Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1.
Influenza virus polymerase complex is a heterotrimer consisting of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Of these, only PB1, which has been implicated in RNA chain elongation, possesses the four conserved motifs (motifs I, II, III, and IV) and the four invariant amino acids (one in each motif) found among all viral RNA-dependent RNA or RNA-dependent DNA polymerases. We have modified an assay system developed by Huang et al. (T.-J. Huang, P. Palese, and M. Krystal, J. Virol. 64:5669-5673, 1990) to reconstitute the functional polymerase activity in vivo. Using this assay, we have examined the requirement of each of these motifs of PB1 in polymerase activity. We find that each of these invariant amino acids is critical for PB1 activity and that mutation in any one of these residues renders the protein nonfunctional. We also find that in motif III, which contains the SSDD sequence, the signature sequence of influenza virus RNA polymerase, SDD is essentially invariant and cannot accommodate sequences found in other RNA viral polymerases. However, conserved changes in the flanking sequences of SDD can be partially tolerated. These results provide the experimental evidence that influenza virus PB1 possesses a similar polymerase module as has been proposed for other RNA viruses and that the core SDD sequence of influenza virus PB1 represents a sequence variant of the GDN in negative-stranded nonsegmented RNA viruses, GDD in positive-stranded RNA virus and double-stranded RNA viruses, or MDD in retroviruses.
Biswas
S K
SK
Department of Microbiology and Immunology, Jonsson Comprehensive Cancer Center, University of California at Los Angeles School of Medicine 90024-1747.
Nayak
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*; D014779:Virus Replication
D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D015500:Chloramphenicol O-Acetyltransferase; D012324:RNA-Dependent RNA Polymerase