Nucleotide sequence changes in the polymerase basic protein 2 gene of temperature-sensitive mutants of influenza A virus.

Influenza A viruses bearing temperature-sensitive (ts) mutations are restricted in replication in the respiratory tract of animals and humans and are therefore attenuated. Nucleotide sequences were determined for the RNA segment coding for the polymerase basic protein 2 (PB2) from a panel of 12 influenza A/Udorn/307/72 (H3N2) ts viruses, previously characterized to have a ts mutation in the PB2 gene. Each of the viruses with a ts mutation in the PB2 gene had a single amino acid change located at position 65, 100, 112, 174, 298, 310, 386, 391, 556, or 658 of the PB2 protein. The sites of the single mutations were scattered throughout the length of the protein and occurred in regions that are highly conserved among the influenza A virus PB2 predicted amino acid sequences. Interestingly, the substitution of aspartic acid for asparagine at position 556 was found to lie within a region that has homology with cap-binding motifs of human and yeast proteins. Taken together, the findings of lesion sites in the A/Udorn/307/72 PB2 gene and the three reported amino acid changes at positions 265, 417, and 512 for A/AA/6/60, A/WSN/33, and A/FPV/Ros/34 ts PB2 genes, respectively, indicate that the PB2 gene can sustain a viable ts mutation at different sites. This information will allow us to construct cloned cDNA copies of the A/Udorn/307/72 PB2 gene mutagenized at specific sites. Different configurations of two or more ts mutations may be incorporated into the cDNA PB2 gene constructs. We have a host-range reassortant virus that should permit rescue of in vitro-produced transcripts of the PB2 gene into infectious virus. The rescue of these mutated PB2 RNA segments into an infectious influenza A virus may lead to the development of live attenuated reassortant virus vaccines that are satisfactorily attenuated, genetically stable, and immunogenic in humans.