The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA3cRNA3vRNA), but its transcriptional activity (vRNA3mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNAbinding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.Influenza A virus is a negative-strand RNA virus containing eight segments of single-stranded RNA as its genome (39). The RNA genome is transcribed and replicated by the viral RNA-dependent RNA polymerase in the cell nucleus (21). The viral RNAs (vRNA) are transcribed into mRNAs and replicated through a cRNA intermediate to produce more vRNA molecules. Synthesis of these three RNA species requires different modes of initiation and termination (reviewed in references 23 and 34). Synthesis of mRNAs is primed by short capped RNA fragments that are generated from cellular pre-mRNAs by endonucleolytic cleavage. Consequently, viral mRNA molecules contain a 9-to 17-nucleotide (nt) capped host-derived RNA sequence at their 5Ј ends. On the other hand, the synthesis of cRNA and vRNA molecules is initiated in a primer-independent manner, resulting in triphosphorylated 5Ј ends. Synthesis of mRNAs is prematurely terminated 16 to 17 nucleotides from the 5Ј end of the vRNA template at a sequence of 5 to 7 uridines that acts as a polyadenylation signal (30,47,49). The poly(A) tail is synthesized by the viral RNA polymerase by repeated copying of the U sequence (47). During the synthesis of cRNA molecules, the polyadenylation signal is ignored, resulting in full-length copies of vRNA.All three reactions, i.e., vRNA3mRNA (transcription), vRNA3cRNA (first step of replication), and cRNA3vRNA (second step of replication) are catalyzed b...

Avian influenza A H5N1 viruses similar to those that infected humans in Hong Kong in 1997 continue to circulate in waterfowl and have reemerged in poultry in the region, raising concerns that these viruses could reappear in humans. The currently licensed trivalent inactivated influenza vaccines contain hemagglutinin (HA) and neuraminidase genes from epidemic strains in a background of internal genes derived from the vaccine donor strain, A/Puerto Rico/8/34 (PR8). Such reassortant candidate vaccine viruses are currently not licensed for the prevention of human infections by H5N1 influenza viruses. A transfectant H5N1/PR8 virus was generated by plasmid-based reverse genetics. The removal of the multibasic amino acid motif in the HA gene associated with high pathogenicity in chickens, and the new genotype of the H5N1/PR8 transfectant virus, attenuated the virus for chickens and mice without altering the antigenicity of the HA. A Formalin-inactivated vaccine prepared from this virus was immunogenic and protected mice from subsequent wild-type H5N1 virus challenge. This is the first successful attempt to develop an H5N1 vaccine seed virus resembling those used in currently licensed influenza A vaccines with properties that make it a promising candidate for further evaluation in humans.

mRNAs are capped at their 5-end by a unique cap structure containing N 7 -methyl guanine. Recognition of the cap structure is of paramount importance in some of the most central processes of gene expression as well as in some viral processes, such as priming of influenza virus transcription. The recent resolution of the structure of three evolutionary unrelated cap binding proteins, the vaccinia viral protein VP39, the eukaryotic translation factor eIF4E, and the nuclear cap-binding protein CBP20 showed that the recognition of the cap structure is achieved by the same general mechanism, i.e. by "sandwiching" of the N 7 -methyl guanine of the cap structure between two aromatic amino acid residues. The purpose of the present study was to test whether a similar cap recognition mechanism had independently evolved for the RNA polymerase of influenza virus. Combining in vivo and in vitro methods, we characterized two crucial aromatic amino acids, Phe 363 and Phe 404 , in the PB2 subunit of the viral RNA polymerase that are essential for cap binding. The aromaticity of these two residues is conserved in influenza A, B, and C and even in the divergent Thogoto virus PB2 subunits. Thus, our results favor a similar mechanism of cap binding by the influenza RNA polymerase as in the evolutionary unrelated VP39, eIF4E, and CBP20.Eukaryotic RNA polymerase II transcripts (mRNAs and small nuclear RNAs) are modified at their 5Ј extremity by the addition of an N 7 -methyl guanine cap (m 7 G)

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.The RNA-dependent RNA polymerase complex of influenza A virus consists of three subunits, polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). All three subunits are required for the transcription (viral RNA [vRNA] 3 mRNA) and replication (vRNA 3 cRNA 3 vRNA) of the eight segments of the negative single-stranded vRNA genome (3). The PB1 subunit forms the core of the complex and is responsible for polymerase activity. The PB2 subunit is involved in generation of capped RNA primers for the initiation of transcription by binding the cap structures of host pre-mRNAs prior to their endonucleolytic cleavage by PB1. The PA subunit is essential for both transcription and replication, but its exact role in the replication cycle of the virus is not clear. Recently, we reported that a histidine-to-alanine mutation at position 510 of PA (H510A) inhibited the endonucleolytic cleavage of capped RNAs, suggesting that PA might be directly involved in the generation of capped primers (4). PA can also induce a generalized proteolysis of both viral and host proteins, but the significance of this observation remains to be determined (9,13,17,19).In a recent report (4), a recombinant A/WSN/33 virus with an R638A mutation in the PA protein that produced pinheadsized plaques on MDBK cells was described. The R638A PA protein was expressed at levels similar to those of the wild-type PA in transfected cells, suggesting that neither the stability nor the proteolytic activity, believed to be associated with PA, was significantly affected. In functional assays, a recombinant polymerase with the R638A mutation in its PA subunit was able to transcribe and replicate a vRNA-like chloramphenicol acetyltransferase (CAT) RNA as determined by measuring CAT activity and RNA levels in transfected cells. Intriguingly, in this transient assay system, the R638A mutant polymerase apparently synthesized increased amounts of CAT vRNA (about a fivefold increase compared to the wild type) but reduced amounts of CAT mRNA (about a fivefold decrease compared to the wild type). In the context of viral infection, however, we did not observe a significant difference in the ratios of vRNA, cRNA, and mRNA for PA, HA, or NA in a primer extension assay (results not shown), suggesting that this discrepancy might be due to the artificial nature of the transient assay used previously. Further studies, employing in vitro tests to assay the promoter binding, endonuclease, and transcription initiation (with ApG or capped RNA) activities of the R638A recombin...

Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells.