Search

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.

We report here the nucleotide sequence of the L gene of avian pneumovirus (APV). This is the second pneumovirus L gene and the second avian paramyxovirus L gene, following that of Newcastle disease virus, to be sequenced. The APV L gene is 6099 nucleotides long and encodes a single large ORF of 2004 amino acids. This makes the APV L protein the smallest to be described for any nonsegmented, negative-strand RNA virus. The protein contains six linear non-contiguous domains, a putative ATP-binding site and four polymerase motifs previously described for the L proteins of negative-strand RNA viruses. Phylogenetic analysis of domain III of 14 different L proteins suggests the pneumoviruses to be as distant in evolutionary terms from the other members of the Paramyxoviridae as are the Filoviridae.

The nucleocapsid protein (N) gene of two subgroup A and one subgroup B strains of avian pneumovirus has been cloned and sequenced. The gene of all three isolates comprised 1197 nucleotides (nt), which formed a single major open reading frame, potentially encoding a protein of 391 amino acid residues. The N gene of the two subgroup A isolates differed by only 1 nt but differed by 282 (24%) nt and 35 (11%) amino acids from the B isolate. The predicted protein was identical in length to that of human, bovine and ovine respiratory syncytial viruses, the amino acid identity being approximately 41% overall but with some regions of identity > 90%.

The nucleotide sequence of the gene encoding the matrix protein of a subgroup B avian pneumovirus has been determined. The gene shows 73.5% homology with that of a subgroup A virus, with most differences occurring in the third codon position. Comparison with pneumovirus matrix proteins shows that the APV matrix protein retains the hydrophobic domain common to the others. The analysis indicates that the matrix protein gene can be used to differentiate the two APV subgroups.

The sequences of the genes encoding the putative attachment (G) proteins of pathogenic (strain J3666) mouse lung-passaged and nonpathogenic (strain 15) tissue culture-passaged strains of pneumonia virus of mice (PVM) have been determined. In both cases the major polypeptide was synthesised from the second open reading frame (ORF), a feature also found in the G gene of respiratory syncytial (RS) virus, another pneumovirus. However, the ORFs of the G genes of the two PVM strains were initiated at different nucleotide positions in the mRNA and comparison of hydrophobicity profiles revealed the presence of the putative amino-terminal cytoplasmic domain in the strain J3666 G protein and its absence in the predicted G protein of PVM strain 15. In common with the G protein of RS virus, the gene product of both PVM strains contained a high serine, threonine, and proline content. Indirect immunofluorescence analysis of BSC-1 cells expressing the G gene products confirmed the surface location of the proteins. Thus, the absence of a cytoplasmic domain does not interfere with the translocation of the G protein of PVM strain 15. In vitro translation of mRNA from the two PVM genes directed the synthesis of a larger polypeptide with the G gene of PVM strain J3666 than was seen with strain 15 G gene. In addition, a second protein was seen with strain J3666 mRNA which was the same size as the strain 15 G protein.