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European Journal of Cell Biology journal homepage: www.elsevier.com/locate/ejcb
Adaptive mutation PB2 D701N promotes nuclear import of influenza vRNPs in mammalian cells Hanna Sediri a , Folker Schwalm a , Gülsah Gabriel b , Hans-Dieter Klenk a,∗ a b
Institute of Virology, Philipps Universität, Marburg, Germany Heinrich-Pette-Institut für Experimentelle Virologie, Hamburg, Germany
a r t i c l e
i n f o
Keywords: Influenza virus Nuclear import PB2
a b s t r a c t The segmented genome of influenza viruses is translocated into the nucleus to initiate transcription and replication. The gene segments are present as viral ribonucleoprotein (vRNP) particles composed of RNA, multiple copies of the nucleoprotein (NP), and the polymerase subunits PB1, PB2 and PA. The PB2 subunit and each NP monomer contain a nuclear localisation signal (NLS) that binds to importin-␣. To throw light on the role of the NLSs of NP and PB2 in nuclear transport, we have analysed the effect of mutation D701N, responsible for the exposure of the NLS domain of PB2, on the intracellular localisation of vRNPs. We show that exposure of PB2 NLS significantly enhances the amount of vRNPs present in the nucleus. These observations suggest that entry of vRNPs into the nucleus depends on controlled interplay of the NLSs of PB2 and NP with the nuclear import machinery. © 2015 Published by Elsevier GmbH.
1. Introduction Influenza-A-viruses have a wide host range comprising wild aquatic birds, poultry and many mammals including man. Occasionally the viruses are transmitted from one species to another, but these transmissions are usually transient. However, because of their high genetic flexibility, influenza viruses may adapt in some instances to the new host. When adaptation occurs in man, the virus may give rise to a pandemic. Host specificity is determined by the interaction of the viral proteins with many host factors required for and interfering with virus replication. A shift in host specificity depends on adaptive mutations of the viral proteins that optimise these interactions in the new host (Cauldwell et al., 2014; Gabriel and Fodor, 2014; Schrauwen and Fouchier, 2014). Influenza-A-viruses are enveloped particles that contain a segmented single-stranded RNA genome of negative polarity. Each of the eight gene segments is present as a viral ribonucleoprotein (vRNP) composed of the RNA, multiple copies of the nucleoprotein (NP), and the polymerase trimer formed by the subunits PA, PB1
Abbreviations: CHX, cycloheximide; LMB, leptomycin B; NLS, nuclear localisation signal; NP, nucleoprotein; TPCK, tosyl phenylalanyl chloromethyl ketone; vRNP, viral ribonucleoprotein; WT, wild-type; w/o, without. ∗ Corresponding author at: Institute of Virology, Hans-Meerwein Straße 2, 35043 Marburg, Germany. Tel.: +49 6421 286 6191. E-mail address: [email protected] (H.-D. Klenk).
and PB2. The vRNPs show a double helix conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase at the other end (Coloma et al., 2009; Arranz et al., 2012). Infection of a cell is initiated by receptor-mediated endocytosis of the virus particle. Fusion of the viral envelope with the endosomal membrane leads to the release of incoming vRNPs into the cytoplasm and to their transport into the nucleus where replication and transcription take place. After primary transcription and translation newly synthesized NP and polymerase subunits are also transported into the nucleus to amplify transcription and replication and form progeny vRNPs. These RNPs are then exported to the cytoplasm and assembled at the cell membrane into budding virus progeny (Fig. 1). Thus, during the viral life cycle the polymerase is transported twice from the cytoplasm into the nucleus: first it is bound to incoming vRNP of infecting virus, and then newly synthesized polymerase subunits are imported in unbound form (Hutchinson and Fodor, 2012; Resa-Infante and Gabriel, 2013). The polymerase complex is an important determinant of host specificity. Over the years, several mutations in the PB2 subunit have been identified that mediate adaptation of an avian virus to a mammalian host. The most prominent ones are mutations E627K and D701N that enhance polymerase activity and replication in mammalian cells, as well as pathogenicity in animal models (Subbarao et al., 1993; Gabriel et al., 2005; Steel et al., 2009; CzudaiMatwich et al., 2014; Zhang et al., 2014). Several modes of action
http://dx.doi.org/10.1016/j.ejcb.2015.05.012 0171-9335/© 2015 Published by Elsevier GmbH.
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Fig. 1. Replication cycle of influenza-A-viruses. Infection of a cell is initiated by receptor-mediated endocytosis of the virus particle (step 1). After fusion of the viral envelope with the endosomal membrane (steps 2–3) the incoming vRNPs are released into the cytoplasm and then transported into the nucleus (step 4) where replication and transcription take place (step 5). After primary transcription and translation newly synthesized NP and polymerase subunits are transported from the cytoplasm to the nucleus to amplify transcription and replication (step 6). Each new vRNA is associated with NP proteins and the polymerase complex to form progeny vRNPs. These RNPs are then exported to the cytoplasm and assembled into virus particles (steps 7–8) budding from the plasma membrane (step 9). Modified from Neumann et al., 2009.
have been proposed for mutation E627K. Very recently, Weber et al. have shown that incoming vRNPs are directly impaired by RIG-I, and that this viral inhibition is impeded by the presence of mutation E627K (Weber et al., 2015). Mutation D701N is localised in the NLS domain of PB2 and plays an important role in adaptation of the protein to the nuclear import machinery. The mutation from an aspartate present in avian isolates to asparagine present in mammalian strains induces a conformational change in the Cterminal part of PB2 which allows interaction with importin-␣, by exposing the NLS domain (Tarendeau et al., 2007; Gabriel et al., 2008). It has been shown that mutation D701N enhances the nuclear import of newly synthesized PB2 (Gabriel et al., 2008), believed to be present in monomeric form (Fodor and Smith, 2004; Deng et al., 2005). Furthermore, there is evidence that the nuclear import of vRNPs is driven by NP associated to the viral RNA (O’Neill et al., 1995; Cros et al., 2005). However, it is not known if mutation D701N present on the PB2 subunit is as well involved in the transport of vRNP. It was therefore of interest to test if nuclear entry of vRNP is affected by this mutation. We show here that mutation D701N in the PB2 subunit enhances the nuclear localisation of vRNPs in mammalian cells. Therefore mutation D701N is not only promoting the nuclear import of newly synthesized PB2 protein, but also of PB2 bound to incoming vRNPs. These observations demonstrate that the polymerase plays an important role in the nuclear import of vRNP.
2. Results 2.1. Immunodetection of incoming vRNP in infected cells NP is highly conserved among influenza viruses. This allowed us to use an antibody known to cross-react with NP of several
strains. This antibody was used for immunostaining of 293T cells that were transfected with a single plasmid encoding NP or with plasmids encoding PB2, PA, PB1, NP and a plasmid transcribed into RNA of negative polarity (vRNP). Unbound NP protein and NP associated with vRNPs is detected in the nucleus (Fig. 2A). No staining was observed when cells were transfected with plasmids encoding for PB1, PB2, PA, HA, M and NS segments (Fig. 2B). These observations demonstrate that the antibody specifically recognizes NP and that there is no cross-reaction with the other viral proteins. To detect incoming vRNPs, cells were pre-treated with cycloheximide (CHX) and leptomycin-B (LMB). As translation is blocked under these conditions, only NP on incoming vRNP is present. By blocking the nuclear export machinery, transport of incoming vRNP from the nucleus to the cytoplasm is also prevented (Weber et al., 2015). Fig. 3 shows A549 cells infected with H9N2 WT virus in the presence or absence of CHX-LMB. In the absence of inhibitors, the viral life cycle is not obstructed, and translation of new viral mRNA occurs. Therefore, newly synthesized NP is present in the nucleus, resulting in strong nuclear staining (Fig. 3A left panel). In contrast, CHX-LMB interrupts the replication cycle after the primary transcription step. Thus, only incoming vRNP is detected, indicated by a dot-like staining pattern predominantly in the cytoplasm (Fig. 3A right panel). To complement the immunofluorescence data, infected cells were harvested at 4, 6 and 8 h post infection, and levels of NP production were monitored by Western Blot (Fig. 3B). The results show that treatment of the cells with CHX-LMB completely aborted the synthesis of NP protein, supporting the immunofluorescence observations. It has to be pointed out that the amounts of NP present on incoming vRNP are too low to be detected by Western blot analysis. Taken together these results demonstrate that NP staining and CHX-LMB treatment allow the detection of incoming vRNP. Furthermore, we observe that incoming vRNPs of H9N2 WT virus are located predominantly in the cytoplasm.
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Fig. 2. Detection of NP and vRNP in transfected cells. (A) 293T cells were transfected with plasmids encoding NP or with plasmids encoding NP in combination with PB1, PA, PB2, and RNA of negative polarity (ribonucleoprotein complex (vRNP)). At 48 h post-transfection, cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI. (B) 293T cells were transfected with plasmids encoding PB1, PA, PB2, HA, M or NS. At 48 h post-transfection, cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI.
2.2. Mutation D701N promotes nuclear import of incoming H9N2 and H7N7 vRNPs Mutation D701N has been shown to enhance the interaction with human but not with avian importins, which led to an increased nuclear localisation of newly synthesized PB2 in mammalian cells (Gabriel et al., 2008). It was therefore of interest to investigate if mutation D701N also plays a role in import of incoming vRNP. To this end, A549 cells were infected in presence of CHX-LMB with H9N2 WT virus or with virus containing the adaptive mutation D701N in the PB2 subunit. After 1 and 6 h of infection, cells were fixed, and immunodetection of NP was performed (Fig. 4A). At 1 h post infection with H9N2 WT virus, vRNPs were present only in the cytoplasm. At 6 h post infection, some vRNPs were present in the nucleus, but most vRNPs remained in the cytoplasm close to the cell surface. In contrast, vRNPs of H9N2 D701N virus were completely shifted from the cytoplasm to the nucleus at 6 h post infection (Fig. 4A). These results show clearly that PB2 mutation D701N promotes nuclear transport of incoming H9N2 vRNPs in A549 cells. We also analysed the effect of mutation D701N on vRNP import of H7N7 virus. Again, nuclear vRNP import has been compared with
H7N7 WT virus and H7N7 D701N virus (Fig. 4B). The results resembled those obtained with H9N2 virus. At 1 h post infection, vRNPs of both H7N7 WT and mutant virus were located predominantly in the cytoplasm, but showing a more diffuse pattern as compared to H9N2 virus. At 6 h post infection, there was again a shift of vRNPs from the cytoplasm to the nucleus, which was quite clear with H7N7 mutant virus and less distinct with H7N7 WT virus (Fig. 4B). In conclusion, these data illustrate that mutation D701N promotes nuclear transport of incoming vRNP of both H9N2 and H7N7 viruses.
3. Discussion It has been known from previous studies that adaptive mutation D701N enhances polymerase activity and virus replication as well as pathogenicity in a mammalian host (Gabriel et al., 2005, 2007; Steel et al., 2009). Furthermore, there is evidence that this is accomplished by promotion of the nuclear entry of newly synthesized PB2 monomers (Gabriel et al., 2008). We show now that this mutation also facilitates nuclear transport of vRNPs released from infecting virus.
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Fig. 3. Detection of incoming vRNPs of H9N2 WT virus in CHX-LMB treated cells. (A) A549 cells were or were not pre-treated with CHX-LMB for 1 h at 37 ◦ C, infected with H9N2 WT virus and incubated for 6 h (MOI 1) in the presence or absence of the inhibitors. Cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI. (B) A549 cells were or were not pre-treated with CHX-LMB and infected with H9N2 WT virus for 8 h (MOI 1). Cells were harvested 4, 6 and 8 h post infection. Cell lysates were analysed by SDS-PAGE and Western Blot using virus-specific antibodies.
Structural analysis of the C-terminal domain of PB2 has thrown light on the mechanism triggered by mutation D701N. It could be shown that an aspartic acid residue at position 701 forms a salt bridge with an arginine residue at position 753 at the C-terminal end of PB2. This salt bridge binds the C-terminal amino acids containing a NLS at positions 735–752 tightly to the main body of the protein. Mutation D701N disrupts the salt bridge resulting in the exposure of the NLS that can now bind to importin-␣ mediating the transport through the nuclear pore (Tarendeau et al., 2007).
Recent studies in which the structure of the polymerase complex has been elucidated are compatible with this view. They indicated that PB2 has retained some mobility when complexed with PA and PB1, and that its C-terminal domain may be flexible enough to allow interaction with importin-␣ (Reich et al., 2014). These observations support the data presented here indicating that exposure of the C-terminal NLS of PB2 is essential for targeting vRNPs to the nucleus. It has previously been shown that adaptation to a mammalian host by mutation D701N involves a shift in the specificity of
Fig. 4. Mutation PB2 D701N enhances the nuclear import of H9N2 and H7N7 vRNPs. A549 cells were pre-treated with CHX-LMB and infected with (A) H9N2 WT and H9N2 D701N viruses or (B) H7N7 WT and H7N7 D701N viruses for 1 h or 6 h (MOI 1). Cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). WT virus contains PB2 701D and D701N virus contains PB2 701N.
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Fig. 5. Model of entry of incoming vRNP in mammalian cells. The exposed NLS domain of PB2 701N is recognized by importin-␣. This interaction brings the vRNPs into an orientation that facilitates entry into the pore. Binding of importin-␣ to the NP NLSs drives the vRNP then through the channel. If vRNPs contain PB2 701D with unexposed NLS they are not correctly positioned, and transport through the pore is impeded.
PB2 monomers from importin-␣3 to importin-␣1 and -␣7 (Gabriel et al., 2011). Whether vRNP-importin binding is also linked to such a shift remains to be seen. With a length of 30–110 nm (depending on the size of the vRNA) and a diameter of 10–20 nm, vRNPs outsize PB2 monomers by more than an order of magnitude (Whittaker et al., 1996). It is therefore reasonable to assume that, although the classical importin pathway is used in both cases, the nuclear entry mechanisms are quite different. In vRNPs, PB2 is complexed with NP which has long been known to be actively involved in the transport process. NP has an N-terminal unconventional NLS (Wang et al., 1997) and a classical bipartite NLS (Weber et al., 1998). It has been shown previously that NP NLSs mediate nuclear entry of vRNP complexes lacking the polymerase proteins (O’Neill et al., 1995; Cros and Palese, 2003; Cros et al., 2005). These observations are in line with the findings reported here where vRNP containing PB2 701D with unexposed NLS is present in moderate amounts in the nucleus. We have also seen here, however, that exposure of the NLS in PB2 701N leads to a distinct vRNP increase in the nucleus. This observation is particularly surprising, since vRNP particles are composed of dozens of NP units containing NLSs at the protein surface (Ye et al., 2006; Ng et al., 2008). vRNPs are therefore studded with an excess of NP NLSs that out-number by far the single PB2 NLS. Yet vRNPs containing PB2 701N are efficiently transported into the nucleus, whereas entry of vRNPs with PB2 701D is limited. Since both vRNPs differ only by this mutation, it is clear that the PB2 NLS dominates the NP NLS. We conclude from these observations that the transport of vRNPs is controlled by a hierarchical interplay between the NLSs of PB2 and NP with the import machinery: First, binding of the PB2 NLS to importin-␣ forces vRNPs into a head-down orientation that allows entry into the nuclear pore. Passage through the channel of the pore is then driven by sequential importin-␣ binding to NP NLSs along the vRNP shaft (Fig. 5). This model does not depend on adaptive NPmutations that enhance importin-␣ binding and nuclear import of newly synthesized NP (Gabriel et al., 2005, 2007). Whether such mutations have an effect on vRNP entry remains to be seen.
4. Materials and methods 4.1. Cells 293 T human embryonic kidney, Madin-Darby canine kidney (MDCK), and A549 human lung adenocarcinoma epithelial cell lines were cultivated in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% foetal calf serum (FCS; Gibco), glutamine, penicillin, and streptomycin (Gibco). All cell cultures were kept at 37 ◦ C and 5% CO2 . For infection, growth medium was changed to infection medium containing 0.3% bovine serum albumin (BSA; PAA) instead of FCS. 4.2. Viruses The influenza viruses used in this study were A/quail/Shantou/ 2061/2000 (H9N2) virus (kindly provided by Y. Guan, Hong-Kong University, Hong-Kong China) and A/Seal/Massachusetts/35/1980 (H7N7) (Gabriel et al., 2005). Influenza viruses were propagated in MDCK cells in infection medium containing 1 g/ml tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). Cell supernatants were cleared by low-speed centrifugation and stored at −80 ◦ C until titration by plaque assay. 4.3. Plasmids and transfection To clone all eight genes of A/quail/Shantou/2061/2000 (H9N2), each segment was amplified by RT-PCR from isolated RNA using the QIAamp viral RNA minikit (Qiagen) and ligated into plasmid pHW2000. Mutation D701N was introduced into the plasmid coding for the PB2 subunit by site-directed mutagenesis using the QuickChange II site-directed mutagenesis kit® (Agilent) according to the manufacturer’s protocol. All transfections were performed using the ProFection Mammalian Transfection System (Promega) according to the manufacturers protocol. Presence of mutations
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was checked by sequencing. Primer sequences are available on request.
4.4. Antibodies Polyclonal antibodies against A/chicken/Rostock/34 (H7N1) virus were derived from rabbits. A mouse monoclonal antibody against the Influenza A virus nucleoprotein (NP) was purchased from Abcam. Species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Dako.
4.5. Generation of recombinant viruses Recombinant H9N2 viruses were generated by reverse genetics (Hoffmann et al., 2000). Briefly, all plasmids (1 g) encoding gene segments were transfected in 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions for 6 h. Then transfection medium was replaced by fresh DMEM medium containing TPCK-trypsin (1 g/ml). After 48 h incubation, supernatant was collected, treated for 1 h with TPCK-trypsin (10 g/ml) at 37 ◦ C, and then transferred on MDCK cells for another 48–72 h incubation at 37 ◦ C, 5% CO2 for virus propagation. Virus stocks were stored at −80 ◦ C until titration by plaque assay. All experiments with recombinant H9N2 wild-type and mutant viruses were approved by the relevant German authorities (the Regierungspräsidium Giessen) and conducted in biosafety level 3 facilities at the Institute of Virology of the University of Marburg.
4.6. Plaque assay Virus titres were determined by plaque assay with Avicel overlay as described (Matrosovich et al., 2006). Briefly, MDCK cells were inoculated with 10-fold serial dilutions of each sample and incubated with Avicel overlay containing 1 g/ml TPCKtrypsin for 48 h. Following this incubation, cells were fixed, permeabilized, and immunostained using antiserum raised against A/Quail/Shantou/2061/2000 (H9N2) virus and horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) followed by final incubation with the peroxidase substrate TrueBlue (KPL).
4.7. Immunofluorescence analysis A549 cells were pre-treated for 1 h at 37 ◦ C with cycloheximide (CHX, Sigma) and leptomycine B (LMB, Sigma) in infection medium at final concentrations of 50 g/ml and 16 nM, respectively. Cells were inoculated with virus at a multiplicity of infection (MOI) of 10 for 1 h, the inoculum was removed, and the cells were incubated in infection medium containing CHX-LMB for 1 h to 6 h. Cells were fixed with 4% paraformaldehyde dissolved in PBS, permeabilized for 30 min with 0.3% Triton X-100 (Sigma) dissolved in PBS, and blocked for 1 h at room temperature with 2% bovine serum albumine (BSA), 5% glycerol, 0.2% Tween 20 dissolved in PBS. Primary mouse monoclonal antibodies recognizing NP protein (1:200) were diluted in blocking solution, and cells were stained for 1 h at room temperature. After three times washing with PBS, cells were incubated for 45 min at room temperature with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse secondary antibodies (1:200) supplemented with 4,4-diamidino-2-phenylindole (DAPI, 1:10.000). Cells were washed three times with PBS, and coverslips were mounted using Fluoroprep solution (bioMérieux). Stained cell samples were examined using a Leica SP5 confocal microscope.
4.8. SDS-PAGE and Western blotting A549 cells were or were not pre-treated with CHX-LMB. Cells were inoculated with virus at a MOI 1 for 1 h. The inoculum was then removed, and the cells were incubated in infection medium containing or not containing CHX-LMB for 8 h. Cell lysates were harvested at 4, 6 and 8 h post infection. To analyse expression of NP in infected cells, cell lysates were subjected to SDS-PAGE and Western blot analysis as described previously (Böttcher-Friebertshauser et al., 2010). Pellets were resuspended in reducing SDS sample buffer, heated 2 times at 100 ◦ C for 10 min, and subjected to SDSPAGE and Western blot analysis. Proteins were detected with polyclonal anti-A/chicken/Rostock/34 (H7N1) antibodies at dilution 1:6000. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (SFB 593 TP-B1) and the European Commission (FP7 project FLUPHARM). References Arranz, R., Coloma, R., Chichon, F.J., Conesa, J.J., Carrascosa, J.L., Valpuesta, J.M., Ortin, J., Martin-Benito, J., 2012. The structure of native influenza virion ribonucleoproteins. Science 338 (6114), 1634–1637. Böttcher-Friebertshauser, E., Freuer, C., Sielaff, F., Schmidt, S., Eickmann, M., Uhlendorff, J., Steinmetzer, T., Klenk, H.D., Garten, W., 2010. Cleavage of influenza virus hemagglutinin by airway proteases TMPRSS2 and HAT differs in subcellular localization and susceptibility to protease inhibitors. J. Virol. 84 (11), 5605–5614. Cauldwell, A.V., Long, J.S., Moncorge, O., Barclay, W.S., 2014. Viral determinants of Influenza A virus host range. J. Gen. Virol. 95 (Pt 6), 1193–1210. Coloma, R., Valpuesta, J.M., Arranz, R., Carrascosa, J.L., Ortin, J., Martin-Benito, J., 2009. The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog. 5 (6), e1000491. Cros, J.F., Garcia-Sastre, A., Palese, P., 2005. An unconventional NLS is critical for the nuclear import of the Influenza A virus nucleoprotein and ribonucleoprotein. Traffic 6 (3), 205–213. Cros, J.F., Palese, P., 2003. Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses. Virus Res. 95 (1–2), 3–12. Czudai-Matwich, V., Otte, A., Matrosovich, M., Gabriel, G., Klenk, H.D., 2014. PB2 mutations D701N and S714R promote adaptation of an influenza H5N1 virus to a mammalian host. J. Virol. 88 (16), 8735–8742. Deng, T., Sharps, J., Fodor, E., Brownlee, G.G., 2005. 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. J. Virol. 79 (13), 8669–8674. Fodor, E., Smith, M., 2004. The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the Influenza A virus RNA polymerase complex. J. Virol. 78 (17), 9144–9153. Gabriel, G., Abram, M., Keiner, B., Wagner, R., Klenk, H.D., Stech, J., 2007. Differential polymerase activity in avian and mammalian cells determines host range of influenza virus. J. Virol. 81 (17), 9601–9604. Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H.D., Stech, J., 2005. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc. Natl. Acad. Sci. U. S. A. 102 (51), 18590–18595. Gabriel, G., Fodor, E., 2014. Molecular determinants of pathogenicity in the polymerase complex. In: Compans, R.W., Oldstone, M.B.A. (Eds.), Influenza Pathogenesis and Control – Volume I. Springer International Publishing, pp. 35–60. Gabriel, G., Herwig, A., Klenk, H.D., 2008. Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of Influenza A virus. PLoS Pathog. 4 (2), e11. Gabriel, G., Klingel, K., Otte, A., Thiele, S., Hudjetz, B., Arman-Kalcek, G., Sauter, M., Shmidt, T., Rother, F., Baumgarte, S., Keiner, B., Hartmann, E., Bader, M., Brownlee, G.G., Fodor, E., Klenk, H.D., 2011. Differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus. Nat. Commun. 2 (81), 156. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Webster, R.G., 2000. A DNA transfection system for generation of Influenza A virus from eight plasmids. Proc. Natl. Acad. Sci. U. S. A. 97 (11), 6108–6113. Hutchinson, E.C., Fodor, E., 2012. Nuclear import of the Influenza A virus transcriptional machinery. Vaccine 30 (51), 7353–7358. Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H.-D., 2006. New low-viscosity overlay medium for viral plaque assays. Virol. J. 3 (1), 63. Neumann, G., Noda, T., Kawaoka, Y., 2009. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459 (7249), 931–939.
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European Journal of Cell Biology journal homepage: www.elsevier.com/locate/ejcb
Adaptive mutation PB2 D701N promotes nuclear import of influenza vRNPs in mammalian cells Hanna Sediri a , Folker Schwalm a , Gülsah Gabriel b , Hans-Dieter Klenk a,∗ a b
Institute of Virology, Philipps Universität, Marburg, Germany Heinrich-Pette-Institut für Experimentelle Virologie, Hamburg, Germany
a r t i c l e
i n f o
Keywords: Influenza virus Nuclear import PB2
a b s t r a c t The segmented genome of influenza viruses is translocated into the nucleus to initiate transcription and replication. The gene segments are present as viral ribonucleoprotein (vRNP) particles composed of RNA, multiple copies of the nucleoprotein (NP), and the polymerase subunits PB1, PB2 and PA. The PB2 subunit and each NP monomer contain a nuclear localisation signal (NLS) that binds to importin-␣. To throw light on the role of the NLSs of NP and PB2 in nuclear transport, we have analysed the effect of mutation D701N, responsible for the exposure of the NLS domain of PB2, on the intracellular localisation of vRNPs. We show that exposure of PB2 NLS significantly enhances the amount of vRNPs present in the nucleus. These observations suggest that entry of vRNPs into the nucleus depends on controlled interplay of the NLSs of PB2 and NP with the nuclear import machinery. © 2015 Published by Elsevier GmbH.
1. Introduction Influenza-A-viruses have a wide host range comprising wild aquatic birds, poultry and many mammals including man. Occasionally the viruses are transmitted from one species to another, but these transmissions are usually transient. However, because of their high genetic flexibility, influenza viruses may adapt in some instances to the new host. When adaptation occurs in man, the virus may give rise to a pandemic. Host specificity is determined by the interaction of the viral proteins with many host factors required for and interfering with virus replication. A shift in host specificity depends on adaptive mutations of the viral proteins that optimise these interactions in the new host (Cauldwell et al., 2014; Gabriel and Fodor, 2014; Schrauwen and Fouchier, 2014). Influenza-A-viruses are enveloped particles that contain a segmented single-stranded RNA genome of negative polarity. Each of the eight gene segments is present as a viral ribonucleoprotein (vRNP) composed of the RNA, multiple copies of the nucleoprotein (NP), and the polymerase trimer formed by the subunits PA, PB1
Abbreviations: CHX, cycloheximide; LMB, leptomycin B; NLS, nuclear localisation signal; NP, nucleoprotein; TPCK, tosyl phenylalanyl chloromethyl ketone; vRNP, viral ribonucleoprotein; WT, wild-type; w/o, without. ∗ Corresponding author at: Institute of Virology, Hans-Meerwein Straße 2, 35043 Marburg, Germany. Tel.: +49 6421 286 6191. E-mail address: [email protected] (H.-D. Klenk).
and PB2. The vRNPs show a double helix conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase at the other end (Coloma et al., 2009; Arranz et al., 2012). Infection of a cell is initiated by receptor-mediated endocytosis of the virus particle. Fusion of the viral envelope with the endosomal membrane leads to the release of incoming vRNPs into the cytoplasm and to their transport into the nucleus where replication and transcription take place. After primary transcription and translation newly synthesized NP and polymerase subunits are also transported into the nucleus to amplify transcription and replication and form progeny vRNPs. These RNPs are then exported to the cytoplasm and assembled at the cell membrane into budding virus progeny (Fig. 1). Thus, during the viral life cycle the polymerase is transported twice from the cytoplasm into the nucleus: first it is bound to incoming vRNP of infecting virus, and then newly synthesized polymerase subunits are imported in unbound form (Hutchinson and Fodor, 2012; Resa-Infante and Gabriel, 2013). The polymerase complex is an important determinant of host specificity. Over the years, several mutations in the PB2 subunit have been identified that mediate adaptation of an avian virus to a mammalian host. The most prominent ones are mutations E627K and D701N that enhance polymerase activity and replication in mammalian cells, as well as pathogenicity in animal models (Subbarao et al., 1993; Gabriel et al., 2005; Steel et al., 2009; CzudaiMatwich et al., 2014; Zhang et al., 2014). Several modes of action
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Fig. 1. Replication cycle of influenza-A-viruses. Infection of a cell is initiated by receptor-mediated endocytosis of the virus particle (step 1). After fusion of the viral envelope with the endosomal membrane (steps 2–3) the incoming vRNPs are released into the cytoplasm and then transported into the nucleus (step 4) where replication and transcription take place (step 5). After primary transcription and translation newly synthesized NP and polymerase subunits are transported from the cytoplasm to the nucleus to amplify transcription and replication (step 6). Each new vRNA is associated with NP proteins and the polymerase complex to form progeny vRNPs. These RNPs are then exported to the cytoplasm and assembled into virus particles (steps 7–8) budding from the plasma membrane (step 9). Modified from Neumann et al., 2009.
have been proposed for mutation E627K. Very recently, Weber et al. have shown that incoming vRNPs are directly impaired by RIG-I, and that this viral inhibition is impeded by the presence of mutation E627K (Weber et al., 2015). Mutation D701N is localised in the NLS domain of PB2 and plays an important role in adaptation of the protein to the nuclear import machinery. The mutation from an aspartate present in avian isolates to asparagine present in mammalian strains induces a conformational change in the Cterminal part of PB2 which allows interaction with importin-␣, by exposing the NLS domain (Tarendeau et al., 2007; Gabriel et al., 2008). It has been shown that mutation D701N enhances the nuclear import of newly synthesized PB2 (Gabriel et al., 2008), believed to be present in monomeric form (Fodor and Smith, 2004; Deng et al., 2005). Furthermore, there is evidence that the nuclear import of vRNPs is driven by NP associated to the viral RNA (O’Neill et al., 1995; Cros et al., 2005). However, it is not known if mutation D701N present on the PB2 subunit is as well involved in the transport of vRNP. It was therefore of interest to test if nuclear entry of vRNP is affected by this mutation. We show here that mutation D701N in the PB2 subunit enhances the nuclear localisation of vRNPs in mammalian cells. Therefore mutation D701N is not only promoting the nuclear import of newly synthesized PB2 protein, but also of PB2 bound to incoming vRNPs. These observations demonstrate that the polymerase plays an important role in the nuclear import of vRNP.
2. Results 2.1. Immunodetection of incoming vRNP in infected cells NP is highly conserved among influenza viruses. This allowed us to use an antibody known to cross-react with NP of several
strains. This antibody was used for immunostaining of 293T cells that were transfected with a single plasmid encoding NP or with plasmids encoding PB2, PA, PB1, NP and a plasmid transcribed into RNA of negative polarity (vRNP). Unbound NP protein and NP associated with vRNPs is detected in the nucleus (Fig. 2A). No staining was observed when cells were transfected with plasmids encoding for PB1, PB2, PA, HA, M and NS segments (Fig. 2B). These observations demonstrate that the antibody specifically recognizes NP and that there is no cross-reaction with the other viral proteins. To detect incoming vRNPs, cells were pre-treated with cycloheximide (CHX) and leptomycin-B (LMB). As translation is blocked under these conditions, only NP on incoming vRNP is present. By blocking the nuclear export machinery, transport of incoming vRNP from the nucleus to the cytoplasm is also prevented (Weber et al., 2015). Fig. 3 shows A549 cells infected with H9N2 WT virus in the presence or absence of CHX-LMB. In the absence of inhibitors, the viral life cycle is not obstructed, and translation of new viral mRNA occurs. Therefore, newly synthesized NP is present in the nucleus, resulting in strong nuclear staining (Fig. 3A left panel). In contrast, CHX-LMB interrupts the replication cycle after the primary transcription step. Thus, only incoming vRNP is detected, indicated by a dot-like staining pattern predominantly in the cytoplasm (Fig. 3A right panel). To complement the immunofluorescence data, infected cells were harvested at 4, 6 and 8 h post infection, and levels of NP production were monitored by Western Blot (Fig. 3B). The results show that treatment of the cells with CHX-LMB completely aborted the synthesis of NP protein, supporting the immunofluorescence observations. It has to be pointed out that the amounts of NP present on incoming vRNP are too low to be detected by Western blot analysis. Taken together these results demonstrate that NP staining and CHX-LMB treatment allow the detection of incoming vRNP. Furthermore, we observe that incoming vRNPs of H9N2 WT virus are located predominantly in the cytoplasm.
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Fig. 2. Detection of NP and vRNP in transfected cells. (A) 293T cells were transfected with plasmids encoding NP or with plasmids encoding NP in combination with PB1, PA, PB2, and RNA of negative polarity (ribonucleoprotein complex (vRNP)). At 48 h post-transfection, cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI. (B) 293T cells were transfected with plasmids encoding PB1, PA, PB2, HA, M or NS. At 48 h post-transfection, cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI.
2.2. Mutation D701N promotes nuclear import of incoming H9N2 and H7N7 vRNPs Mutation D701N has been shown to enhance the interaction with human but not with avian importins, which led to an increased nuclear localisation of newly synthesized PB2 in mammalian cells (Gabriel et al., 2008). It was therefore of interest to investigate if mutation D701N also plays a role in import of incoming vRNP. To this end, A549 cells were infected in presence of CHX-LMB with H9N2 WT virus or with virus containing the adaptive mutation D701N in the PB2 subunit. After 1 and 6 h of infection, cells were fixed, and immunodetection of NP was performed (Fig. 4A). At 1 h post infection with H9N2 WT virus, vRNPs were present only in the cytoplasm. At 6 h post infection, some vRNPs were present in the nucleus, but most vRNPs remained in the cytoplasm close to the cell surface. In contrast, vRNPs of H9N2 D701N virus were completely shifted from the cytoplasm to the nucleus at 6 h post infection (Fig. 4A). These results show clearly that PB2 mutation D701N promotes nuclear transport of incoming H9N2 vRNPs in A549 cells. We also analysed the effect of mutation D701N on vRNP import of H7N7 virus. Again, nuclear vRNP import has been compared with
H7N7 WT virus and H7N7 D701N virus (Fig. 4B). The results resembled those obtained with H9N2 virus. At 1 h post infection, vRNPs of both H7N7 WT and mutant virus were located predominantly in the cytoplasm, but showing a more diffuse pattern as compared to H9N2 virus. At 6 h post infection, there was again a shift of vRNPs from the cytoplasm to the nucleus, which was quite clear with H7N7 mutant virus and less distinct with H7N7 WT virus (Fig. 4B). In conclusion, these data illustrate that mutation D701N promotes nuclear transport of incoming vRNP of both H9N2 and H7N7 viruses.
3. Discussion It has been known from previous studies that adaptive mutation D701N enhances polymerase activity and virus replication as well as pathogenicity in a mammalian host (Gabriel et al., 2005, 2007; Steel et al., 2009). Furthermore, there is evidence that this is accomplished by promotion of the nuclear entry of newly synthesized PB2 monomers (Gabriel et al., 2008). We show now that this mutation also facilitates nuclear transport of vRNPs released from infecting virus.
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Fig. 3. Detection of incoming vRNPs of H9N2 WT virus in CHX-LMB treated cells. (A) A549 cells were or were not pre-treated with CHX-LMB for 1 h at 37 ◦ C, infected with H9N2 WT virus and incubated for 6 h (MOI 1) in the presence or absence of the inhibitors. Cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI. (B) A549 cells were or were not pre-treated with CHX-LMB and infected with H9N2 WT virus for 8 h (MOI 1). Cells were harvested 4, 6 and 8 h post infection. Cell lysates were analysed by SDS-PAGE and Western Blot using virus-specific antibodies.
Structural analysis of the C-terminal domain of PB2 has thrown light on the mechanism triggered by mutation D701N. It could be shown that an aspartic acid residue at position 701 forms a salt bridge with an arginine residue at position 753 at the C-terminal end of PB2. This salt bridge binds the C-terminal amino acids containing a NLS at positions 735–752 tightly to the main body of the protein. Mutation D701N disrupts the salt bridge resulting in the exposure of the NLS that can now bind to importin-␣ mediating the transport through the nuclear pore (Tarendeau et al., 2007).
Recent studies in which the structure of the polymerase complex has been elucidated are compatible with this view. They indicated that PB2 has retained some mobility when complexed with PA and PB1, and that its C-terminal domain may be flexible enough to allow interaction with importin-␣ (Reich et al., 2014). These observations support the data presented here indicating that exposure of the C-terminal NLS of PB2 is essential for targeting vRNPs to the nucleus. It has previously been shown that adaptation to a mammalian host by mutation D701N involves a shift in the specificity of
Fig. 4. Mutation PB2 D701N enhances the nuclear import of H9N2 and H7N7 vRNPs. A549 cells were pre-treated with CHX-LMB and infected with (A) H9N2 WT and H9N2 D701N viruses or (B) H7N7 WT and H7N7 D701N viruses for 1 h or 6 h (MOI 1). Cells were fixed and immunostained using an antibody against NP and a FITC-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). WT virus contains PB2 701D and D701N virus contains PB2 701N.
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Fig. 5. Model of entry of incoming vRNP in mammalian cells. The exposed NLS domain of PB2 701N is recognized by importin-␣. This interaction brings the vRNPs into an orientation that facilitates entry into the pore. Binding of importin-␣ to the NP NLSs drives the vRNP then through the channel. If vRNPs contain PB2 701D with unexposed NLS they are not correctly positioned, and transport through the pore is impeded.
PB2 monomers from importin-␣3 to importin-␣1 and -␣7 (Gabriel et al., 2011). Whether vRNP-importin binding is also linked to such a shift remains to be seen. With a length of 30–110 nm (depending on the size of the vRNA) and a diameter of 10–20 nm, vRNPs outsize PB2 monomers by more than an order of magnitude (Whittaker et al., 1996). It is therefore reasonable to assume that, although the classical importin pathway is used in both cases, the nuclear entry mechanisms are quite different. In vRNPs, PB2 is complexed with NP which has long been known to be actively involved in the transport process. NP has an N-terminal unconventional NLS (Wang et al., 1997) and a classical bipartite NLS (Weber et al., 1998). It has been shown previously that NP NLSs mediate nuclear entry of vRNP complexes lacking the polymerase proteins (O’Neill et al., 1995; Cros and Palese, 2003; Cros et al., 2005). These observations are in line with the findings reported here where vRNP containing PB2 701D with unexposed NLS is present in moderate amounts in the nucleus. We have also seen here, however, that exposure of the NLS in PB2 701N leads to a distinct vRNP increase in the nucleus. This observation is particularly surprising, since vRNP particles are composed of dozens of NP units containing NLSs at the protein surface (Ye et al., 2006; Ng et al., 2008). vRNPs are therefore studded with an excess of NP NLSs that out-number by far the single PB2 NLS. Yet vRNPs containing PB2 701N are efficiently transported into the nucleus, whereas entry of vRNPs with PB2 701D is limited. Since both vRNPs differ only by this mutation, it is clear that the PB2 NLS dominates the NP NLS. We conclude from these observations that the transport of vRNPs is controlled by a hierarchical interplay between the NLSs of PB2 and NP with the import machinery: First, binding of the PB2 NLS to importin-␣ forces vRNPs into a head-down orientation that allows entry into the nuclear pore. Passage through the channel of the pore is then driven by sequential importin-␣ binding to NP NLSs along the vRNP shaft (Fig. 5). This model does not depend on adaptive NPmutations that enhance importin-␣ binding and nuclear import of newly synthesized NP (Gabriel et al., 2005, 2007). Whether such mutations have an effect on vRNP entry remains to be seen.
4. Materials and methods 4.1. Cells 293 T human embryonic kidney, Madin-Darby canine kidney (MDCK), and A549 human lung adenocarcinoma epithelial cell lines were cultivated in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% foetal calf serum (FCS; Gibco), glutamine, penicillin, and streptomycin (Gibco). All cell cultures were kept at 37 ◦ C and 5% CO2 . For infection, growth medium was changed to infection medium containing 0.3% bovine serum albumin (BSA; PAA) instead of FCS. 4.2. Viruses The influenza viruses used in this study were A/quail/Shantou/ 2061/2000 (H9N2) virus (kindly provided by Y. Guan, Hong-Kong University, Hong-Kong China) and A/Seal/Massachusetts/35/1980 (H7N7) (Gabriel et al., 2005). Influenza viruses were propagated in MDCK cells in infection medium containing 1 g/ml tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). Cell supernatants were cleared by low-speed centrifugation and stored at −80 ◦ C until titration by plaque assay. 4.3. Plasmids and transfection To clone all eight genes of A/quail/Shantou/2061/2000 (H9N2), each segment was amplified by RT-PCR from isolated RNA using the QIAamp viral RNA minikit (Qiagen) and ligated into plasmid pHW2000. Mutation D701N was introduced into the plasmid coding for the PB2 subunit by site-directed mutagenesis using the QuickChange II site-directed mutagenesis kit® (Agilent) according to the manufacturer’s protocol. All transfections were performed using the ProFection Mammalian Transfection System (Promega) according to the manufacturers protocol. Presence of mutations
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was checked by sequencing. Primer sequences are available on request.
4.4. Antibodies Polyclonal antibodies against A/chicken/Rostock/34 (H7N1) virus were derived from rabbits. A mouse monoclonal antibody against the Influenza A virus nucleoprotein (NP) was purchased from Abcam. Species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Dako.
4.5. Generation of recombinant viruses Recombinant H9N2 viruses were generated by reverse genetics (Hoffmann et al., 2000). Briefly, all plasmids (1 g) encoding gene segments were transfected in 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions for 6 h. Then transfection medium was replaced by fresh DMEM medium containing TPCK-trypsin (1 g/ml). After 48 h incubation, supernatant was collected, treated for 1 h with TPCK-trypsin (10 g/ml) at 37 ◦ C, and then transferred on MDCK cells for another 48–72 h incubation at 37 ◦ C, 5% CO2 for virus propagation. Virus stocks were stored at −80 ◦ C until titration by plaque assay. All experiments with recombinant H9N2 wild-type and mutant viruses were approved by the relevant German authorities (the Regierungspräsidium Giessen) and conducted in biosafety level 3 facilities at the Institute of Virology of the University of Marburg.
4.6. Plaque assay Virus titres were determined by plaque assay with Avicel overlay as described (Matrosovich et al., 2006). Briefly, MDCK cells were inoculated with 10-fold serial dilutions of each sample and incubated with Avicel overlay containing 1 g/ml TPCKtrypsin for 48 h. Following this incubation, cells were fixed, permeabilized, and immunostained using antiserum raised against A/Quail/Shantou/2061/2000 (H9N2) virus and horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) followed by final incubation with the peroxidase substrate TrueBlue (KPL).
4.7. Immunofluorescence analysis A549 cells were pre-treated for 1 h at 37 ◦ C with cycloheximide (CHX, Sigma) and leptomycine B (LMB, Sigma) in infection medium at final concentrations of 50 g/ml and 16 nM, respectively. Cells were inoculated with virus at a multiplicity of infection (MOI) of 10 for 1 h, the inoculum was removed, and the cells were incubated in infection medium containing CHX-LMB for 1 h to 6 h. Cells were fixed with 4% paraformaldehyde dissolved in PBS, permeabilized for 30 min with 0.3% Triton X-100 (Sigma) dissolved in PBS, and blocked for 1 h at room temperature with 2% bovine serum albumine (BSA), 5% glycerol, 0.2% Tween 20 dissolved in PBS. Primary mouse monoclonal antibodies recognizing NP protein (1:200) were diluted in blocking solution, and cells were stained for 1 h at room temperature. After three times washing with PBS, cells were incubated for 45 min at room temperature with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse secondary antibodies (1:200) supplemented with 4,4-diamidino-2-phenylindole (DAPI, 1:10.000). Cells were washed three times with PBS, and coverslips were mounted using Fluoroprep solution (bioMérieux). Stained cell samples were examined using a Leica SP5 confocal microscope.
4.8. SDS-PAGE and Western blotting A549 cells were or were not pre-treated with CHX-LMB. Cells were inoculated with virus at a MOI 1 for 1 h. The inoculum was then removed, and the cells were incubated in infection medium containing or not containing CHX-LMB for 8 h. Cell lysates were harvested at 4, 6 and 8 h post infection. To analyse expression of NP in infected cells, cell lysates were subjected to SDS-PAGE and Western blot analysis as described previously (Böttcher-Friebertshauser et al., 2010). Pellets were resuspended in reducing SDS sample buffer, heated 2 times at 100 ◦ C for 10 min, and subjected to SDSPAGE and Western blot analysis. Proteins were detected with polyclonal anti-A/chicken/Rostock/34 (H7N1) antibodies at dilution 1:6000. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (SFB 593 TP-B1) and the European Commission (FP7 project FLUPHARM). References Arranz, R., Coloma, R., Chichon, F.J., Conesa, J.J., Carrascosa, J.L., Valpuesta, J.M., Ortin, J., Martin-Benito, J., 2012. The structure of native influenza virion ribonucleoproteins. Science 338 (6114), 1634–1637. Böttcher-Friebertshauser, E., Freuer, C., Sielaff, F., Schmidt, S., Eickmann, M., Uhlendorff, J., Steinmetzer, T., Klenk, H.D., Garten, W., 2010. Cleavage of influenza virus hemagglutinin by airway proteases TMPRSS2 and HAT differs in subcellular localization and susceptibility to protease inhibitors. J. Virol. 84 (11), 5605–5614. Cauldwell, A.V., Long, J.S., Moncorge, O., Barclay, W.S., 2014. Viral determinants of Influenza A virus host range. J. Gen. Virol. 95 (Pt 6), 1193–1210. Coloma, R., Valpuesta, J.M., Arranz, R., Carrascosa, J.L., Ortin, J., Martin-Benito, J., 2009. The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog. 5 (6), e1000491. Cros, J.F., Garcia-Sastre, A., Palese, P., 2005. An unconventional NLS is critical for the nuclear import of the Influenza A virus nucleoprotein and ribonucleoprotein. Traffic 6 (3), 205–213. Cros, J.F., Palese, P., 2003. Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses. Virus Res. 95 (1–2), 3–12. Czudai-Matwich, V., Otte, A., Matrosovich, M., Gabriel, G., Klenk, H.D., 2014. PB2 mutations D701N and S714R promote adaptation of an influenza H5N1 virus to a mammalian host. J. Virol. 88 (16), 8735–8742. Deng, T., Sharps, J., Fodor, E., Brownlee, G.G., 2005. 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. J. Virol. 79 (13), 8669–8674. Fodor, E., Smith, M., 2004. The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the Influenza A virus RNA polymerase complex. J. Virol. 78 (17), 9144–9153. Gabriel, G., Abram, M., Keiner, B., Wagner, R., Klenk, H.D., Stech, J., 2007. Differential polymerase activity in avian and mammalian cells determines host range of influenza virus. J. Virol. 81 (17), 9601–9604. Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H.D., Stech, J., 2005. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc. Natl. Acad. Sci. U. S. A. 102 (51), 18590–18595. Gabriel, G., Fodor, E., 2014. Molecular determinants of pathogenicity in the polymerase complex. In: Compans, R.W., Oldstone, M.B.A. (Eds.), Influenza Pathogenesis and Control – Volume I. Springer International Publishing, pp. 35–60. Gabriel, G., Herwig, A., Klenk, H.D., 2008. Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of Influenza A virus. PLoS Pathog. 4 (2), e11. Gabriel, G., Klingel, K., Otte, A., Thiele, S., Hudjetz, B., Arman-Kalcek, G., Sauter, M., Shmidt, T., Rother, F., Baumgarte, S., Keiner, B., Hartmann, E., Bader, M., Brownlee, G.G., Fodor, E., Klenk, H.D., 2011. Differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus. Nat. Commun. 2 (81), 156. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Webster, R.G., 2000. A DNA transfection system for generation of Influenza A virus from eight plasmids. Proc. Natl. Acad. Sci. U. S. A. 97 (11), 6108–6113. Hutchinson, E.C., Fodor, E., 2012. Nuclear import of the Influenza A virus transcriptional machinery. Vaccine 30 (51), 7353–7358. Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H.-D., 2006. New low-viscosity overlay medium for viral plaque assays. Virol. J. 3 (1), 63. Neumann, G., Noda, T., Kawaoka, Y., 2009. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459 (7249), 931–939.
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