Parvovirus genome: nucleotide sequence of H-1 and mapping of its genes by hybrid-arrested translation.

The nucleotide sequence of the parvovirus H-1 has been determined by the chain-terminating method of Sanger. The sequence is 5,176 nucleotides long. Two large open reading frames (1 and 2) and two smaller open reading frames (3 and 4) of potential importance were identified in the plus-strand sequence. Promoter sequences are located at map positions 4 and 38 when map positions are expressed as percent of genome length from the 3' end of the virion minus strand. The locations for the genes for the parvovirus capsid proteins and a 76,000-dalton noncapsid protein (NCVP1) were mapped by hybrid-arrested translation. The gene for the capsid proteins VP1 and VP2' is located in the 5' half of the virus genome. The gene for NCVP1 is located in the 3' half of the viral DNA.     

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.     

The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.     

Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region.

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5' end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3' half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5' half (including the capsid proteins) are more heterogeneous.     

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.     

The nucleotide sequence of cowpea mosaic virus B RNA

The complete sequence of the bottom component RNA (B RNA) of cowpea mosaic virus (CPMV) has been determined. Restriction enzyme fragments of double-stranded cDNA were cloned in M13 and the sequence of the inserts was determined by a combination of enzymatic and chemical sequencing techniques. Additional sequence information was obtained by primed synthesis on first strand cDNA. The complete sequence deduced is 5889 nucleotides long excluding the 3' poly(A), and contains an open reading frame sufficient to code for a polypeptide of mol. wt. 207 760. The coding region is flanked by a 5' leader sequence of 206 nucleotides and a 3' non-coding region of 82 residues which does not contain a polyadenylation signal.     

The envelope-associated 22K protein of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polytranscript

We recently determined that respiratory syncytial virus (strain A2) encodes a fourth unique envelope-associated virion protein that has molecular weight of approximately 24,000, as estimated by gel electrophoresis. The nucleotide sequence of the mRNA encoding this novel protein has now been determined from five cDNA clones, including three that contain the complete mRNA sequence. The complete mRNA sequence is 957 nucleotides, exclusive of polyadenylate, and contains two partially overlapping open reading frames. The 5'-proximal open reading frame is favored for utilization by the criteria of the location and sequence of its translational start site. Furthermore, the calculated molecular weight of the encoded protein, 22,153, is in agreement with the previous estimate of 24,000 for the authentic protein identified by hybrid selection and in vitro translation. The sequence of the predicted protein, now designated the 22K protein, contains 194 amino acids, is relatively hydrophilic, and appears to be the most basic of the respiratory syncytial virus proteins. The mRNA also contains a second, internal open reading frame which would encode a protein of 90 amino acids. However, no evidence for this translation product is known. The first nine nucleotides in the mRNA sequence, 5'-GGGGCAAAU, are identical to the conserved sequence identified previously at the 5' termini of seven other respiratory syncytial viral mRNAs; the sequence at the 3' end of the 22K mRNA, 5'. . . AGUUAUUU-polyadenylate, contains the elements of the previously identified 3'-terminal consensus sequence for respiratory syncytial virus mRNAs, AGUUAA(N)1-4-polyadenylate (P. L. Collins, Y. T. Huang, and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 81:7683-7687). In addition, we present and describe the intergenic sequence of a dicistronic RNA derived from readthrough of the F and 22K protein genes.     

Kelp fly virus: a novel group of insect picorna-like viruses as defined by genome sequence analysis and a distinctive virion structure

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.     

Identification of the initiation site of poliovirus polyprotein synthesis.

The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVP00. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VP0, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of the tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of the poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.     

Complete nucleotide sequence of genomic RNA of the potato M-virus

The complete nucleotide sequence of potato virus M genomic RNA has been determined to be 8534 nucleotides (with the exception of the poly(A) tail at the 3' end). The sequence contains six large open reading frames coding for proteins of mol. wt. 223206, 25438, 11893, 6793, 33906, and 12183 (in 5'----3' direction). According to its primary sequence analysis the 223K protein ORF codes for a virus RNA replicase. The in vitro translation product of 34K protein gene precipitates by the antisera against the RVM indicating that the 34K protein is the virus coat protein. The general aspects of carla- and potexvirus gene organization are discussed.     

Molecular cloning and sequence of the gene for p9Ka. A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein

The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.     

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.     

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.     

Conservation of protein coding potential in the long terminal repeats of exogenous and endogenous mouse mammary tumor viruses

In vitro protein synthesis and DNA sequence analysis indicate that mouse mammary tumor virus differs from other well-characterized retroviruses in that the long terminal repeat region of the provirus has the capacity to encode proteins. Different exogenously transmitted mouse mammary tumor virus strains and endogenous proviral units conserved this open reading frame feature in the long terminal repeat despite a variation in nucleotide sequence. The proteins encoded by the different long terminal repeats were clearly related, but showed minor variations in size and tryptic peptide maps. In each case, the largest in vitro product had a molecular weight of about 36,000 to 37,000, suggesting that the open reading frame sequences must extend for approximately 1,000 nucleotides beginning at the extreme 5' end of the long terminal repeat. The fact that the reading frame was conserved among these viruses argues in favor of an in vivo function for the open reading frame protein.