[Show abstract][Hide abstract] ABSTRACT: The RNA-dependent RNA polymerase complex of influenza A virus consists of three subunits, PB1, PB2 and PA. To investigate the function of the PA subunit, we mutated evolutionarily conserved amino acids to alanines in the C-terminal third of PA of influenza A/WSN/33 that shows the highest degree of conservation between PA proteins of influenza A, B and C viruses. A H510A mutation inhibited the endonuclease cleavage of capped RNAs, K539A downregulated replication, while R638A resulted in severe attenuation of viral growth in cell culture by promoting the synthesis of defective interfering (DI) RNAs. Our results show that PA is required for both transcription and replication and that, in addition to its role in endonuclease cleavage, it might also act as an elongation factor during viral RNA synthesis. To investigate the function of PB2 in cap binding, we mutated all evolutionarily conserved aromatic amino acids of PB2 of influenza A/WSN/33. Our results suggest that F363 and F404 bind the cap structure of mRNA in an aromatic sandwich as in the evolutionarily unrelated VP39, eIF4E and CBP20.
International Congress Series 06/2004; 1263:25-28. DOI:10.1016/j.ics.2004.02.021
[Show abstract][Hide abstract] ABSTRACT: mRNAs are capped at their 5'-end by a unique cap structure containing N7-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 N7-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, Phe363 and Phe404, 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 05/2003; 77(8):5017-20. DOI:10.1128/JVI.77.8.5017-5020.2003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U-A replacing G-C at nucleotide positions 12'-11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.
Journal of General Virology 04/2003; 84(Pt 3):507-15. DOI:10.1099/vir.0.18795-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 (vRNA-->cRNA-->vRNA), but its transcriptional activity (vRNA-->mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNA-binding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.
Journal of Virology 09/2002; 76(18):8989-9001. DOI:10.1128/JVI.76.18.8989-9001.2002 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Polyadenylation of eukaryotic mRNAs occurs by cleavage of the pre-mRNA downstream of a conserved AAUAAA hexamer, followed by polyadenylation of the upstream cleavage product by a poly(A) polymerase in a template-independent manner. By contrast, polyadenylation of influenza virus mRNA molecules is performed by the viral RNA polymerase by reiterative copying of a U5–7 sequence near the 5′ end of the vRNA template. Methods: We used both in vitro and in vivo transcription assays to demonstrate that replacement of the viral U6 poly(A) site with a A6 sequence in vRNA results in transcription products with poly(U) tails. A recombinant influenza A/WSN/33 virus has been generated, by using a helper virus-dependent rescue method, in which the U6 poly(A) site of the neuraminidase (NA) gene has been modified so that the virus expressed a poly(U)-tailed NA mRNA. The growth properties of the virus were characterised in cell culture and in an animal model. A plasmid-based transcription/replication assay was used to study whether influenza RNA polymerase transcripts are substrates for cleavage and polyadenylation by the eukaryotic 3′ end processing machinery. Results and Conclusions: Our studies show, firstly, that the U5–7 sequence near the 5′ end of vRNA acts directly as a template for poly(A) addition. Secondly, we show that a recombinant virus expressing a poly(U)-tailed mRNA for the NA gene is attenuated in cell culture and in mice, suggesting a novel strategy for designing live attenuated influenza virus vaccines. Thirdly, we show that a poly(U)-tailed NA influenza RNA polymerase transcripts containing a eukaryotic poly(A) site, could be processed by the cellular polyadenylation machinery even although such molecules are not synthesized by the host RNA polymerase II.
International Congress Series 10/2001; 1219:427-434. DOI:10.1016/S0531-5131(01)00645-8
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 07/2001; 75(11):5410-5. DOI:10.1128/JVI.75.11.5410-5415.2001 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The poly(A) tail of influenza virus mRNAs is synthesized by the viral RNA polymerase by reiterative copying of a U5-7 sequence near the 5' end of the viral RNA (vRNA) template. We have engineered a vRNA molecule by replacing its viral U6 poly(A) site with a negative-sense eukaryotic polyadenylation signal. The vRNA was transcribed by the viral RNA polymerase and the transcription product was processed by the cellular 3' end processing machinery in vivo. According to the current model, 3' end processing of eukaryotic pre-mRNAs is coupled to cellular RNA polymerase II (pol II) transcription; thus only RNAs synthesized by pol III are believed to be polyadenylated efficiently. Our results show that the cellular polyadenylation machinery is nevertheless able to recognize and process RNA transcripts that are not synthesized by pol II, indicating that synthesis by pol II is not an absolute requirement for 3' end processing in vivo.