The P gene of human parainfluenza virus type 1 encodes P and C proteins but not a cysteine-rich V protein.

The nucleotide sequence of the P gene of human parainfluenza virus type 1 (PIV1) was determined from cloned cDNA copies of the mRNA. By analogy with the gene organization of Sendai virus, two open reading frames in the mRNA sense of the gene were identified as coding sequences for the P protein (568 amino acids with an estimated molecular weight of 64,655) and the C protein (204 amino acids with an estimated molecular weight of 24,108). Comparison of the deduced amino acid sequences of the P and C proteins of PIV1 with those of Sendai virus showed a high degree of homology. However, a sequence for the cysteine-rich V protein, which was considered a common feature of other paramyxoviruses, was interrupted by the presence of multiple stop codons. The sequence analysis of three P-gene-specific cDNA clones generated from genomic RNA by polymerase chain reaction and one additional clone generated from mRNA confirmed that the coding sequence for the cysteine-rich region is silent in the PIV1 gene and thus is not translated into protein. Two potential editing sites with the consensus sequence 3'UUYUCCC were found in the PIV1 P gene at positions 564 to 570 and 1430 to 1436. However, examination of the PIV1 mRNA population by a primer extension method indicated that neither of these sites is utilized. These results indicate that the PIV1 P gene has a coding strategy different from those of other paramyxovirus P genes.     

Mapping of antigenic domains of Sendai virus nucleocapsid protein expressed in Escherichia coli.

Several nonoverlapping epitopes were mapped on the primary sequence of the Sendai virus NP protein. After a complete cDNA clone of the Sendai virus NP gene was expressed in Escherichia coli, deletion constructs were used to generate a series of overlapping NP fragments deleted at their C termini. Immunoblot analyses with 11 monoclonal antibodies identified four antigenic sites. All of these sites resided in the C-terminal half of NP and were also the only sites detected with a polyclonal serum. These findings confirm and extend the evidence that the C terminus of the NP protein represents the domain exposed on the surface of the nucleocapsid. One of the monoclonal antibodies reacted with a site, comprising only 6 amino acids, lying with a hinge between an alpha-helix and a beta-strand in the predicted secondary structure of NP. Since this antibody is a potent inhibitor of in vitro viral RNA synthesis (K. L. Deshpande and A. Portner, Virology 139:32-42, 1984), the epitope may be critical to the flexibility of the NP molecule that makes the RNA template accessible during RNA synthesis.     

Nucleotide sequence of the bovine parainfluenza 3 virus genome: its 3'' end and the genes of NP, P, C and M proteins.

We present the nucleotide sequence of bovine parainfluenza 3 virus (BPIV3) genome from its 3' end to the opening region of the F gene, through the NP, P plus C, and M genes. Comparison of the sequence with those reported for other paramyxoviruses indicated that BPIV3 was most similar to human parainfluenza 3 virus (HPIV3), and also very similar to Sendai virus in the structural make-up of its genome and the amino acid sequences of its gene products, suggesting that these three viruses constitute a paramyxovirus subgroup from which Newcastle disease and measles viruses are separable. In BPIV3 and Sendai virus, the NP and M proteins, the main structural elements, were more highly conserved than the functionally important P and C proteins. This tendency was also observed even in BPIV3 and HPIV3. Virus-specific amino acid sequences of the NP and M proteins were found at the carboxyl and amino terminal regions, respectively. BPIV3 M mRNA was found to have aberrations in its poly A attachment site.     

Editing of the Sendai virus P/C mRNA by G insertion occurs during mRNA synthesis via a virus-encoded activity.

Two forms of the Sendai virus P/C mRNA have been predicted: one an exact copy of the viral genome, and the other with a single G insertion within a run of three G's. We directly cloned the mRNA or portions of it containing the insertion site and screened the resulting colonies with oligonucleotides that could distinguish the presence of three or four G's at this position. We found that 31% of the mRNAs did in fact contain the predicted insertion, whereas the viral genomes contained no heterogeneity at this position. A smaller fraction (7%) of the mRNA contained two to eight G's inserted at this position. The insertions also took place during RNA synthesis in vitro with purified virions but were not detected when the mRNA was expressed in vivo via a vaccinia virus recombinant. When the Sendai virus- and vaccinia virus-derived P/C mRNAs were coexpressed in the same cells under conditions in which each could be distinguished, those from the Sendai genome were altered as before, but those from the vaccinia virus genome remained unaltered. The activity that alters the mRNA is therefore likely to be coded for by the virus and cannot function in trans.     

Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small

The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA. We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs. DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene. Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich. The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.     

Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.