The mumps virus nucleocapsid mRNA sequence and homology among the Paramyxoviridae proteins

The nucleotide sequence of mumps virus nucleocapsid protein (NP) mRNA has been determined from two overlapping cDNA clones and confirmed by partial sequencing of the mRNA and the genome. The mRNA contains 1844 nucleotides excluding poly(A) and encodes a protein of 553 amino acids with a calculated molecular weight of 61,792. A comparison of the mumps virus nucleocapsid protein sequence with that of other paramyxoviruses revealed a moderate degree of homology (33.1%) with the Newcastle disease virus (NDV) only. The nucleocapsid proteins of all paramyxoviruses studied to date, excluding that of the genus pneumovirus, have a conserved sequence of six amino acids (Ser-Tyr-Ala-Met-Gly-Val) except that of NDV which has a mismatch of two amino acids (Ser-Phe-Ala-Met-Gly-Met) in that sequence. In addition, there is another conserved region of seven amino acids (Phe-Ala-Pro-Gly-X-Tyr-Pro) in the nucleocapsid proteins of mumps virus, Sendai virus and parainfluenza virus type 3. The nucleocapsid proteins of measles virus and canine distemper virus (CDV) also have this conserved region but with three conservative amino acid changes.     

Molecular cloning of the NP and L genes of simian virus 5: identification of highly conserved domains in paramyxovirus NP and L proteins.

We have molecularly cloned and determined the nucleotide sequence of the 3' and 5' regions of the genomic RNA of the paramyxovirus simian virus 5 (SV5), including the 3' leader sequence, nucleocapsid protein (NP) gene, large (L) protein gene, and 5' anti-genomic leader (trailer) sequence. The vRNA 3' proximal leader sequence contains 55 nucleotides. The NP gene is 1725 nucleotides in length and encodes a negatively charged protein consisting of 509 residues (MW 56,534). A comparison of the amino acid sequences of 10 paramyxovirus NP proteins indicates a region of high sequence identity near the middle of the protein, and a C-terminal region which is enriched in negatively charged residues. Overall, the SV5 NP protein showed the highest degree of sequence identity with the NP proteins of parainfluenza type 2 virus (58%) and mumps virus (56%). The L gene extends 6804 nucleotides and encodes a positively charged protein consisting of 2255 residues (MW 255,923). The 5' proximal region of the vRNA consists of a 31 nucleotide trailer RNA. The SV5 L protein sequence showed 62% overall identity with the parainfluenza type 2 L protein. Although little overall sequence identity was found between the SV5 and other paramyxovirus L protein sequences, short stretches of extensive amino acid identity were found near the middle of each of the known paramyxovirus L protein sequences, and these common regions may represent sites important for enzymatic activity.     

Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein

To obtain information about the structure and evolution of the nucleocapsid (N) protein of the coronavirus mouse hepatitis virus (MHV), we determined the entire nucleotide sequences of the N genes of MHV-A59, MHV-3, MHV-S, and MHV-1 from cDNA clones. At the nucleotide level, the N gene sequences of these viral strains, and that of MHV-JHM, were more than 92% conserved overall. Even higher nucleotide sequence identity was found in the 3' untranslated regions (3' UTRs) of the five strains, which may reflect the role of the 3' UTR in negative-strand RNA synthesis. All five N genes were found to encode markedly basic proteins of 454 or 455 residues having at least 94% sequence identity in pairwise comparisons. However, amino acid sequence divergences were found to be clustered in two short segments of N, putative spacer regions that, together, constituted only 11% of the molecule. Thus, the data suggest that the MHV N protein is composed of three highly conserved structural domains connected to each other by regions that have much less constraint on their amino acid sequences. The first two conserved domains contain most of the excess of basic amino acid residues; by contrast, the carboxy-terminal domain is acidic. Finally, we noted that four of the five N genes contain an internal open reading frame that potentially encodes a protein of 207 amino acids having a large proportion of basic and hydrophobic residues.     

Sequence characterization of the membrane protein gene of paramyxovirus simian virus 5

The complete nucleotide sequence of the membrane (M) protein gene of the paramyxovirus simian virus 5 (SV5) was determined from cDNA clones of viral mRNAs. The M gene boundaries were determined by (i) primer extension sequencing on M mRNA; (ii) nuclease S1 analysis; and (iii) primer extension sequencing on viral genomic RNA. The M gene mRNA consisted of 1371 templated nucleotides. It contains a single large open reading frame that can encode a protein of 377 amino acids with a predicted Mr = 42,253. The authenticity of the predicted M protein coding sequence was confirmed by synthesis of the M protein from mRNA synthesized from cDNA. The predicted M amino acid sequence indicated it is an overall hydrophobic protein carrying a net positive charge. Alignment of the SV5 protein amino acid sequence with the M protein sequences of other paramyxoviruses indicated that these viruses fall into the following two groups: (1) SV5, mumps virus, and Newcastle disease virus; or (2) Sendai, parainfluenza virus type 3, measles virus, and canine distemper virus, with mumps virus M sequence being the most closely related to SV5.     

Divergence and conservation of the genomic RNAs of Taiwan and Hawaii strains of papaya ringspot potyvirus

Summary. The complete nucleotide sequence of the genome of a Taiwan isolate of papaya ringspot potyvirus (PRSV YK) was determined from three overlapping cDNA clones and by direct RNA sequencing. Comparison was made with the reported Hawaii isolate of PRSV HA. Both genomes are 10 326 nucleotides long, excluding the poly(A)-tail. They encode a polyprotein of 3 344 amino acids with a 5′ leader of 85 nucleotides and a 3′ non-translated region of 209 nucleotides. The two genomes share an overall nucleotide identity of 83.4% and an amino acid identity of 90.6%. The 3′ non-translated regions show 92.3% identity. The first 23 nucleotides of the leaders are identical, while the remaining parts of the leaders only show 51.6% identity. The P1 protein genes of the two isolates are very different, with 70.9% nucleotide identity and 66.7% encoded amino acids identity. However, the other viral proteins of the two virus isolates are similar, with a 82.5–89.8% nucleotide identity of their genes and 91.2–97.6% amino acid identity, indicating that they are strains of the same potyvirus. Analysis of the ratios of nucleotide differences to the actual amino acid changes revealed that there are only 2.63 nucleotide changes for each amino acid change in the P1 protein, whereas for the other proteins 4.0–16.4 nucleotide changes are required for each amino acid replacement. The P1 protein has 58% of all the differences of polyprotein. The unusual variation in the leader sequences and the P1 proteins suggests that the two PRSV strains were derived from different evolutionary pathways in different geographic areas. Received February 5, 1996 Accepted September 17, 1996     

Conserved structures among the nucleocapsid proteins of the paramyxoviridae: complete nucleotide sequence of human parainfluenza virus type 3 NP mRNA

The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5' terminal sequence of PIV-3 NP mRNA (5'-AGGATTAAAG-3') was similar to the conserved sequence (formula; see text) found at the 5' termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3' terminal tetranucleotide (5'-TAAG-3') preceding the poly (A) tail.     

Complete nucleotide sequence of the matrix protein mRNA of vesicular stomatitis virus (New Jersey serotype)

The complete nucleotide sequence of the mRNA of the matrix (M) protein of vesicular stomatitis virus [New Jersey serotype, VSV(NJ)] was derived from a cDNA clone and mRNA. The mRNA is 758 nucleotides long (excluding polyadenylic acid) and encodes a protein of 229 amino acids. The predicted amino acid sequence was compared with that of the corresponding protein of Indiana serotype [VSV(IND)] and a fish rhabdovirus, spring viremia of carp virus (SVCV). An amino acid identity of 62% was found between the M proteins of VSV(NJ) and VSV(IND) while only 24% was present between VSV(NJ) and SVCV. A highly basic NH2-terminal domain followed by a proline-proline-X-tyrosine sequence was present in all the three M polypeptides. Except for the L gene sequence, the complete nucleotide sequence of the four genes of VSV(NJ) are now known. The comparison of the amino acid sequences between the Indiana and New Jersey serotypes demonstrates a high degree of homology between these genes except for the phosphoprotein gene, NS.     

Conserved and non-conserved regions of the outer coat protein, VP2, of the Australian bluetongue serotype 1 virus, revealed by sequence comparison to the VP2 of North American BTV serotype 10

The complete nucleotide sequence of the outer coat protein, VP2, of the Australian BTV serotype 1 was determined and found to be 2940 nucleotides in length. An open reading frame of 961 amino acids was found, defining a protein of 112,115 Daltons having a charge of +15 at neutral pH. This coding region was flanked by 5' and 3' non-coding regions of 17 and 37 nucleotides, respectively. When compared to VP2 of the North American BTV serotype 10 (Purdy et al., 1985, J. Virol. 55, 826-830) a homology of 52% at the nucleotide level, and 40% at the amino acid level, was observed. Significant conservation of amino acid sequence was found in eleven distinct regions; the overall hydropathy of the protein, as well as 9 of its 12 cysteine residues, was conserved. The importance of this conservation, in relation to similar observations for the other outer coat protein, VP5, is discussed.     

Nucleotide sequence of the genome segment encoding nonstructural protein NS1 of bluetongue virus serotype 20 from Australia

The nucleotide sequence of the genome segment (S6) encoding the nonstructural protein NS1 of an Australian isolate of bluetongue virus serotype 20 (BTV 20) has been determined from a series of overlapping cDNA clones synthesized using two terminal 15-mer oligonucleotides as primers. The gene consists of 1769 nucleotides with an open reading frame between nucleotides 35 and 1690 encoding a protein of 552 amino acids (molecular weight 64,506 Da; net charge –2 at pH 7). Comparison of the nucleotide and deduced amino acid sequence of this genome segment with cognate segments of isolates of BTV 1 from Australia and South Africa, and BTV 10 and BTV 17 from the United States, revealed homologies of 98%, 80%, 79%, and 79%, respectively, at the nucleotide level and 98%, 90%, 89%, and 90% identity, respectively, at the amino acid level. The data indicate that the evolutionary divergence between NS1 genes of two different Australian BTV serotypes (BTV 20 and BTV 1) is less than that between isolates of the same (BTV 1) or different serotypes from different geographical locations.The EMBL Data Library sequence accession Code is X56735 BLUETONGUE VIRUS RNA SEGMENT 6.     

Genomic location and nucleotide sequence of a serotype 3 porcine adenovirus hexon gene

Summary.  The putative hexon gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and the nucleotide sequence determined. The genomic location of the PAV3 hexon gene was determined and an open reading frame (ORF) encoding a polypeptide of 939 amino acids identified. Comparison of the nucleotide sequence of the putative PAV3 hexon gene with the sequence of the HAV2 hexon gene returned an overall identity of approximately 63%. A stop codon 144 nucleotides upstream and a start codon 18 nucleotides downstream of the ORF were identified and comparison with the HAV genome demonstrated that their positions corresponded to the stop site of the pVI gene and start site of the 23K gene, respectively. To confirm the correct start codon of the putative PAV3 hexon gene, the acceptor splice site for the putative PAV3 hexon gene was determined from cDNA and found to be between the two guanines immediately upstream of the first ATG in the ORF. Comparison with the HAV2 hexon protein showed overall identity of approximately 65%, with higher identity in the carboxy-terminus of approaching 76% over 380 amino acids. Multiple alignment of the PAV3 hexon amino acid sequence with other known HAV and animal adenovirus hexon sequences indicated that conservation is generally maintained but that identity is much lower within the loop structures of the protein. Received September 28, 1998 Accepted January 27, 1999     

Cloning and sequencing of peach rosette mosaic virus RNA1

The complete nucleotide sequence of peach rosette mosaic nepovirus (PRMV) RNA1 has been determined. A grapevine isolate of PRMV from Michigan was propagated and purified and cDNA clones representing 99. 5% of the RNA1 were constructed. The cDNA and direct RNA sequence analysis revealed a RNA species of 8004 nucleotides, excluding a 3' polyadenylated tail. The 5'- and 3'-untranslated regions were 52 and 1474 nucleotides, respectively. Computer analysis of the PRMV RNA1 nucleotide sequence unveiled a single long open reading frame of 6477 nucleotides, which is capable of encoding a 240 kDa polyprotein. Analysis of the predicted amino acid sequence of RNA1 revealed amino acid motifs characteristic of a replicase, proteinase, NTP-binding protein and a proteinase cofactor. The order and identity of these putative proteins are consistent with other nepoviruses. Analysis of PRMV RNA1 further distinguishes the taxonomic subdivisions within the nepovirus group, confirms the subgroup three status of PRMV and lays the groundwork for a replicase-mediated resistance strategy.     

Structure of the glycoprotein gene of sonchus yellow net virus, a plant rhabdovirus

The nucleotide sequence of the glycoprotein (G) gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined from viral genomic and mRNA cDNA clones. The G cistron is 2045 nucleotides (nt) long and the G protein mRNA open reading frame (ORF), as determined from the cDNA sequence, contains 1896 nt and encodes a protein of 632 amino acids. Immunoblots with antibodies elecited against the purified glycoprotein from virus particles reacted with a fusion protein produced in Escherichia coli, indicating that the cloned ORF encodes the G protein. The 5' end of the G protein mRNA corresponds to nt 5111, relative to the 3' end of the viral (minus sense) genome, as determined by primer extension from mRNA isolated from infected plants, and extends to nt position 7155 on the genomic RNA. A 34-nt untranslated 5' leader sequence and a 115-nt untranslated 3' end flank the ORF on the mRNA. The gene junctions on either side of the G gene on the genomic RNA are identical to those previously described for other SYNV genes and are similar to sequences separating genes of animal rhabdoviruses. The predicted molecular weight of the G protein is 70,215 Da, a value less than the 77,000 Da estimated for the glycosylated G protein from virus particles. The deduced amino acid sequence of the SYNV G protein shares little direct relatedness with the G proteins of other rhabdoviruses, but appears to contain a similar signal sequence, a transmembrane anchor domain, and glycosylation signals. In addition, the SYNV G protein contains a putative nuclear targeting site near the carboxy terminus, which may be involved in transit to the nuclear membrane prior to morphogenesis.     

Complete nucleotide and deduced amino acid sequence of genome segment 5 encoding the outer capsid protein, VP5, of a U.S. isolate of bluetongue virus serotype 11.

The complete nucleotide sequence of the RNA genome segment coding for the outer capsid protein, VP5, of the United States prototypic strain of bluetongue virus (BTV) serotype 11 was determined from two overlapping cDNA clones. The genome segment was found to be 1638 nucleotides in length with a single open reading frame coding for a 526 amino acid protein of MW 59,278 and having a net charge of -4.0 at neutral pH. Comparisons of the predicted amino acid sequence of VP5 of BTV 11 with those of the United States serotypes 2, 10, and 13 and two isolates of BTV 1 from Australia and South Africa confirmed earlier reports that VP5 is a conserved protein with no clear regions of variability. A computer generated consensus sequence suggested VP5 of BTV 2 to be representative of the average VP5 sequences reported thus far.