Differential use of importin-α isoforms governs cell tropism and host adaptation of influenza virus.

Influenza A viruses are a threat to humans due to their ability to cross species barriers, as illustrated by the 2009 H1N1v pandemic and sporadic H5N1 transmissions. Interspecies transmission requires adaptation of the viral polymerase to importin-α, a cellular protein that mediates transport into the nucleus where transcription and replication of the viral genome takes place. In this study, we analysed replication, host specificity and pathogenicity of avian and mammalian influenza viruses, in importin-α-silenced cells and importin-α-knockout mice, to understand the role of individual importin-α isoforms in adaptation.

Correlation between polymerase activity and pathogenicity in two duck H5N1 influenza viruses suggests that the polymerase contributes to pathogenicity.

The influenza RNA polymerase is known to be important in pathogenicity and adaptation of avian influenza viruses to mammalian hosts. However, the molecular mechanisms responsible are only partly understood. Here we investigated the role of the polymerase in two different, closely related, H5N1 influenza viruses - a high pathogenic, A/duck/Fujian/01/2002 (FJ) strain and a low pathogenic, A/duck/Guangxi/53/2002 (GX) strain.

A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization.

We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N-hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described.

The N-terminal region of the PA subunit of the RNA polymerase of influenza A/HongKong/156/97 (H5N1) influences promoter binding.

Background: The RNA polymerase of influenza virus is a heterotrimeric complex of PB1, PB2 and PA subunits which cooperate in the transcription and replication of the viral genome. Previous research has shown that the N-terminal region of the PA subunit of influenza A/WSN/33 (H1N1) virus is involved in promoter binding.

Methodology/principal Findings: Here we extend our studies of the influenza RNA polymerase to that of influenza strains A/HongKong/156/97 (H5N1) and A/Vietnam/1194/04 (H5N1).

Role of initiating nucleoside triphosphate concentrations in the regulation of influenza virus replication and transcription.

The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA.

Differential role of the influenza A virus polymerase PA subunit for vRNA and cRNA promoter binding.

The RNA polymerase of influenza A virus is a heterotrimeric complex of PB1, PB2 and PA subunits that is required for transcription and replication of the viral genome. Here, we demonstrate a differential requirement of the PA subunit for binding to the vRNA and cRNA promoters--specifically, PA is more important for binding to the cRNA than the vRNA promoter. Furthermore, five point mutations were identified in the L163-I178 region of PA, which resulted in an inhibition of polymerase activity when provided with a cRNA compared to vRNA promoter.

Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication.

Both transcription and replication of the influenza virus RNA genome are catalysed by a virus-specific RNA polymerase. Recently, an in vitro assay, based on the synthesis of pppApG, for the initiation of replication by recombinant RNA polymerase in the absence of added primer was described. Here, these findings are extended to show that adenosine, AMP and ADP can each substitute for ATP in reactions catalysed by either recombinant ribonucleoprotein or RNA polymerase complexes with either model virion RNA (vRNA) or cRNA promoters.

Role of ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex.

The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin beta3, importin beta3, or importin 5, as an interactor of the PB1 subunit.

Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding.

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA.

A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase.

The influenza A virus RNA-dependent RNA polymerase consists of three subunits PB1, PB2 and PA. The 5' and 3' terminal sequences of the viral RNA (vRNA) form the viral promoter and are bound by the PB1 subunit. The putative promoter-binding sites of the PB1 subunit have been mapped in previous studies but with contradictory results.

Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication.

Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed.

Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses.

Influenza virus RNA polymerase is a heterotrimeric complex consisting of PB1, PB2, and PA subunits. These polymerase subunits accumulate in the nucleus of infected cells. We report here that PB2, from both human and avian influenza viruses, could also localize to mitochondria in transfected cells.

In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex.

Influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex of PB1, PB2, and PA. We show that the individually expressed PB2 subunit can be assembled with the coexpressed PB1-PA dimer in vitro into a transcriptionally active complex. Furthermore, we demonstrate that a model viral RNA promoter can bind to the PB1-PA dimer prior to assembly with PB2.

Recognition of mRNA cap structures by viral and cellular proteins.

Most cellular and eukaryotic viral mRNAs have a cap structure at their 5' end that is critical for efficient translation. Cap structures also aid in mRNA transport from nucleus to cytoplasm and, in addition, protect the mRNAs from degradation by 5' exonucleases. Cap function is mediated by cap-binding proteins that play a key role in translational control.

Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates.

The RNA-dependent RNA polymerase of influenza A virus is responsible for both transcription and replication of negative-sense viral RNA. It is thought that a "switching" mechanism regulates the transition between these activities. We demonstrate that, in the presence of preexisting viral RNA polymerase and nucleoprotein (NP), influenza A virus synthesizes both mRNA (transcription) and cRNA (replication) early in infection.

Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical for replication.

Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A "corkscrew" secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses.

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.

Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding.

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.

A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs.

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 RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter.

The RNA polymerase of the influenza virus is responsible for the transcription and replication of the segmented RNA viral genome during infection of host cells. Polymerase function is known to be strictly dependent on interaction with its RNA promoter, but no attempts to investigate whether the virion RNA (vRNA) promoter stabilizes the polymerase have been reported previously. Here we tested whether the vRNA promoter protects the polymerase against heat inactivation.

Differential activation of influenza A virus endonuclease activity is dependent on multiple sequence differences between the virion RNA and cRNA promoters.

Influenza virus endonuclease activity was studied in vitro with model virion RNA (vRNA) and cRNA molecules. We show that endonuclease activity can be partially rescued by transplanting vRNA-like promoter features into the model cRNA promoter. This study defines three distinctive features within the vRNA promoter--absent in the cRNA promoter--that are required for endonuclease cleavage.