Respiratory syncytial virus envelope glycoprotein (G) has a novel structure.

Amino acid sequence of human respiratory syncytial virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino acid microsequencing of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated in vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane insertion at 41 residues from the N-terminus. There was no N-terminal signal sequence and the hydrophilic N-terminal 20 residues probably represent the cytoplasmic tail of the protein. The N-terminally oriented membrane insertion was somewhat analogous to paramyxovirus hemagglutinin-neuraminidase (HN) and influenza neuraminidase (NA). The protein was moderately hydrophilic and rich in hydroxy-amino acids. It was both N- and O-glycosylated with the latter contributing significantly to the net molecular mass 90K.     

Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.     

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.     

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.     

Sequence determination of the Sendai virus HN gene and its comparison to the influenza virus glycoproteins

The nucleotide sequence of the Sendai virus (SV) HN (hemagglutinin-neuraminidase) gene was determined. The deduced primary structure of the protein showed only one hydrophobic domain likely to represent the transmembrane region, but at its N terminus. Since the SV F protein is anchored in the membrane at its C terminus, the two SV glycoproteins are thus membrane-anchored in opposite orientations, similar to the two influenza virus (FLU) glycoproteins. Amino acid sequence comparisons of the SV HN and the FLU HA and NA proteins revealed homologies between 100 amino acids of the hemagglutinin region of the FLU HA protein and the C terminus of the SV HN, and between 200 amino acids of the neuraminidase region of the FLU NA and the central region of SV HN. Alignment of the neuraminidase, hemagglutinin, and fusion regions shared by these glycoproteins suggest the structure of a possible ancestral gene.     

mRNA sequence and deduced amino acid sequence of the mumps virus small hydrophobic protein gene.

The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.     

Analysis of the Sendai virus M gene and protein.

The nucleotide sequence of the Sendai virus M (matrix or membrane) gene region was determined from cloned genomic DNA, and the limits of the M mRNA were determined by S1 nuclease mapping. The M mRNA is 1,173 nucleotides long and contains a single long open reading frame coding for a protein of 348 amino acids. The amino acid sequences of the N- and C-terminal peptides of the M protein were obtained by mass spectrometric analysis and correspond to those predicted from the open reading frame, with the N terminus modified in vivo by cleavage of the initiating methionine and acetylation of the following amino acid. The amphiphilic nature of the M protein structure is discussed.     

Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.     

Structure of full-length cDNA coding for sulfatide activator, a Co-beta-glucosidase and two other homologous proteins: two alternate forms of the sulfatide activator

Full-length cDNA clones have been isolated for an mRNA which codes for four different but homologous proteins--a sulfatide activator protein, a co-beta-clucosidase, and two other proteins of similar structures. The primary structure as deduced from the nucleotide sequence is highly homologous to the precursor of the rat Sertoli cell sulfated glycoprotein 1. The full-length clone was 2,734-bp long, starting from 8 bases above the initiator ATG and terminating with a poly A tail. The nucleotide sequence confirmed an earlier prediction based on the amino acid sequence that a previously published sequence contained errors. On the other hand, the amino acid sequence now closely agrees with the recent revised sequence published by the same group except for several amino acids near the N-terminus. Two alternate forms of the sulfatide activator were detected, differing from each other by the presence or absence of 3-amino acid insertion.     

Primary structure of sorghum malate dehydrogenase (NADP) deduced from cDNA sequence. Homology with malate dehydrogenase (NAD)

Malate dehydrogenase (NADP) (NADP-MDH) is an important enzyme of the photosynthetic CO2 fixation pathway of C4 plants. We have isolated two clones from a sorghum lambda gt11 cDNA library (CM3, 932 bp, and CM7, 1441 bp). Nucleotide sequence analysis of the cDNAs CM3 and CM7 showed the existence of two NADP-MDH mRNA species encoding different enzyme subunits. Microsequencing of the N-terminus of the mature protein indicated that a specific cleavage of 13 amino acids occurred during the purification steps of the enzyme. The full-length cDNA CM7 contains a large open reading frame encoding an NH2-terminal transit peptide of 40 amino acids and a mature protein of 389 amino acids (42.207 kDa). Alignment of the NADP-MDH sequence with those of several malate dehydrogenases revealed some similarities with NAD-MDHs.     

Interacting domains of the HN and F proteins of newcastle disease virus

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.     

Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3

Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells.     

Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein (PSP-A)

Pulmonary surfactant is composed mainly of phospholipid and two groups of apoproteins. One of these apoproteins is a family of glycoproteins (pulmonary surfactant-associated protein A, PSP-A). We have isolated and sequenced a cDNA clone encoding for rat PSP-A and the full amino acid sequence has been deduced from the nucleotide sequence. The sequence of 56 amino acids at the N-terminus of PSP-A isolated from rats treated with silica was determined independently, and there is complete agreement with the sequence deduced from the cDNA. Isolated rat alveolar type II cells contain two species of mRNA for this protein.     

Human endo-alpha1,2-mannosidase is a Golgi-resident type II membrane protein

The cDNA for human endo-alpha1,2-mannosidase was reconstructed using two independent EST-clones and its properties characterized. The 2837 bp cDNA construct contained a 1389 bp open reading frame (ORF) encoding for 462 amino acids and an approximately 53.6 kDa protein, respectively. Hydrophobicity analysis of this amino acid sequence, as well as proteolytic degradation studies, indicate that the enzyme is a type II protein, anchored in the membrane via a 19 amino-acid long apolar sequence close to the N-terminus. Human endo-alpha1,2-mannosidase displays a high degree of sequence identity with the catalytic domain of the homologous rat liver endo-enzyme, but differs substantially in the N-terminal peptide region, which includes the transmembrane domain. No sequence similarity exists with other processing alpha-glycosidases. Based on sequence information provided by the 2837 bp construct, the cDNA consisting of the complete 1389 bp ORF was amplified by RT-PCR using human fibroblast RNA. Incubation of E. coli lysates with this cDNA, previously modified for boost translation by codon optimization, resulted in the synthesis of an approximately 52 kDa protein which degraded [(14)C]Glc(3)-Man(9)-GlcNAc(2) efficiently, indicating that the catalytic domain of the enzyme folds correctly under cell-free conditions. Transfection of the endo-alpha1,2-mannosidase wild-type cDNA into COS 1 cells resulted in a moderate (approximately 1.5-fold) but reproducible increase of activity compared with control cells, whereas >18-fold increase in activity was measured after expression of a chimera containing green-fluorescent-protein (GFP) attached to the N-terminus of the endo-alpha1,2-mannosidase polypeptide. This, together with the observation that GFP-endo-alpha1,2-mannosidase is expressed as a Golgi-resident type II protein, points to enzyme-specific parameters directing folding and membrane anchoring, as well as Golgi-targeting, not being affected by fusion of GFP to the endo-alpha1,2-mannosidase N-terminus.     

cDNA and genomic DNA sequence of the 21.3 kDa subunit of NADH:ubiquinone reductase (complex I) from Neurospora crassa.

The primary structure of a nuclear-encoded subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the protein. The sequence correlates to a protein of 200 amino acids and a molecular mass of 21349 Da. The protein is synthesized without a cleavable presequence. It contains two alpha-helices predicted to traverse the bilayer and is a constituent of the membrane part of complex I.     

Genomic DNA sequence for human C-reactive protein

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.     

Amino acid sequence of human respiratory syncytial virus nucleocapsid protein.

Amino acid sequence of the human respiratory syncytial (RS) virus nucleocapsid (NC) protein, deduced from the DNA sequence of a recombinant plasmid, is presented. The cDNA plasmid (pRSB11) has 1412 bp of RS viral NC sequence and lacks six nucleotides of the 5' end of mRNA. There is a single long open reading frame encoding 467 amino acids. This 51540 dal protein is rich in basic amino acids and has no homologies with other known viral capsid proteins.     

Use of the deoxyinosine-containing probe to isolate and sequence cDNA encoding the fusion (F) glycoprotein of Sendai virus (HVJ)

A synthetic 20-mer based on the known amino acid (aa) sequence of the N-terminus of Sendai virus F1 polypeptide was synthesized. Using this dI-probe, which contained deoxyinosines at all six ambiguous codon positions, we isolated clones carrying cDNAs for the F mRNA of Sendai virus. Nucleotide (nt) sequence analysis revealed a long open reading frame (ORF) that encodes a protein of 565 aa. Thus, this type of dI-probes should prove useful for selecting cDNA clones, when the aa sequence is known and is characterized by high codon redundancy.