Adaptive mutations in the nuclear export protein of human-derived H5N1 strains

にの The nuclear export protein NEP (NS2) of the highly pathogenic human-derived H5N1 strain には A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase にば activity in human cells in a concentration-dependent manner. While low NEP levels enhance にぱ the polymerase activity, high levels act inhibitory. To gain insights into the underlying にひ mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after ぬど reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP ぬな resides in the C-terminal moiety and that removal of the last three amino acids completely ぬに abrogates this activity. Moreover, compared to full length NEP, the C-terminal moiety alone ぬぬ exhibited significantly higher activity and seemed to be deregulated, since even highest ぬね concentration did not result in an inhibition of polymerase activity. To determine transient ぬの interactions between the Nand C-terminal domains in cis, we fused both ends of NEP to a ぬは split click-beetle luciferase and performed fragment complementation assays. With ぬば decreasing temperature, increased luciferase activity was observed, suggesting that ぬぱ intramolecuar binding between Cand N-terminal domains is preferentially stabilized at low ぬひ temperatures. This stabilizing effect was significantly reduced with the adaptive mutation ねど M16I or a combination of adaptive mutations (M16I, Y41C and E75G), which further increase ねな polymerase activity also at 34°C. We therefore propose a model, in which the N-terminal ねに moiety of NEP exerts an inhibitory function by backfolding to the C-terminal domain. In this ねぬ model, adaptive mutations in NEP decrease binding between the Cand N-terminal domains, ねね thereby allowing the protein to “open up” and become active already at low temperature. ねの ねは Introduction ねば Zoonotic transmissions of avian influenza A viruses pose a constant threat to the human ねぱ population and can cause severe pandemics associated with high numbers of fatal cases ねひ mainly due to the lack of a preexisting immunity (1-3). Because of effective species barriers, のど only in rare cases avian viruses are able to establish a new lineage in humans. To overcome のな this species barrier, changes in receptor specificity, stability and glycosylation of the viral のに on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 3 hemagglutinin (HA) as well as adaptation to the host immune response are required (1, 4-6). のぬ In addition, avian viruses have to overcome the poor polymerase activity in human cells (1, 7, のね 8). The basic underlying mechanisms for the impaired polymerase activity are unknown and のの are possibly due to incompatibility with cellular factors required for efficient replication (9-12). のは Recent evidence suggests that in the case of avian H5N1 viruses efficient replication of のば genomic RNA (vRNA) is affected in human cells (13). のぱ のひ Several adaptive mutations in the polymerase, consisting of the subunits PA, PB1 and PB2, はど of avian H5N1 viruses are known to be required to overcome the impaired replication はな efficiency in human cells (7, 8). Although the single mutation E627K in PB2 is sufficient to はに increase polymerase activity to levels comparable to human influenza A virus strains (13-17), はぬ approximately 40% of the human-derived H5N1 strains, including A/Thailand/1(KAN-1)/04 はね maintained the avian 627E signature (8). This suggests that other mutations in the はの polymerase subunits are required to increase the polymerase activity in human cells. Indeed, はは several adaptive mutations in H5N1 strains were identified in all three polymerase subunits はば and, surprisingly, also in the nuclear export protein (NEP) (7, 8, 13). はぱ はひ NEP is encoded by segment 8, consists of 121 amino acids and is translated from mRNA ばど that represents a splice product of the NS1 coding mRNA. It has been suggested to mediate ばな nuclear export of newly assembled viral ribonucleoproteins (RNPs) by bridging the ばに interaction of the viral matrix protein M1, which is associated to the vRNPs, and the nuclear ばぬ export protein Crm1 (18-20). Structural investigations revealed that NEP consists of an Nばね terminal and C-terminal domain, both harboring two α-helices (N1, N2 and C1 and C2; Fig. ばの 1A) (20, 21). The two nuclear export sequences (NESs) that mediate the interaction with ばは CRM1 are located in the first and second N-terminal α-helix of NEP (18, 22). The N-terminal ばば domain is proposed to be highly flexible and solvent exposed (21). In contrast, the C-terminal ばぱ domain that crystallizes as a homo dimer comprises the M1 binding site and adopts a rigid, ばひ protease-resistant hairpin structure (20). NEP was also found to interact with the く subunit of ぱど on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 4 the F1Fo-ATPase, which appears to be important for influenza virion formation and budding ぱな (23). ぱに ぱぬ In addition, NEP has been discovered as a novel polymerase-associated co-factor that ぱね enhances the synthesis of cRNA and vRNA of human H1N1 strains, while mRNA ぱの transcription is suppressed (24, 25). Intriguingly, avian H5N1 polymerases seem to be ぱは especially susceptible to the polymerase activity-enhancing properties of NEP that harbor an ぱば adaptive mutation (13). This is based on the observation that single adaptive mutations (e.g. ぱぱ M16I, Y41C or E75G) in NEP of human-derived H5N1 viruses are sufficient to greatly ぱひ enhance the avian H5N1 polymerase activity in human cells (13). Remarkably, besides ひど stimulating cRNA and vRNA synthesis, those human adapted NEP also strongly enhance ひな mRNA transcription of avian H5N1 viruses. ひに ひぬ However, the molecular mechanism how a single adaptive mutation in NEP increases ひね polymerase activity of avian H5N1 polymerases in human cells is unclear (8, 13). We provide ひの experimental evidence that the N-terminal domain of NEP regulates the polymerase-activity ひは enhancing activity that is localized in the C-terminus of this protein. Furthermore, ひば intramolecular folding studies suggest that the adaptive mutation M16I in NEP diminishes the ひぱ interaction between the Nand C-terminus, thereby allowing the exposure of the C-terminus, ひひ which results in increased polymerase activity. などど などな Material and Methods などに Plasmid construction. などぬ The pCAGGS expression plasmids encoding the three polymerase subunits, NP and NEP などね (designated avNEP) of the avian H5N1 precursor virus (AvianPr) and the human-derived などの A/Thailand/1(KAN-1)/04 NEP, harboring the adaptive mutation M16I (designated huNEP) are などは described in Mänz et al. (13). To create pCAGGS-NEP-Trp, the mutations Y41C and E75G などば were introduced via quickchange PCR using pCAGGS-huNEP as a template. The pCAGGS などぱ on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 5 expression plasmids encoding GFPand RevM10-fused to huNEP were obtained by PCR などひ cloning of the GFP and RevM10 (18, 26) coding region into pCAGGS Strep-NEP (13). ななど pCAGGS expression vectors coding for the various GFP and RevM10 fusion proteins were ななな obtained by PCR amplification of the desired sequences and subsequent cloning of the ななに amplification products into pCAAGS-GFP-huNEP and pCAGGS-RevM10-huNEP, ななぬ respectively, and digested with NotI/XhoI. For cloning of pCAGGS-GFP-P, the DNA fragment ななね obtained after digestion of pCAGGS Flag-P (27) was cloned into pCAGGS-GFP-huNEP ななの using the same restriction sites. pCAGGS plasmids encoding NLuc-huNEP and huNEP-CLuc ななは were created by a two step assembly PCR using plasmids pCBG-C-FKBP, pFRB-CBR-N ななば (28) and pCAGGS-huNEP. pCAGGS-NLuc-huNEP-CLuc was generated by digesting ななぱ pCAGGS-huNEP-CLuc with EcoRV/XhoI and subsequent cloning into pCAGGS-NLuc-huNEP. ななひ In an analogous manner pCAGGS-NLuc-avNEP-CLuc, pCAGGS-NLuc-NEP-TRP-CLuc and なにど pCAGGS-NLuc-huNEPH1N1-CLuc were obtained. NLuc-CLuc was generated by XmaI-digestion of なにな pCAGGS-NLuc-huNEP-CLuc and religation. なにに なにぬ Reconstitution of the influenza virus polymerase activity. なにね HEK293T cells were transiently transfected with a transfection mixture containing plasmids なにの encoding PB1-, PB2-, PAand NP of AvianPr, a polymerase I (Pol I)-driven plasmid なには transcribing an influenza A virus-like RNA coding for the reporter protein firefly luciferase to なにば monitor viral polymerase activity as well as with expression plasmids coding for the indicated なにぱ NEP derivate. The minigenome RNA was flanked by non-coding sequences of segment 8 of なにひ FluA (13). The transfection mixture also contained a plasmid constitutively expressing Renilla なぬど luciferase, which served to normalize variation in transfection efficiency. The reporter activity なぬな was determined 24 h post transfection and normalized using the Dual-Glo Luciferase Assay なぬに System (Promega). なぬぬ なぬね Fragment complementation assays. なぬの on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 6 HEK293T cells were transiently transfected with plasmids (1000ng) either encoding NEP なぬは variants flanked by the respective fragments of the click beetle luciferase or Nand Cなぬば terminal click beetle luciferase fused by a linker sequence (intramolecular binding assay). なぬぱ Cells were lysed 24 hours post transfection and lysates were warmed to 37°C for 30 min. なぬひ Subsequently, luciferase activity was measured after stepwise cooling down the lysates (10 なねど min) to the indicated temperature using the Dual-Glo Luciferase Assay system (Promega). なねな To determine intermolecular binding between NEP, HEK293T cells were transiently なねに transfected with plasmids coding for NLuc-huNEP and huNEP-CLuc (each 200ng). Cell なねぬ extract was prepared and luciferase activity was measured using the Dual-Glo Luciferase なねね Assay system (Promega). なねの なねは Rev-dependent export assay. なねば HEK293T cells (4x10 cells) were transiently transfected with 700 ng plasmids pdm128 (29) なねぱ and the indicated plasmids (each 100 ng) coding for the RevM10 fusion proteins using なねひ Lipofectamine (Invitrogen) as recommend by the manufacture and 24 hours post なのど transfection, cell pellet were obtained in PBS by centrifugation (1000 rpm) for 10 min at 4°C. なのな Cat protein levels were determined by ELISA (CAT-ELISA, Roche). なのに なのぬ Primer extension analysis. なのね For determination of transcript levels in virus-infected or transiently transfected HEK293T なのの cells, cells were seeded in 6-well plates. Infection was carried out with infection media なのは (Dulbecco’s modified Eagle’s medium supplemented with 0.2 % BSA, 2 mM L-glutamine and なのば 1 % penicillin/streptomycin). At the indicated time point post-infection or post transfection なのぱ (24h), cells were collected in Trizol and RNA was purified according to the manufacturer’s なのひ protocol (Invitrogen). Primer extension analysis was performed using specific primers for the なはど NA segment (mRNA, cRNA and vRNA) and cellular 5sRNA as described (13). なはな なはに Single-cycle replication assay. なはぬ on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 7 HEK293T cells were cultured in 6-well plates and infected at an MOI of 2 in infection media なはね (Dulbecco's Modified Eagle Medium containing 0.3% bovine serum albumine). After one hour なはの incubation at 34°C, unbound viral particles were inactivated by treating the cells with PBS なはは (pH 2) for 45 seconds, subsequently washed and further incubated in 2 ml infection media. なはば Viral titers at the indicated time points were determined by plaque assay. なはぱ なはひ Results and Discussion なばど The C-terminal moiety of NEP possesses polymerase activity-enhancing property なばな To define the polymerase activity-enhancing domain of the human adapted NEP (huNEP) of なばに the H5N1 strain A/Thailand/1(KAN-1)/04, we created GFP-NEP fusion constructs with Nand なばぬ Cterminal deletions in NEP (Fig. 1A). We then analyzed their ability to stimulate polymerase なばね activity of the putative avian precursor virus of KAN-1, designated AvianPr (13), in human なばの cells. Consistent with our previous findings (13), co-transfection of 25ng of expression なばは plasmid coding for GFP fused human adapted NEP (huNEP) harboring the adaptive mutation なばば M16I (GFP-huNEP), increased the polymerase activity by more than 10 fold, whereas なばぱ transfection of 250ng of the same expression plasmid led to a nearly complete inhibition of なばひ polymerase activity (Fig. 1B-D). In contrast, expression of GFP fused to the phosphoprotein なぱど of Borna Disease Virus (GFP-P) had no inhibitory effect on the avian H5N1 polymerase at なぱな high plasmid concentrations (Fig. 1B-D). なぱに Expression of GFP fused to the N-terminal domain of huNEP consisting of amino acids 1-49 なぱぬ (GFP-huNEP-N) did not increase polymerase activity and, compared to GFP-P, we observed なぱね at the most a 2 fold decrease in viral polymerase activity upon expression at higher なぱの concentrations (Fig. 1B). In contrast, co-expression of GFP-NEP-C (GFP fused to amino なぱは acids 50 to 121 of NEP) resulted in 400-fold higher polymerase activity (Fig. 1C). Of note, なぱば GFP-NEP-C stimulated the polymerase activity at expression levels at which GFP-huNEP なぱぱ abrogated polymerase activity (Fig. 1C, relative activity with 500 ng GFP-NEP-C compared なぱひ to 250 ng GFP-huNEP). なひど on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 8 The last three amino acids of NEP are highly conserved amongst influenza A viruses and なひな rescue of influenza A viruses coding for NEP lacking the last three amino acids failed なひに (Schwemmle et al., unpublished data). Consistently, expression of GFP fused to huNEP なひぬ lacking the last three C-terminal amino acids (GFP-huNEP1-118) (25ng plasmid) failed to なひね stimulate the polymerase activity. Moreover, increasing amounts of GFP-huNEP1-118 なひの expressing plasmid only slightly decreased the polymerase activity (3 fold compared to GFPなひは P, Fig. 1D). As predicted, GFP-NEP-C lacking the C-terminal 3 amino acids was found to be なひば inactive and failed to stimulate the avian H5N1 polymerase activity in human cells (data not なひぱ shown). なひひ Together, these results indicate that the polymerase enhancing function of H5N1 NEP is にどど located in the C-terminal domain of NEP and that the integrity of the C-terminus is crucial for にどな both the polymerase activity-enhancing function of NEP as well as the inhibitory effect at にどに higher protein concentrations. This is consistent with observations by others that the NEP of にどぬ laboratory strains A/WSN/33 (24) or A/Puerto Rico/8/34 (30) with N-terminal deletions can にどね still activate viral replication to a certain extent. Although the precise role of the C-terminal にどの three amino acids remains to be shown, it is tempting to speculate that these amino acids にどは may stabilize the interaction with other viral proteins such as the polymerase subunits. にどば To confirm our finding that only the C-terminal domain of NEP is sufficient to stimulate にどぱ polymerase activity, we reconstituted the avian H5N1 polymerase using an authentic viral にどひ segment (segment 6) and performed primer extension analysis to visualize the levels of viral になど transcripts as described in (13). Consistent with previous findings (13), co-transfection of になな 75ng GFP-huNEP-encoding plasmids resulted in a substantial increase in vRNA and cRNA になに levels, while expression of mRNA was diminished (Fig. 1E and F). As expected, increasing になぬ amount of plasmid (750ng) resulted in abrogation of viral RNA synthesis. Expression of になね either GFP-huNEP-N or GFP-P did not alter viral RNA levels (Fig. 1E and F). In contrast, になの expression of GFP-NEP-C resulted in the increase of all 3 RNA species (Fig. 1F). At highest になは plasmid concentrations, however, mRNA transcript levels decreased (Fig. 1F). Together, になば on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 9 these results confirm that NEP-C is constitutively active and sufficient to stimulate the activity になぱ of the avian H5N1 polymerase. になひ To determine the effect of the Nand C-terminal moieties of NEP on the polymerase ににど enhancing function of full length NEP we co-expressed increasing amounts of either GFPににな huNEP-N or GFP-NEP-C in the presence of GFP-huNEP. Interestingly, GFP-NEP-C did not ににに further enhance polymerase activity in the presence of GFP-huNEP (Fig. 1G). As expected, ににぬ co-expression of GFP-huNEP-N did not alter polymerase-enhancing activity of GFP-huNEP. ににね ににの Intramolecular folding of NEP is influenced by the adaptive mutation M16I にには As shown in Fig. 1C, the C-terminal domain of NEP is constitutively active in the absence of ににば the N-terminal domain. We therefore speculated that binding between the Nand C-terminal ににぱ domain might regulate the polymerase activity-enhancing activity of the C-terminal moiety. ににひ To determine intramolecular interactions between the Nand C-terminal moieties, we fused にぬど both ends of NEP to split click beetle luciferase halves (Fig. 2A) to perform fragment にぬな complementation assays. We reasoned that back-folding of the Nto the C-terminus would にぬに bring the split click beetle luciferase moieties into close proximity and allow reversible にぬぬ reconstitution of active luciferase, especially at lower temperatures were weak affinities are にぬね preferentially stabilized (Fig. 2B). In contrast, a conformation that prevents the close にぬの proximity of split click beetle luciferase moieties would result in no reporter activity. To にぬは determine transient interactions between the Nand C-terminal moieties, HEK293T cells にぬば were transfected with plasmids encoding avian H5N1 NEP (avNEP) or huNEP fused to both にぬぱ Nand C-terminal click beetle luciferase moieties (NLuc-avNEP-CLuc, NLuc-huNEP-CLuc) and にぬひ cell extracts were prepared 24 hours post transfection at 4°C. To monitor temperature にねど dependent differences, these extracts were warmed up to 37°C for 30 min and subsequently にねな cooled down to 34, 31, 28, 25, 22 and 19°C (20 min each temperature). At each temperature にねに a fraction of the cell extract was used to determine the luciferase activity. The luciferase にねぬ activity of each fusion protein observed at 37°C was set to 100%. As shown in Fig. 2B, with にねね decreasing temperatures, luciferase activity for both avian (NLuc-avNEP-CLuc) and humanにねの on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 10 adapted (NLuc-huNEP-CLuc) NEP increased. Interestingly, the temperature dependent にねは increase in luciferase activity was significantly delayed for NLuc-huNEP-CLuc, suggesting that にねば the M16I mutation reduces the affinity between the Nand C-terminal moieties. As expected, にねぱ split click beetle luciferase moieties fused to each other via a flexible linker (NLuc-CLuc) にねひ showed a slight decrease in activity (Fig. 2B). This is consistent with the observations by にのど others (28) that the click beetle luciferase activity itself is not affected by changes in にのな temperature. にのに To confirm that the signals we measured in the split luciferase assay did result from an にのぬ intramolecular and not intermolecular interaction between two NEPs, we transiently にのね expressed both huNEP fused to the N-terminal domain of click beetle luciferase (NLucにのの huNEP) and huNEP fused to the C-terminal domain of click beetle luciferase (huNEP-CLuc) にのは (Fig. 2A) and compared the luciferase activity obtained with cell extract containing NLucにのば huNEP-CLuc only. While the expression levels of the fusion proteins were comparable, the にのぱ luciferase activity observed after expression of both NLuc-huNEP and huNEP-CLuc was にのひ significantly lower (>50 fold) than with NLuc-huNEP-CLuc only (Fig. 2C), indicating that the にはど activity observed with the latter protein reflects indeed an intramolecular interaction. にはな Next, we subjected NEP derived from the pandemic H1N1 isolate A/Hamburg/4/2009 (NLucにはに huNEPH1N1-CLuc) to the split click beetle luciferase assay and compared it to the activity にはぬ observed with the NLuc-huNEP-CLuc. As shown in Fig. 2D, the temperature dependent にはね changes in luminescence measured for NLuc-huNEP-CLuc and NLuc-huNEPH1N1-CLuc are similar, にはの indicating that the intramolecular affinity of human adapted H5N1 is comparable to that of a にはは bona fide human NEP. にはば NEP is known to bind to cellular nuclear export proteins including Crm-1 (20) by virtue of its にはぱ two nuclear export sequences (NES) that are localized in the N-terminal α-helical domains にはひ N1 and N2 (18, 22). We therefore speculated that M16I-mediated change in the kinetic of にばど huNEP to obtain its tertiary conformation might also favor the interaction of the exposed Nにばな terminus of NEP with Crm-1, resulting in export competent protein complexes. To compare にばに the export activity of avian and human-adapted NEP, we fused HIV-RevM10 (18, 26), which にばぬ on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 11 is deficient in binding to Crm-1 (31), to either avNEP (avNEP-M10), huNEP (huNEP-M10) or にばね NEP-C (NEP-C-M10) (Fig. 2E) and co-expressed these proteins in the presence of a CATにばの encoding mRNA harboring the rev-responsive element (RRE) (29). As expected, the CAT にばは protein levels were low in cells expressing NEP-C-M10 compared to huNEP-M10 expressing にばば cells (Fig. 2F), since there is no NES located in the C-terminal part of NEP (Fig. 2E). にばぱ However, compared to huNEP-M10, the CAT protein levels in cells expressing avNEP-M10 にばひ were significantly lower, despite similar expression of the fusion proteins (Fig. 2F). To rule にぱど out that the adaptive mutation M16I located within the NES directly increases the affinity to にぱな Crm-1, we fused RevM10 to the N-termini of either avNEP (avNEP-N-M10) or huNEP にぱに (huNEP-N-M10) (Fig. 2E) and determined their nuclear export activity. Expression of either にぱぬ fusion protein resulted in comparable CAT protein levels (Fig. 2G). This suggests that the にぱね adaptive mutation M16I does not increase the affinity to Crm-1. Together, these results にぱの support the model that the adaptive mutation M16I causes a conformational change of NEP にぱは that allows enhanced interaction with Crm-1 and likely the exposure to the C-terminal moiety. にぱば Therefore the mutation M16I might not only increase the viral polymerase activity but also にぱぱ enhance nuclear export of vRNPs, a feature that might be important in the adaptation にぱひ process of avian H5N1 viruses to human cells. にひど にひな NEP with three adaptive mutations shows increased activity. にひに In addition to M16I, further single adaptive mutations in NEP of human-derived H5N1 にひぬ isolates, including Y41C and E75G (Fig. 3A), were found to stimulate avian H5N1 にひね polymerases in human cells (13). To test whether the combination of adaptive mutations にひの further enhances the stimulatory activity of avian NEP, we expressed the triple mutant GFPにひは NEP-TRP, harboring the adaptive mutations M16I, Y41C and E75G (Fig. 3A) in the にひば polymerase reconstitution assay. Compared to GFP-huNEP, expression of GFP-NEP-TRP にひぱ further increased the polymerase activity and exhibited decreased inhibitory activity at higher にひひ concentrations (Fig. 3B), suggesting that these mutations might indeed enhance the activity ぬどど in an additive manner. However, the polymerase activity observed in the presence of the Cぬどな on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 12 terminus of NEP with (GFP-NEP-C-E75G) and without (GFP-NEP-C) the adaptive mutation ぬどに E75G was comparable (Fig. 3B). This lack of increased polymerase activity with GFP-NEPぬどぬ C-E75G might suggest that the adaptive mutation E75G (and most likely Y41C) functions in ぬどね a similar manner as M16I, namely by decreasing intramolecular interactions between the Nぬどの and C-terminal domains of NEP. We therefore fused both ends of NEP-TRP to split click ぬどは beetle luciferase halves (NLuc-NEP-TRP-CLuc) and determined the relative luminescence at ぬどば decreasing temperatures. This revealed that NLuc-NEP-TRP-CLuc showed no increase in ぬどぱ luciferase activity at each temperature tested compared to NLuc-huNEP-CLuc (Fig. 3C). This ぬどひ indicates that the presence of these three adaptive mutations prevents detectable ぬなど intramolecular folding events that would result in the close proximity of the split click beetle ぬなな luciferase moieties and reconstitution of the luciferase. ぬなに ぬなぬ Adaptive mutations in NEP provide a replication advantage also at lower temperature ぬなね The relatively low temperature of the human upper respiratory tract of ca. 32°C in the upper ぬなの trachea to ca. 35.5°C in the subsegmental bronchi (32) represent a major hurdle for the ぬなは establishment of an infection by avian H5N1 viruses, especially since the replication ぬなば machinery of avian influenza A viruses are adapted to the temperature of the avian intestinal ぬなぱ tract of ca. 39-41°C (33, 34). In contrast, circulating human influenza A viruses predominantly ぬなひ infect the human upper respiratory airways with a characteristic mean temperature of about ぬにど 34°C (32). Since the fragment complementation assay revealed that the intramolecular ぬにな binding between Nand C-terminal domains of avian NEP preferentially occur at low ぬにに temperature (Fig. 2B) thereby possibly preventing the exposure of the polymerase activityぬにぬ enhancing C-terminal domain, we reasoned that the adaptive mutation M16I increases the ぬにね poor replication of avian polymerases not only at 37°C, but also at low temperature of 34°C. ぬにの To demonstrate this, we first reconstituted the avian H5N1 polymerase in human 293T cells ぬには at 34°C without NEP. As expected, at the lower temperature the avian H5N1 polymerase ぬにば showed only residual activity, compared to reconstitution at 37°C (Fig. 3D). Remarkably, at ぬにぱ 34°C, in the presence of 10ng of either huNEP or NEP-TRP the polymerase activities ぬにひ on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 13 increased ca. 74and 164-fold, respectively, whereas avNEP had only a minor 2-fold ぬぬど stimulatory effect. At 37°C, huNEP and NEP-TRP stimulated the polymerase activity ca. 30ぬぬな fold, while avian NEP increased the polymerase activity 4-fold. ぬぬに These results suggest that NEP of avianH5N1 viruses harboring a human-specific adaptive ぬぬぬ mutation might increase the replication efficiency especially at lower temperature. To show ぬぬね this in the context of viral infection, HEK293T cells kept at 34°C or 37°C were infected with ぬぬの either avian H5N1 with an NS segment coding for NEP lacking (AvianPr) (13) or harboring ぬぬは the adaptive mutation M16I (AvianPr-NS-KAN-1) (13) with an MOI of 5. At 1, 2, 2.5, and 3 ぬぬば hours post infection, transcript levels were determined by primer extension analysis. As ぬぬぱ shown in Fig. 3E, infection with AvianPr-NS-KAN-1 resulted in increased transcript levels of ぬぬひ mainly cand mRNA compared to cells infected with AvianPr at 34°C. As shown previously ぬねど (13), similar differences in transcript levels were observed after infection at 37°C, although ぬねな the total viral transcript levels are significantly higher compared to infections at 34°C. ぬねに Consistently, determination of the single cycle growth characteristics revealed that 293T cells ぬねぬ infected with AvianPr-NS-KAN-1 released significantly higher numbers of infectious particles ぬねね at 34°C compared to 293T cells infected with AvianPr (Fig. 3F). In summary these results ぬねの support the concept that a single adaptive mutation in NEP can significantly contribute to ぬねは overcome the temperature restriction avian viruses are faced with in the human upper ぬねば respiratory tract. ぬねぱ ぬねひ Based on our data, we propose that the N-terminus of NEP is a regulatory domain whose ぬのど conformation relative to the C-terminal domain determines the protein’s activity. The Cぬのな terminal domain comprising the two α 異helices C1 and C2 (Fig. 4) harbors the polymerases ぬのに activity-enhancing property of NEP. Given the rigid nature of the C-terminus (20), the Nぬのぬ terminus may act as a “lid" which opens to expose the surfaces of NEP required for co-factor ぬのね activity (Fig.4). Backfolding of the N-terminus to the C-terminus to a closed conformation ぬのの might be also required for the inhibitory effect of NEP on the viral polymerase activity ぬのは observed at higher protein concentrations. In this respect, the absence of an inhibitory effect ぬのば on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 14 of the NEP mutant NEP-TRP could be due to the reduced affinity between the Nand Cぬのぱ terminal moieties of this protein. ぬのひ The fact that avian influenza viruses replicate preferentially at high temperatures of 39-41°C ぬはど might explain why avian NEP favors a “closed” conformation. A high temperature at the site ぬはな of replication lowers the affinity between Nand C-terminal domains, thereby increasing the ぬはに polymerase-enhancing activity of avian NEP. However, when avian influenza viruses cross ぬはぬ the species barrier to humans the temperature at the site of replication is lowered to ca. ぬはね 34°C, leading to a stronger affinity between the Nand C-termini. As a consequence, NEP ぬはの exists in a non-stimulatory form that precludes the stimulation of the already low polymerase ぬはは activity of avian influenza viruses in human cells. We therefore speculate that NEP of human ぬはば influenza viruses adapted to lower temperatures by reducing the affinities between the Nぬはぱ and C-termini thereby maintaining or even increasing the level of polymerase-stimulatory ぬはひ activity after species transmission. This view is supported by the observation that NEP of the ぬばど pandemic 2009 H1N1 virus (13) as well as the laboratory strain A/WSN/33 (data not shown) ぬばな strongly enhance the avian influenza polymerase activity in human cells in contrast to avian ぬばに NEP lacking adaptive mutations. ぬばぬ Based on its importance to stimulate viral replication, it is tempting to speculate that the ぬばね conformational change of NEP is tightly regulated. Recent determination of the viral ぬばの phosphoproteome revealed that NEP is phosphorylated (35) at highly conserved serine ぬばは residues in the N-terminal a-helix N1 (36). This change in surface charge may also alter the ぬばば intramolecular affinity and thus polymerase-activity enhancing function of NEP. Alternatively, ぬばぱ since the RevM10-dependent export assay suggests that the “open” conformation of NEP ぬばひ might also increase nuclear export of vRNPs, phosphorylation of NEP might be required for ぬぱど this activity only. ぬぱな ぬぱに Acknowledgement ぬぱぬ We thank Georg Kochs and Peter Staeheli for critically reading the manuscript and Kristina ぬぱね Göpfert for technical assistance. This study was funded by the Bundesministerium für ぬぱの on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 15 Bildung und Forschung (FluResearchNet) and the Deutsche Forschungsgesellschaft (SCHW ぬぱは 632/11-2). P.R. is recipient of a Studienstiftung des deutschen Volkes fellowship. ぬぱば ぬぱぱ References: ぬぱひ 1. Horimoto T, Kawaoka Y. 2005. Influenza: lessons from past pandemics, warnings ぬひど from current incidents. Nat Rev Microbiol 3:591-600. ぬひな 2. Taubenberger JK, Kash JC. 2011. Insights on influenza pathogenesis from the ぬひに grave. Virus research 162:2-7. ぬひぬ 3. Gao HN, Lu HZ, Cao B, Du B, Shang H, Gan JH, Lu SH, Yang YD, Fang Q, Shen ぬひね YZ, Xi XM, Gu Q, Zhou XM, Qu HP, Yan Z, Li FM, Zhao W, Gao ZC, Wang GF, ぬひの Ruan LX, Wang WH, Ye J, Cao HF, Li XW, Zhang WH, Fang XC, He J, Liang WF, ぬひは Xie J, Zeng M, Wu XZ, Li J, Xia Q, Jin ZC, Chen Q, Tang C, Zhang ZY, Hou BM, ぬひば Feng ZX, Sheng JF, Zhong NS, Li LJ. 2013. Clinical Findings in 111 Cases of ぬひぱ Influenza A (H7N9) Virus Infection. The New England journal of medicine. ぬひひ 4. Taubenberger JK, Kash JC. 2010. Influenza virus evolution, host adaptation, and ねどど pandemic formation. Cell host & microbe 7:440-451. ねどな 5. Imai M, Kawaoka Y. 2012. The role of receptor binding specificity in interspecies ねどに transmission of influenza viruses. Current opinion in virology 2:160-167. ねどぬ 6. Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, ねどね Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus ねどの AD, Fouchier RA. 2012. Airborne transmission of influenza A/H5N1 virus between ねどは ferrets. Science 336:1534-1541. ねどば 7. Naffakh N, Tomoiu A, Rameix-Welti MA, van der Werf S. 2008. Host restriction of ねどぱ avian influenza viruses at the level of the ribonucleoproteins. Annual review of ねどひ microbiology 62:403-424. ねなど 8. Mänz B, Schwemmle M, Brunotte L. 2013. Adaptation of avian influenza A virus ねなな polymerase in mammals to overcome the host species barrier. Journal of virology. ねなに on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 16 9. Mehle A, Doudna JA. 2008. An inhibitory activity in human cells restricts the function ねなぬ of an avian-like influenza virus polymerase. Cell host & microbe 4:111-122. ねなね 10. Moncorge O, Mura M, Barclay WS. 2010. Evidence for avian and human host cell ねなの factors that affect the activity of influenza virus polymerase. Journal of virology ねなは 84:9978-9986. ねなば 11. Gabriel G, Herwig A, Klenk HD. 2008. Interaction of polymerase subunit PB2 and ねなぱ NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS ねなひ pathogens 4:e11. ねにど 12. Bortz E, Westera L, Maamary J, Steel J, Albrecht RA, Manicassamy B, Chase G, ねにな Martinez-Sobrido L, Schwemmle M, Garcia-Sastre A. 2011. Hostand strainねにに specific regulation of influenza virus polymerase activity by interacting cellular ねにぬ proteins. mBio 2. ねにね 13. Mänz B, Brunotte L, Reuther P, Schwemmle M. 2012. Adaptive mutations in NEP ねにの compensate for defective H5N1 RNA replication in cultured human cells. Nature ねには communications 3:802. ねにば 14. Kim JH, Hatta M, Watanabe S, Neumann G, Watanabe T, Kawaoka Y. 2010. Role ねにぱ of host-specific amino acids in the pathogenicity of avian H5N1 influenza viruses in ねにひ mice. The Journal of general virology 91:1284-1289. ねぬど 15. Subbarao EK, London W, Murphy BR. 1993. A single amino acid in the PB2 gene ねぬな of influenza A virus is a determinant of host range. Journal of virology 67:1761-1764. ねぬに 16. Salomon R, Franks J, Govorkova EA, Ilyushina NA, Yen HL, Hulse-Post DJ, ねぬぬ Humberd J, Trichet M, Rehg JE, Webby RJ, Webster RG, Hoffmann E. 2006. The ねぬね polymerase complex genes contribute to the high virulence of the human H5N1 ねぬの influenza virus isolate A/Vietnam/1203/04. J Exp Med 203:689-697. ねぬは 17. Hatta M, Gao P, Halfmann P, Kawaoka Y. 2001. Molecular basis for high virulence ねぬば of Hong Kong H5N1 influenza A viruses. Science 293:1840-1842. ねぬぱ on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 17 18. O'Neill RE, Talon J, Palese P. 1998. The influenza virus NEP (NS2 protein) ねぬひ mediates the nuclear export of viral ribonucleoproteins. The EMBO journal 17:288ねねど 296. ねねな 19. Paterson D, Fodor E. 2012. Emerging Roles for the Influenza A Virus Nuclear Export ねねに Protein (NEP). PLoS pathogens 8:e1003019. ねねぬ 20. Akarsu H, Burmeister WP, Petosa C, Petit I, Muller CW, Ruigrok RW, Baudin F. ねねね 2003. Crystal structure of the M1 protein-binding domain of the influenza A virus ねねの nuclear export protein (NEP/NS2). The EMBO journal 22:4646-4655. ねねは 21. Lommer BS, Luo M. 2002. Structural plasticity in influenza virus protein NS2 (NEP). ねねば The Journal of biological chemistry 277:7108-7117. ねねぱ 22. Huang S, Chen J, Chen Q, Wang H, Yao Y, Chen J, Chen Z. 2013. A second ねねひ CRM1-dependent nuclear export signal in the influenza A virus NS2 protein (NEP) ねのど contributes to the nuclear export of viral ribonucleoproteins. Journal of virology ねのな 87:767-778. ねのに 23. Gorai T, Goto H, Noda T, Watanabe T, Kozuka-Hata H, Oyama M, Takano R, ねのぬ Neumann G, Watanabe S, Kawaoka Y. 2012. F1Fo-ATPase, F-type protonねのね translocating ATPase, at the plasma membrane is critical for efficient influenza virus ねのの budding. Proceedings of the National Academy of Sciences of the United States of ねのは America 109:4615-4620. ねのば 24. Robb NC, Smith M, Vreede FT, Fodor E. 2009. NS2/NEP protein regulates ねのぱ transcription and replication of the influenza virus RNA genome. The Journal of ねのひ general virology 90:1398-1407. ねはど 25. Bullido R, Gomez-Puertas P, Saiz MJ, Portela A. 2001. Influenza A virus NEP ねはな (NS2 protein) downregulates RNA synthesis of model template RNAs. Journal of ねはに virology 75:4912-4917. ねはぬ 26. Malim MH, McCarn DF, Tiley LS, Cullen BR. 1991. Mutational definition of the ねはね human immunodeficiency virus type 1 Rev activation domain. Journal of virology ねはの 65:4248-4254. ねはは on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 18 27. Schmid S, Mayer D, Schneider U, Schwemmle M. 2007. Functional ねはば characterization of the major and minor phosphorylation sites of the P protein of ねはぱ Borna disease virus. Journal of virology 81:5497-5507. ねはひ 28. Villalobos V, Naik S, Bruinsma M, Dothager RS, Pan MH, Samrakandi M, Moss ねばど B, Elhammali A, Piwnica-Worms D. 2010. Dual-color click beetle luciferase ねばな heteroprotein fragment complementation assays. Chemistry & biology 17:1018-1029. ねばに 29. Hope TJ, Huang XJ, McDonald D, Parslow TG. 1990. Steroid-receptor fusion of the ねばぬ human immunodeficiency virus type 1 Rev transactivator: mapping cryptic functions ねばね of the arginine-rich motif. Proceedings of the National Academy of Sciences of the ねばの United States of America 87:7787-7791. ねばは 30. Perez JT, Zlatev I, Aggarwal S, Subramanian S, Sachidanandam R, Kim B, ねばば Manoharan M, Tenoever BR. 2012. A Small-RNA Enhancer of Viral Polymerase ねばぱ Activity. Journal of virology 86:13475-13485. ねばひ 31. Fornerod M, Ohno M, Yoshida M, Mattaj IW. 1997. CRM1 is an export receptor for ねぱど leucine-rich nuclear export signals. Cell 90:1051-1060. ねぱな 32. McFadden ER, Jr., Pichurko BM, Bowman HF, Ingenito E, Burns S, Dowling N, ねぱに Solway J. 1985. Thermal mapping of the airways in humans. J Appl Physiol 58:564ねぱぬ 570. ねぱね 33. Hatta M, Hatta Y, Kim JH, Watanabe S, Shinya K, Nguyen T, Lien PS, Le QM, ねぱの Kawaoka Y. 2007. Growth of H5N1 influenza A viruses in the upper respiratory tracts ねぱは of mice. PLoS pathogens 3:1374-1379. ねぱば 34. Massin P, van der Werf S, Naffakh N. 2001. Residue 627 of PB2 is a determinant of ねぱぱ cold sensitivity in RNA replication of avian influenza viruses. Journal of virology ねぱひ 75:5398-5404. ねひど 35. Richardson JC, Akkina RK. 1991. NS2 protein of influenza virus is found in purified ねひな virus and phosphorylated in infected cells. Archives of virology 116:69-80. ねひに 36. Hutchinson EC, Fodor E. 2012. Nuclear import of the influenza A virus ねひぬ transcriptional machinery. Vaccine 30:7353-7358. ねひね on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 19 FIG. 1. The polymerase-activity enhancing function of NEP resides in its C-terminus ねひの (A) Cartoon depicting Nand C-terminal truncation mutants of NEP fused to GFP. Black bar ねひは represents the location of the adaptive mutation M16I. ねひば (B-D) To determine the stimulatory activity of the indicated NEP variants on the avian H5N1 ねひぱ polymerase, HEK293T cells were transiently transfected with expression plasmids coding for ねひひ the corresponding PB2, PB1, PA and NP proteins of avian H5N1 (AvianPr), a human のどど polymerase I-driven vRNA-luciferase reporter plasmid, a renilla-expressing plasmid and the のどな indicated concentrations of NEP expression plasmids GFP-huNEP (B), GFP-NEP-C (C) and のどに GFP-huNEP1-118 (D). Omission of PB1 (-PB1) was used as a negative control. Luciferase のどぬ reporter activity was normalized to Renilla activity to address variations in transfection のどね efficiency. Normalized reporter activities obtained after co-transfection of an expression のどの plasmid (25 ng) coding for GFP fused to the Borna Disease Virus phosphoprotein P (GFP-P) のどは were set to 100%. Protein levels of the GFP fusion proteins were determined by Western blot のどば using GFP-specific antibodies. Detection of tubulin served as a loading control. Error bars のどぱ indicate the standard deviations of three independent experiments. Student’s t-test was のどひ performed to determine the P value. *P<0.05, **P<0.01, ***P<0.001. のなど (E and F) Polymerase reconstitution assay using segment 6 of AvianPr in the presence of のなな NEP mutant proteins. Determination of the mRNA, cRNA and vRNA levels by primer のなに extension analysis obtained after reconstitution of the AvianPr in the presence of the のなぬ indicated GFP fusion constructs using specific primers for segment 6. Determination of the のなね 5sRNA levels served as an internal loading control. Omission of PB1 (-PB1) was used as a のなの negative control. のなは (G) Effect of co-expression of the indicated amounts of GFP-huNEP-N and GFP-NEP-C on のなば the polymerase enhancing capacity of GFP-huNEP. Reporter levels obtained for the のなぱ reconstituted polymerase alone were set to 100%. Error bars indicate standard deviations of のなひ three independent experiments. のにど のにな FIG. 2. Detection of intramolecular changes in NEP. のにに on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 20 (A) Cartoon depicting NEP fused to split click beetle luciferase halves highlighted in black. のにぬ (B) To analyze temperature-dependent intramolecular folding events, HEK293T cells were のにね transfected with plasmids encoding either NLuc-huNEP-CLuc or NLuc-avNEP-Cluc and 24 のにの hours post transfection cell lysates were prepared at 4°C. The extracts were warmed up to のには 37°C for 30 min and subsequently cooled down to 34, 31, 28, 25, 22 and 19°C (20 min each のにば temperature). At each temperature, a fraction of the sample was used to determine luciferase のにぱ activity. The luciferase activity observed at 37°C was set to 100%. Cell extracts containing のにひ NLuc-CLuc (N-Luc and C-Luc fused by linker sequence) served as a control for a のぬど temperature stable luciferase (28). Error bars indicate standard deviations of three のぬな independent experiments. Student’s t-test was performed to determine the P value. *P<0.05, のぬに **P<0.01. The model above the graph depicts the proposed temperature dependent のぬぬ intramolecular interaction of NEP fused to the respective halves of a click beetle luciferase のぬね (filled halves). T, temperature. のぬの (C) Comparison of the interand intramolecular binding efficiencies. Luciferase activity was のぬは determined in extracts of HEK293T cells transiently transfected with plasmids expressing のぬば both NLuc-huNEP and huNEP-CLuc (intermolecular binding) or NLuc-huNEP-CLuc のぬぱ (intramolecular binding). Lower panels show the expression levels of the respective のぬひ constructs determined by Western blot analysis. Detection of actin served as a loading のねど control. Error bars indicate the standard deviations of three independent experiments. のねな (D) Analysis of the temperature-dependent affinity of the Cand N-terminal domains of NEP のねに derived from the pandemic H1N1 isolate A/Hamburg/4/2009 (NLuc-huNEPH1N1-CLuc) in のねぬ comparison to the human adapted H5N1 NEP (NLuc-huNEP-CLuc). Student’s t-test was のねね performed to determine the P value. *P<0.05 のねの (E) Cartoon depicting NEP variants fused to RevM10. The locations of the adaptive mutation のねは M16I and the two NES are indicated. のねば (F and G) Crm1-dependent nuclear export activity of NEP-RevM10 fusion proteins. huNEP のねぱ or avNEP (E) or the corresponding N-terminal fragments comprising amino acids 1 – 49 (F) のねひ fused to the export inactive Rev protein (RevM10) were transiently expressed in HEK293T ののど on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 21 cells together with a plasmid that allowed the synthesis of an intron-containing CAT reporter ののな mRNA harboring a Rev-responsive element. RevM10-NEP-C (amino acids 50-121), lacking ののに the NES domains served as negative controls. Protein levels of the NEP fusion proteins were ののぬ analyzed by Western blot analysis. Detection of tubulin served as a loading control. The ののね export activity was determined by measuring CAT protein levels and normalized to the levels ののの of the NEP fusion proteins. Error bars indicate standard deviations of three independent ののは experiments. ののば ののぱ FIG. 3. Additive effects of adaptive mutations in NEP ののひ (A) Cartoon depicting NEP with three adaptive mutations (highlighted in black) or C-terminal のはど domain of NEP harboring the adaptive mutation E75G fused to split click beetle luciferase のはな halves. のはに (B) Effect of NEP-GFP fusion proteins harboring the single adaptive mutation M16I (GFPのはぬ huNEP), three adaptive mutations M16I, Y41C and E75G (GFP-NEP-TRP), the C-terminal のはね domain of NEP without (GFP-NEP-C) or with the adaptive mutation E75G (GFP-NEP-Cのはの E75G) on the reconstituted avian H5N1 polymerase activity in human cells. Reporter levels のはは obtained after co-transfection of the expression plasmid coding for GFP-P was set to 100%. のはば Amounts of expression plasmid coding for GFP fusion proteins are indicated. Protein levels のはぱ of the GFP fusion proteins were determined by Western blot analysis using GFP-specific のはひ antibodies. Detection of tubulin served as a loading control. Error bars indicate standard のばど deviations of three independent experiments. Student’s t-test was performed to determine のばな the P value. *P<0.05, **P<0.01, ***P<0.001. のばに (C) Comparison of the temperature-dependent changes in luciferase activity of huNEP and のばぬ NEP-TRP fused to split click beetle luciferase halves. The experiments were carried out as のばね decribed in Fig. 2B. Error bars represent standard deviations of three independent のばの experiments. Student’s t-test was performed to determine the P value. *P<0.05. のばは (D) Comparison of the temperature-dependent stimulatory activity of huNEP, avNEP and のばば NEP-TRP. The avian H5N1 polymerase was reconstituted in HEK293T cells in the presence のばぱ on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Reuther et al. Polymerase-activity enhancing property of NEP 22 of the indicated fusion proteins at either 34°C or 37°C. Omission of PB1 was used as a のばひ negative control (background levels). Luciferase reporter activity was normalized to Renilla のぱど activity to address variations in transfection efficiency. The relative induction in reporter のぱな activity was calculated by dividing the normalized luciferase activity by the background のぱに levels. The relative induction in reporter activity at 37°C in the absence of NEP was set to のぱぬ 100%. Error bars indicate standard deviations of three independent experiments. Student’s tのぱね test was performed to determine the P value. *P<0.05. のぱの (E) HEK293T cells were infected with AvianPr or AvianPr-NS-KAN-1 at a MOI of 5. After the のぱは indicated time points post infection (p.i.), cells were lysed and RNA levels were determined のぱば by primer extension analysis using primers specific for segment 6. Determination of the のぱぱ 5sRNA levels served as an internal loading control. のぱひ (F) Comparison of the growth kinetics of AvianPr and AvianPr-NS-KAN-1 by single-cycle のひど replication assay. HEK293T cells were infected at an MOI of 2 and incubated at 34°C. Viral のひな titers at the indicated time points post infection were determined by plaque assay. Student’s のひに t-test was performed to determine the P value. *P<0.05. **P<0.01. のひぬ のひね Fig. 4: Model of the regulation of the polymerase activity-enhancing property of NEP. のひの NEP consists of two predicted N-terminal (N1 and N2) and two C-terminal (C1 and C2) gのひは helices. The N-terminus might regulate the polymerase-enhancing activity of the C-terminus のひば by its proposed property to fold back to the C-terminus. In an "open conformation" the Cのひぱ terminus would be exposed, resulting in the stimulation of the polymerase activity. In a のひひ “closed conformation” the N-terminus would shield the C-terminus, rendering NEP inactive. はどど The indicated human-adapted mutations in NEP, which are found in N1, N2 and C1 of はどな different H5N1 strains (13), would preferentially induce an “open conformation” due to a はどに lower affinity between the Nand C-termini. Avian NEP lacking adaptive mutations might はどぬ favor a “closed conformation”, thereby reducing the polymerase-enhancing activity. はどね はどの はどは on A uust 0, 2017 by gest http/jvi.asm .rg/ D ow nladed fom