Nuclear transport of influenza virus polymerase PA protein (original) (raw)
Related papers
Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit
Journal of General Virology, 1994
The protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A beta-galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.
Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2
The EMBO journal, 1986
In order to re-examine the sub-cellular location of the three influenza A/NT/60/68 polymerase proteins PB1, PB2 and PA in infected cells, specific antisera for each polymerase component have been prepared by immunizing rabbits with polymerase-beta-galactosidase fusion proteins synthesized in Escherichia coli. We show that polymerase PB1, PB2, and PA are predominantly associated with the nucleus of influenza-infected MDCK cells by immunocytochemical techniques. In the case of polymerase PB2 we investigate the possibility that nuclear accumulation is an intrinsic property of the PB2 protein. Using a vaccinia-PB2 recombinant virus, we show that PB2 accumulates intra-nuclearly in monkey CV-1 cells in the absence of any other influenza protein, suggesting it contains an intrinsic nuclear signal.
Journal of Virology, 2004
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.
Virology, 2012
The influenza polymerase complex composed of PA, PB1 and PB2, plays a key role in viral replication and pathogenicity. Newly synthesized components must be translocated to the nucleus, where replication and transcription of viral genomes take place. Previous studies suggest that while PB2 is translocated to the nucleus independently, PA and PB1 subunits could not localize to the nucleus unless in a PA-PB1 complex. To further determine the molecular interactions between the components, we created a panel of 16 hybridoma cell lines, which produce monoclonal antibodies (mAbs) against each polymerase component. We showed that, although PB1 interacts with both PA and PB2 individually, nuclear localization of PB1 is enhanced only when co-expressed with PA. Interestingly, one of the anti-PA mAbs reacted much more strongly with PA when co-expressed with PB1. These results suggest that PA-PB1 interactions induce a conformational change in PA, which could be required for its nuclear translocation.
The PA influenza virus polymerase subunit is a phosphorylated protein
Journal of General Virology, 1998
The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by twodimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [ 32 P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine
Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins
Journal of Virology, 1998
Influenza virus nucleoprotein (NP) is a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. In this study, we investigated the interaction of NP with the viral polymerase protein complex. Using coimmunoprecipitation with monospecific or monoclonal antibodies, we observed that NP interacted with the RNP-free polymerase protein complex in influenza virus-infected cells. In addition, coexpression of the components of the polymerase protein complex (PB1, PB2, or PA) with NP either together or pairwise revealed that NP interacts with PB1 and PB2 but not PA. Interaction of NP with PB1 and PB2 was confirmed by both coimmunoprecipitation and histidine tagging of the NP-PB1 and NP-PB2 complexes. Further, it was observed that NP-PB2 interaction was rather labile and sensitive to dissociation in 0.1% sodium dodecyl sulfate and that the stability of NP-PB2 interaction was regulated by the sequences present at the ...
Journal of Virology, 2006
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 3, importin 3, 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.
The Journal of General Virology, 2011
Avian influenza A viruses often do not propagate efficiently in mammalian cells. The viral polymerase protein PB2 is important for this host restriction, with amino-acid polymorphisms at residue 627 and other positions acting as ‘signatures’ of avian- or human-adapted viruses. Restriction is hypothesized to result from differential interactions (either positive or inhibitory) with unidentified cellular factors. We applied fluorescence recovery after photobleaching (FRAP) to investigate the mobility of the viral polymerase in the cell nucleus using A/PR/8/34 and A/Turkey/England/50-92/91 as model strains. As expected, transcriptional activity of a polymerase with the avian PB2 protein was strongly dependent on the identity of residue 627 in human but not avian cells, and this correlated with significantly slower diffusion of the inactive polymerase in human but not avian nuclei. In contrast, the activity and mobility of the PR8 polymerase was affected much less by residue 627. Sequen...
Involvement of Influenza Virus PA Subunit in Assembly of Functional RNA Polymerase Complexes
Journal of Virology, 2005
The RNA-dependent RNA polymerase of influenza virus consists of three subunits, PB1, PB2, and PA, and synthesizes three kinds of viral RNAs, vRNA, cRNA, and mRNA. PB1 is a catalytic subunit; PB2 recognizes the cap structure for generation of the primer for transcription; and PA is thought to be involved in viral RNA replication. However, the process of polymerase complex assembly and the exact nature of polymerase complexes involved in synthesis of the three different RNA species are not yet clear. ts53 virus is a temperaturesensitive (ts) mutant derived from A/WSN/33 (A. Sugiura, M. Ueda, K. Tobita, and C. Enomoto, Virology 65:363-373, 1975). We confirmed that the mRNA synthesis level of ts53 remains unaffected at the nonpermissive temperature, whereas vRNA synthesis is largely reduced. Sequencing of the gene encoding ts53 PA and recombinant virus rescue experiments revealed that an amino acid change from Leu to Pro at amino acid position 226 is causative of temperature sensitivity. By glycerol density gradient analyses of nuclear extracts prepared from wild-type virus-infected cells, we found that polymerase proteins sediment in three fractions: one (H fraction) consists of RNP complexes, another (M fraction) contains active polymerases but not viral RNA, and the other (L fraction) contains inactive forms of polymerases. Pulse-chase experiments showed that polymerases in the L fraction are converted to those in the M fraction. In ts53-infected cells, polymerases accumulated in the L fraction. These results strongly suggest that PA is involved in the assembly of functional viral RNA polymerase complexes from their inactive intermediates.