Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs.     

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein

Influenza virus PA is a subunit of RNA-dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin-like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa-histidine-tagged M1 expressed in Escherichia coli bound to PA. Hexa-histidine-tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc-Leu-Leu-Val-Tyr-MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.     

The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex

The RNA genome of influenza virus is transcribed and replicated by the viral RNA polymerase complex in the cell nucleus. We have generated green fluorescent protein (GFP)-tagged polymerase subunits to study the assembly of the polymerase complex. Our results show that individually expressed polymerase basic protein 1 (PB1) and polymerase acidic protein (PA) subunits were distributed in both the cytoplasm and the nucleus, while the polymerase basic protein 2 (PB2) subunit accumulated in the nucleus. Although it has been reported that PB1 alone accumulates in the nucleus, we demonstrate that PB1 requires the coexpression of PA for efficient nuclear accumulation. Our results support a model which proposes that PB1 and PA are transported into the nucleus as a complex.     

Defective assembly of influenza A virus due to a mutation in the polymerase subunit PA

The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.     

Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus

Adaptive mutations in viral polymerase, which is composed of PB1, PB2, and PA, of avian influenza viruses are major genetic determinants of the host range. In this study, to elucidate the molecular mechanism of mammalian adaptation of avian viral polymerase, we performed cell-based vRNP reconstitution assays and biochemical analyses using purified recombinant viral polymerase complexes. We found that avian viral polymerase from A/duck/Pennsylvania/10,218/84 (DkPen) enhances the viral polymerase activity in mammalian cells by replacing the PA or PB2 gene with that from human influenza virus A/WSN/33 (WSN). Chimeric constructs between DkPen PA and WSN PA showed that the N-terminal endonuclease domain of WSN PA was essential for the mammalian adaptation of DkPen viral polymerase. We also found that the cap-snatching activity of purified DkPen viral polymerase was more than 5 times weaker than that of WSN in vitro in a PB2 Glu627-dependent manner. However, the cap-snatching activity of DkPen viral polymerase was hardly increased by replacing DkPen PA to WSN PA. These results suggest that the activity of viral genome replication may be enhanced in the DkPen reassortant containing WSN PA.     

A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase promotes the generation of defective interfering RNAs

An R638A mutation of the polymerase acidic protein (PA) subunit of the RNA polymerase of influenza A/WSN/33 virus results in severe attenuation of viral growth in cell culture by promoting the synthesis of defective interfering RNAs. We propose that R638A is an "elongation" mutant that destabilizes PA-RNA template interactions during elongation. A C453R mutation in PA can compensate for this defect, suggesting that amino acids C453 and R638 form part of the same domain.     

Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers

The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.     

Promoter analysis of influenza virus RNA polymerase.

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.     

Involvement of the N-terminal portion of influenza virus RNA polymerase subunit PB1 in nucleotide recognition

The influenza virus PB1 protein functions as a catalytic subunit of the viral RNA-dependent RNA polymerase and contains the highly conserved motifs of RNA-dependent RNA polymerases together with putative nucleotide-binding sites. PB1 also binds to viral genomic RNAs and its replicative intermediates through the promoter regions. The detail function and interplay between functional domains are not clarified although a part of structures and functions of PB1 have been clarified. In this study, we analyzed the function of PB1 subunit in the sense of nucleotide recognition using ribavirin, which is a nucleoside analog and inhibits viral RNA synthesis of many RNA viruses including influenza virus. We screened ribavirin-resistant PB1 mutants from randomly mutated PB1 cDNA library using a mini-replicon assay, and we identified a single mutation at the amino acid position 27 of PB1 as an important residue for the nucleotide recognition.