Molecular cloning and sequence determination of the fusion protein gene of human parainfluenza virus type 1.

Undegraded mRNA transcripts were isolated from human parainfluenza virus type 1 (hPIV-1)-infected LLC-MK2 cells and their size was determined through denaturing agarose electrophoresis. The two predominantly represented mRNA species (1.65 and 1.87 kb) are similar in size to other paramyxoviral mRNAs that encode their respective glycoproteins. The cDNA transcripts corresponding to these two mRNAs were used to construct two size-restricted cDNA libraries. A cDNA clone, containing a 1.87-kb insert, was identified as encoding the hPIV-1 fusion protein by positively hybridizing with a synthetic oligonucleotide mix whose sequence was derived from the conserved sequences of other paramyxoviral F0 genes. The nucleotide sequence of the cDNA insert was determined and found to contain a single, large open reading frame encoding a putative protein of 60,795 Da consisting of 556 amino acids. Comparison of the amino acid sequence with the fusion proteins of other paramyxoviruses enabled the identification of the highly conserved amino acids of the F1 N-terminus. In addition, the positions of the hydrophobic signal and transmembrane regions, cysteine, and proline residues are all conserved. These analyses confirm that the cDNA sequence is that of the F0 protein. The 5' end of the fusion protein mRNA was determined by primer extension to lie 155 bases beyond the 5' end of the cDNA insert.