain were identified following automated Edman degradation. The amino-terminal sequence of the F1 chain was homologous to previously reported F1 sequences from three other paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus). Secondary structure predictions suggest an alpha-helical conformation for the mumps virus F1 amino-terminal sequence. A helical wheel model of the paramyxovirus F1 NH2 terminus is presented which defines conserved and variable arcs of the helix and provides a spatial representation of this critical functional domain of the paramyxovirus fusion protein.     

Molecular cloning,sequence analysis and expression of the gene encoding an antifungal-protein from Aspergillus giganteus

The gene encoding the precusor of a small secretory protein with antifungal activity was isolated from A. giganteus and characterized by restriction mapping, hybridization and nucleotide sequencing. The promoter contains a typical TATA-box at a distance of 135 bp upstream of the open reading frame. The open reading frame is interrupted by two small introns with conserved splice sites. The precursor of the antifungal protein (AFP) consists of 94 amino acids and appears to be processed to the mature AFP of 51 amino acids by a two-step process. Transfer of the gene into A. niger yielded only transformants with a very low expression level, probably because high-expression transformants were counterselected by the antifungal activity of the recombinant protein.     

Molecular cloning and sequence analysis of the human parainfluenza 3 virus mRNA encoding the P and C proteins

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.     

Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens.     

Molecular cloning of the rinderpest virus matrix gene: comparative sequence analysis with other paramyxoviruses

The nucleotide sequence of the gene encoding the matrix or membrane (M) protein of the virulent (Kabete-O) strain of rinderpest virus (RPV) has been determined. The M gene is 1457 nucleotides long with a single, large open reading frame. The derived polypeptide has 335 amino acids, corresponding to a calculated molecular weight of 38,289 and contains both small hydrophobic regions and many basic residues. The predicted amino acid sequence was compared to the M proteins of paramyxoviruses. Sequence comparison and hydropathy profiles among the morbilliviruses revealed that the M protein of RPV exhibits features similar to those of the M protein of MV and CDV. There is 78.2% homology at the amino acid level between the M protein of RPV and MV, and 77.6% between RPV and CDV. This indicates that a high degree of homology exists among the members of the genus Morbillivirus. In contrast, there is only 37.3 and 18% homology between RPV and bovine parainfluenza type 3 (BPV3), and RPV and Newcastle disease virus (NDV) M proteins, respectively. Thus the M proteins of the morbilliviruses are highly conserved whereas the M proteins of the genus Paramyxovirus show more divergence.     

Molecular cloning and sequence analysis of the fusion glycoprotein gene of human parainfluenza virus type 2

A cDNA clone containing a 2.0-kb insert was identified as the human parainfluenza virus type 2 (PI2) fusion glycoprotein gene by hybridizing with a viral RNA probe and a synthetic oligonucleotide derived from a conserved sequence found in other paramyxovirus fusion protein genes. The complete nucleotide sequence of the glycoprotein gene was determined by the dideoxynucleotide sequencing procedure and found to contain a single, large open reading frame encoding a protein of 551 amino acids with a calculated molecular weight of 59,664. Comparison of the P12 fusion protein with those of other paramyxoviruses indicated similarities in overall length, N-terminal signal peptide sequence (amino acids 7 to 25), C-terminal membrane-spanning region (amino acids 486 to 513), and a highly conserved fusion sequence region at the N-terminus of the F1 subunit (amino acids 107 to 132).     

Molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type I

Summary cDNA clones spanning the entire region of the peplomer (S) gene of feline infectious peritonitis virus (FIPV) type I strain KU-2 were obtained and their complete nucleotide sequences were determined. A long open reading frame (ORF) encoding 1464 amino acid residues was found in the gene, which was 12 residues longer than the ORF of the FIPV type II strain 79–1146. The sequences of FIPV type I and mainly FIPV type II were compared. The homologies at the N- (amino acid residues 1–693) and C- (residues 694–1464) terminal halves were 29.8 and 60.7%, respectively. This was much lower than that between FIPV type II and other antigenically related coronaviruses, such as transmissible gastroenteritis virus of swine and canine coronavirus. This supported the serological relatedness of the viruses and confirmed that the peplomer protein of FIPV type I has distinct structural features that differ from those of antigenically related viruses.