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. For efficient virus replication, the polymerase subunit PB2 and the nucleoprotein (NP) of avian viruses required importin-α3, whereas PB2 and NP of mammalian viruses showed importin-α7 specificity. H1N1v replication depended on both, importin-α3 and -α7, suggesting ongoing adaptation of this virus. Thus, differences in importin-α specificity are determinants of host range underlining the importance of the nuclear envelope in interspecies transmission.

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. The polymerase activity of the FJ strain was significantly greater than the GX strain. Experiments with hybrid polymerase constructs - both in vitro and in ribonucleoprotein cell-based assays, suggested that the PA and to a lesser extent the PB2 subunits of the polymerase, were responsible for increased polymerase activity of the high pathogenic strain. However, promoter binding was inversely correlated with polymerase activity implying that excessive promoter binding inhibited polymerase activity by preventing promoter clearance. Overall, we suggest that the influenza polymerase is one of the determinants of pathogenicity of duck H5N1 viruses.

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. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions-at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein-protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs.

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). Both H5N1 strains, originally isolated from patients in 1997 and 2004, showed significantly higher polymerase activity compared with two classical human strains, A/WSN/33 (H1N1) and A/NT/60/68 (H3N2) in vitro. This increased polymerase activity correlated with enhanced promoter binding. The N-terminal region of the PA subunit was the major determinant of this enhanced promoter activity. CONCLUSIONS/SIGNIFICANCE: Overall we suggest that the N-terminal region of the PA subunit of two recent H5N1 strains can influence promoter binding and we speculate this may be a factor in their virulence.

Hsp90 inhibitors reduce influenza virus replication in cell culture.

The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.

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. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3'-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

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. Cross-linking studies suggested that this inhibition was due to a reduction in promoter binding of the mutant polymerases to the cRNA promoter. We conclude that the L163-I178 region of PA is directly or indirectly involved in cRNA promoter binding and suggest a novel function for PA in modulating promoter binding.

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. The use of either adenosine or AMP, rather than ATP, provides a convenient, sensitive and easy assay of replication initiation. Moreover, no pppApG was detected when a PB1-PA dimer, rather than the trimeric polymerase, was used to catalyse synthesis, contrasting with a previous report using baculovirus-expressed influenza RNA polymerase. Overall, it is suggested that the heterotrimeric polymerase is essential for the initiation of replication.

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. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.

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. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.

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. To do this, two in vitro replication assays, which generated transcripts that had 5' triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

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. The aim of the current study was to investigate the function of two evolutionary conserved regions in PB1 - from aa 233 to 249 and 269 to 281, which lie immediately N- and C-terminal, respectively, of a previously proposed binding site for the 3' end of the vRNA promoter. The previously proposed binding site extended from aa 249 to 256 and centred on two phenylalanine residues (F251 and F254). However, the fact that F251 is required for polymerase activity was not confirmed here. Instead, it was proposed that the 233-249 region contains a new 5' vRNA promoter-binding site, and arginine residues crucial for this activity were characterized. However, residues 269-281 were unlikely to be directly involved in promoter binding. These results are discussed in relation to the previous studies and a new model for vRNA promoter binding to the influenza RNA polymerase is presented.

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. Importantly, cells infected with influenza A virus also displayed mitochondrial PB2. We show that an N-terminal motif composed of 120 amino acids is sufficient for localization of PB2 to mitochondria. In particular, leucine residues at positions 7 and 10 were essential for mitochondrial targeting. Recombinant influenza A/WSN/33 viruses expressing PB2 proteins with L7A and/or L10A mutations showed reduced viral titers, but unaffected levels of transcription, replication, and protein expression. The introduction of L7A and/or L10A mutations into recombinant viruses correlated with reduced mitochondrial membrane potential in infected cells, suggesting that mitochondrial localization of PB2 contributes to the preservation of mitochondrial function during influenza virus infection.

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. Our results are consistent with a recently proposed model for the sequential assembly of viral RNA polymerase complex in which the PB1-PA dimeric complex and the PB2 monomer are transported into the nucleus separately and then assembled in the nucleus.

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. Recent structural studies on the cellular cap-binding complex, the eukaryotic translation initiation factor 4E and the vaccinia virus protein 39, suggest that these three evolutionary unrelated cap-binding proteins have evolved a common cap-binding pocket by convergent evolution. In this pocket the positively charged N(7)-methylated guanine ring of the cap structure is stacked between two aromatic amino acids. In this review, the similarities and differences in cap binding by these three different cap-binding proteins are discussed. A comparison with new functional data for another viral cap-binding protein--the polymerase basic protein (PB2) of influenza virus--suggests that a similar cap-binding mechanism has also evolved in influenza virus.

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. We suggest that there may be no switch regulating the initiation of RNA synthesis and present a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP.

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. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5' and 3' ends and (ii) base pairing in the stems of both the 5' and 3' hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3' hairpin loop were crucial for promoter activity in vivo. However, the 3' hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3' hairpin loop remains unknown.