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1 Edinburgh Research Explorer Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements Citation for published version: Elton, D, Medcalf, L, Bishop, K & Digard, P 1999, 'Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements' Virology, vol. 260, no. 1, pp DOI: /viro Digital Object Identifier (DOI): /viro Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Virology Publisher Rights Statement: Copyright 1999 by Academic Press General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 19. Mar. 2019

2 Virology 260, (1999) Article ID viro , available online at on Oligomerization of the Influenza Virus Nucleoprotein: Identification of Positive and Negative Sequence Elements Debra Elton, Elizabeth Medcalf, Konrad Bishop, and Paul Digard 1 Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom Received March 23, 1999; returned to author for final revision May 3, 1999; accepted May 20, 1999 The RNA genome of influenza virus is encapsidated by the virus nucleoprotein (NP) to form ribonucleoprotein (RNP) structures of defined morphology. These structures result from the ability of NP to oligomerise and to bind single-strand RNA. To characterise NP oligomerization, we developed a binding assay using immobilised NP fusion proteins and in vitro translated NP. This system was used to estimate a dissociation constant for NP NP contacts of M. Analysis of NP deletion mutants identified three sequence elements important for oligomerization. Two regions corresponding to the middle and C-terminal thirds of the polypeptide were identified as the minimal sequences capable of promoting NP NP contacts. However, the C-terminal 23 amino-acids of NP inhibited oligomerization, as their removal increased self-association 10-fold. Single codon changes identified amino acids important for the function of these regions. Alanine substitution of R199 decreased binding affinity threefold, whereas alteration of R416 had a more drastic effect, reducing binding 10-fold. In contrast, mutation of F479 increased self-association fivefold. Mutations altering NP oligomerization affected the ability of the polypeptides to support influenza virus gene expression in an in vivo assay. Decreased oligomerization activity correlated with decreased transcriptional function. However, mutations that increased self-association also decreased transcription competence. This indicates that NP contains both positive and negative sequence elements involved in oligomerization and is consistent with the importance of NP NP contacts for the formation of a transcriptionally active RNP Academic Press INTRODUCTION Influenza A viruses are the cause of yearly epidemics and occasional pandemics and despite the availability of efficacious vaccines and antiviral drugs, remain a major concern for public health. The virus possesses a segmented single-strand RNA (ssrna) genome of negative polarity, which is always found in association with viral proteins, in the form of ribonucleoprotein (RNP) complexes (Lamb and Krug, 1996). On infection of cells, the RNP complexes migrate to the nucleus, where expression and replication of the genome segments takes place. Initial transcription produces capped and polyadenylated viral mrna via an unusual process where capped RNA fragments are cleaved from host cell mrnas to be used as primers for virus transcription (Plotch et al., 1981) and where polyadenylation subsequently occurs because the polymerase stutters on a polyuridine stretch near the 5 end of the genomic RNA (vrna) templates (Li and Palese, 1994; Pritlove et al., 1998). As these mechanisms render viral mrna inexact complements of their templates, they cannot serve in turn as substrates for synthesis of progeny vrna. Instead the infecting RNPs are secondarily transcribed via a mechanism thought to use unprimed initiation and 1 To whom reprint requests should be addressed. Fax pd1@mole.bio.cam.ac.uk. necessarily involving suppression of the polyadenylation signal to produce exact complements of the genome segments (Lamb and Krug, 1996). These crnas are also encapsidated to form RNP structures and provide the template for synthesis of new vrna segments, which are used to amplify viral mrna synthesis and are packaged into progeny virions. Four viral proteins are necessary and sufficient to carry out this transcription cycle: three subunits of an RNA dependent RNA polymerase (PB1, PB2, and PA), and a nucleoprotein (Huang et al., 1990). The polymerase carries out all known enzymatic steps, with PB1 catalysing nucleotide addition (Braam et al., 1983; Romanos and Hay, 1984; Biswas and Nayak, 1994), PB2 recognising host-cell cap structures (Ulmanen et al., 1981; Blaas et al., 1982) and possibly providing endonuclease function (Shi et al., 1995; Blok et al., 1996), and PA playing an as yet ill-defined role, possibly during replicative transcription (Mahy et al., 1983). The primary activity associated with the nucleoprotein (NP) is an ss- RNA-binding activity, with little apparent sequence specificity (Scholtissek and Becht, 1971; Kingsbury et al., 1987; Yamanaka et al., 1990; Baudin et al., 1994). This RNAbinding activity is reflected in the stoichiometric quantity of the protein in RNPs, from which it has been estimated that one polypeptide interacts with 20 nucleotides of RNA (Choppin and Compans, 1975). In addition, NP also participates in a variety of macromolecular contacts with viral and cellular proteins. Recent work has shown that it /99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved. 190

3 OLIGOMERIZATION OF INFLUENZA VIRUS NP 191 interacts directly with the PB1 and PB2 subunits of the polymerase (Biswas et al., 1998), while the viral matrix protein binds to RNP complexes (Zvonarjev and Ghendon, 1980), possibly in part through protein protein contacts with NP. NP has also been shown to interact with the cellular protein responsible for recognising nuclear localisation signals, importin (O Neill and Palese, 1995; Wang et al., 1997), and this interaction is capable of driving nuclear uptake of NP RNA complexes (O Neill et al., 1995). NP also binds directly to filamentous actin, and this interaction works in opposition to the nuclear import pathway to cause cytoplasmic accumulation of the protein (Digard et al., 1999). In addition, NP also forms homo-oligomers that are apparently responsible for the higher order structure of RNPs, which occur in the form of helices twisted back into a double helical hairpin, as the structures can persist following removal of the RNA (Pons et al., 1969; Ruigrok and Baudin, 1995). This implies direct protein protein interactions between NP monomers (Zhao et al., 1998; Ruigrok and Baudin, 1995; Prokudina-Kantorovich and Semenova, 1996), which is consistent with the observed cooperative nature of the NP-RNA interaction (Yamanaka et al., 1990). The RNP structure is apparently important for transcription, as although the polymerase complex alone can efficiently transcribe short templates, NP is required for synthesis of longer RNAs, leading to the suggestion that it may act as a processivity factor (Honda et al., 1988). In addition, it has been suggested that the coiled structure of the RNP influences the aberrant transcription events that lead to the generation of defective-interfering RNAs (Jenning et al., 1983). NP may also play a role in the regulation of polymerase activity, as c- and vrna (but not mrna) synthesis depends on a supply of soluble (i.e., not bound to RNA) NP (Beaton and Krug, 1986; Shapiro and Krug, 1988). Also consistent with a regulatory role, a recent study has identified NP point mutations that differentially affect c- and vrna synthesis (Mena et al., 1999). Thus NP is a multifunctional protein of pivotal importance to influenza virus replication whose molecular characterisation is therefore of some interest. Here we report the results of experiments examining the self-association of NP. Using recombinant protein in in vitro binding studies, we show that NP monomers form direct protein protein contacts with an apparent K d of M. We also show that NP contains two discrete regions capable of forming NP NP contacts in isolation, as well as a C-terminal sequence that inhibits self-association. Within each of these regions, we also identify single amino-acid changes that perturb self-association and show that these mutations affect the ability of the polypeptides to support influenza virus gene expression. RESULTS Self-association of NP To examine intermolecular contacts between NP molecules, we tested the ability of in vitro-translated NP to bind to affinity-purified NP fusion proteins. Radiolabelled NP synthesised in rabbit reticulocyte lysate was incubated with MBP-NP or GST-NP, the fusion proteins collected by the addition of amylose- or glutathione Sepharose, respectively, and bound material analysed by SDS PAGE and autoradiography. In both cases, readily detectable quantities of the radiolabelled NP coprecipitated with the NP fusion proteins (Fig. 1a, lanes 3 and 6). The precipitation was specific because NP did not precipitate with MBP or GST alone (lanes 2 and 5). In addition, MBP-NP that had been digested with factor Xa protease to separate the MBP and NP domains failed to precipitate the radiolabelled NP (lane 4). Thus in vitrotranslated NP associates with bacterially expressed NP fusion proteins. We also detected self-association of NP using the yeast two-hybrid system, a genetic assay for protein protein interaction. Very little -galactosidase was expressed in yeast cells singly transformed with plasmids expressing gene fusions between NP and the activation or DNA-binding domains of the GAL4 protein (A and B respectively, Fig. 1b). However, readily detectable activity was obtained when cells were transformed with both plasmids, indicating an interaction between the two NP-fusion proteins (A B, Fig. 1b). Nevertheless because NP binds RNA, the interaction in both systems could possibly be mediated via an RNA intermediate rather than by direct protein protein contacts. Accordingly, we tested the ability of in vitro-translated NP to bind GST-NP under conditions expected to prevent RNA-binding. Addition of 10 g/ml RNaseA to the binding reactions had no effect on the interaction between in vitrotranslated NP and GST-NP (Fig. 1c, cf. lanes 2 and 8). Moreover, the addition of high concentrations of NaCl reduced but did not abolish precipitation of the radiolabelled NP (lanes 3 6). Because NP does not bind RNA at salt concentrations 1 M (Yamanaka et al., 1990), we conclude that the assay reflects a direct protein protein interaction between NP monomers. In addition, protein species with the expected electrophoretic mobilities for dimers, trimers, and higher-order complexes could be generated by incubation of bacterially expressed or in vitro-translated NP with bifunctional chemical cross-linking reagents (data not shown), suggesting that the assay reflects oligomerization of NP and not simple dimerisation. To quantitate the interaction, we titrated increasing concentrations of GST-NP with a constant amount of in vitro-translated NP in the binding assay and determined the distribution of radiolabelled protein between pellet and supernatant fractions. At high concentrations of GST-NP, the majority of the radiolabelled NP was bound with only small quantities remaining in the supernatant

4 192 ELTON ET AL. FIG. 1. Self-association of recombinant NP. (a) Radiolabelled in vitro-translated NP was analysed by SDS PAGE and autoradiography before (T) or after binding to the indicated immobilised unlabelled fusion proteins (MBP-NP/Xa denotes MBP-NP that had been digested with Factor Xa protease to separate the MBP and NP domains). Arrows indicate molecular mass markers (sized in kda) and NP. (b) Self-association of NP in the yeast two-hybrid system. The relative -galactosidase activity obtained from yeast cultures transformed with the indicated mixtures of plasmids encoding full-length NP (or NP C475 [ ]) fused to the GAL-4 activation (A) or DNA-binding (B) domains is plotted. Error bars indicate the range of values obtained from two independent experiments. (c) Role of RNA in NP self-association. In vitro-translated NP was assayed for its ability to bind GST (G) or GST-NP (N) as in (a) ( ) or in the presence of NaCl or RNaseA as indicated. (Fig. 2a, lanes 2 and 3). At intermediate concentrations of GST-NP, the in vitro-translated NP partitioned evenly between supernatant and pellet fractions (lanes 4 and 5), while at low concentrations, the majority remained in the supernatant (lanes 6 9). When the amount of bound NP was quantified and plotted against the concentration of GST-NP, a sigmoidal curve was obtained where essentially all of the radiolabelled NP was precipitated at GST-NP concentrations of 1 M (Fig. 2b). Half-maximum binding was achieved at nm GST-NP (n 4), and because the concentration of radiolabelled NP was substantially lower ( 20 fm), this value can be taken as an estimate of the dissociation constant of NP NP interactions under these conditions. Mutagenic analysis of the NP NP contact points We determined the regions of NP involved in oligomerization by testing the ability of truncated versions of the polypeptide (expressed in reticulocyte lysate) to bind to a fixed concentration (60 nm) of GST-NP. This amount of GST-NP bound readily detectable but subsaturating quantities of radiolabelled NP (Fig. 3a, cf.. lanes 1 and 3), thus providing a sensitive baseline from which to assess changes in the binding ability of mutant polypeptides. Removal of three residues from the C terminus of NP had little effect on self-association (C495, data not shown but summarised in Fig. 4a). However, removal of a further 20 amino acids (to generate C475) apparently enhanced the ability of the protein to oligomerise, as the majority of the mutant polypeptide was bound by 60 nm GST-NP (Fig. 3a, lanes 4 6). When titrated, 10-fold less GST-NP was required to bind equivalent amounts of C475 than WT NP (Fig. 2b), confirming the increased binding ability of the polypeptide. In addition, this increased self-association was not peculiar to the NP:GST-NP interaction, as higher levels of -galactosidase activity were obtained when yeast cells were cotransformed with plasmids expressing a GAL4 DNA-binding domain NP C475 fusion protein and a WT NP-activation domain fusion (Fig. 1b; A B), compared with that obtained from a homozygous mix of WT NP fusions (A B). The increased levels of -galactosidase activity did not arise from fortuitous promoter activation by NP C475 itself, as only background levels of expression were obtained in the absence of the NP-activation domain fusion (Fig. 1b; B). Similar increased levels of self-association in the GST-pulldown assay were obtained with a polypeptide ending at residue 461 (data not shown, but summarised in Fig. 4a). Further C-terminal truncation progressively reduced binding, first to levels similar to that of the WT protein (C358, data not shown but summarised in Fig. 4; C256, Fig. 3a, lanes 7 9), then to undetectable levels (C181, data not shown, summarised in Fig. 4a; C161, Fig. 3a, lanes 10 12). In the converse experiment, 50% of the N terminus of NP could be deleted with little effect on oligomerization, either positive or negative (Fig. 3b, lanes

5 OLIGOMERIZATION OF INFLUENZA VIRUS NP 193 below detectable levels (N357C475, Fig. 3c, lanes 10 12; N371C475, Fig. 4a). The N189 polypeptide could be further C-terminally truncated to residue 358 without destroying binding activity (N189C358, Fig. 3c, lanes 4 6). However, further subdivision of this region of NP into fragments comprising residues or resulted in the loss of detectable self-association (N189C256, Fig. 3c, lanes 7 9; N254C358, data not shown but summarised in Fig. 4a). Therefore the two smallest regions of NP identified as capable of forming NP NP contacts comprise approximately the middle FIG. 2. Titration of the NP:GST-NP interaction. (a) In vitro-translated NP was analysed by SDS PAGE and autoradiography before (T) or after binding to immobilised GST-NP at the indicated concentrations followed by separation into supernatant (S) and pellet (P) fractions. (b) Graphical analysis of NP: GST-NP interactions. Percentages of wildtype (WT) or mutant NP molecules bound at the indicated concentrations of GST-NP are plotted. 1 3, N189; N12, N105, and N254, summarised in Fig. 4). Further deletion of N-terminal sequences again resulted in progressively weaker binding (Fig. 3b, lanes 4 9, N357 and N371), followed by a failure to detectably self-associate (lanes 10 12, N440). However, the smaller N- and C-terminal fragments that retained the ability to bind GST-NP did not contain sequences in common (e.g., C256 and N357), indicating that NP contains at least two regions involved in self-recognition. To further delineate these contact points, we investigated the ability of internal fragments of NP to interact with GST-NP. In the context of polypeptides lacking NP sequences upstream of amino acid 189 or 254, removal of the C-terminal 23 residues did not prevent binding, but neither did it noticeably enhance it (N189C475, Fig. 3c, lanes 1 3; N254C475, data not shown but summarised in Fig. 4a). In contrast, loss of the very C terminus from polypeptides beginning at NP codons 357 or 371 reduced binding to FIG. 3. Deletion analysis of the NP:GST-NP interaction. The indicated in vitro-translated NP deletion mutants were analysed by SDS PAGE and autoradiography before (T) or after binding to GST (G) or GST-NP (N). Equivalent amounts were run in the total and precipitated lanes. (a) Progressive truncation from the C terminus of the protein. (b) Progressive truncation from the N terminus of the protein. (c) Internal fragments of NP. The relative migration of polypeptides between panels do not necessarily correspond because of differences in the percentage of acrylamide in the gels. In addition, the migration of the N440, N189C256, and N357C475 polypeptides is distorted by the large amount of unlabelled globin protein present in the total lanes.

6 194 ELTON ET AL. (NP-1, residues ) and C-terminal (NP-2, ) thirds of the protein (Fig. 4b). This is in contrast to the minimal region of NP that possesses RNA-binding activity, which corresponds to approximately the N-terminal third of the polypeptide (Kobayashi et al., 1994; Albo et al., 1995). To further characterise the NP NP interaction, we analysed the effects of point mutations (to alanine) on selfassociation, concentrating on charged and aromatic residues conserved between the nucleoproteins of influenza subtypes A C and Dhori virus within the NP-1 and NP-2 regions. In addition, although deletion analysis did not provide evidence for the direct involvement of the N-terminal third of NP in oligomerization, we also examined the phenotype of point mutants within this region. The mutant NP molecules were in vitro-translated in rabbit reticulocyte lysate and tested for their ability to bind to a fixed concentration of GST or GST-NP (60 nm) as before. Bound protein was quantified and the results expressed as the percentage precipitation ( 1 standard error, n 3) relative to that seen with the WT polypeptide (Fig. 5). The majority of the mutations, including all those tested within the minimal RNA-binding region of NP, had no significant effect on oligomerization. However, three point mutations were identified in the minimal NP NP contact regions that did substantially affect self-association. Mutation of F479 in the NP-2 region increased binding approximately twofold, while alteration of R199 in the NP-1 region or R416 in the NP-2 region reduced binding around fivefold compared with the WT protein (Fig. 5). When the interaction of the F479 mutant with GST-NP was titrated, 50% of the mutant protein was bound by 40 nm GST-NP, fivefold less than the concentration required for half-maximal binding of WT NP (Fig. 2b). The R199 mutant produced a binding curve that failed to reach saturation, and where, relative to WT in vitro-translated NP, around threefold more GST-NP was required for equivalent levels of precipitation (Fig. 2b). The R416 mutant bound even more poorly and also failed to reach saturation at the concentrations of GST-NP FIG. 4. Summary of the ability of NP fragments to self-associate. (a) The sizes and positions of the indicated mutants are shown by boxes relative to the WT protein. The percentage of input protein bound was determined by densitometry and scored as similar to WT protein (, between 50 and 10% bound), enhanced (, 50% bound), less than WT but detectable (, 10% bound), or undetectable ( ). The smallest fragments identified as capable of forming NP NP contacts are shaded. (b) Functional regions of NP. The minimal regions of NP capable of binding nucleic acid (RNA; Kobayshi et al., 1994, Albo et al., 1995) or NP (NP-1 and NP-2) are indicated by boxes and the amino-acid coordinates given. The hatched box indicates the C-terminal region that inhibits oligomerization. Also shown are the locations of point mutations found to decrease (down arrows) or increase (up arrow) self-association. FIG. 5. Effect of NP point mutations on self-association. In vitrotranslated NP mutants where the indicated amino-acid(s) had been altered to alanine were quantified for their ability to bind to GST-NP (60 nm) relative to the WT protein. Mutants are grouped according to the minimal binding region (Fig. 4b) they lie in, excepting a double mutant containing lesions in both NP-1 and NP-2 regions (R199/R416) and influenza B NP (B-NP). Values plotted are the average and standard error from at least three independent experiments.

7 OLIGOMERIZATION OF INFLUENZA VIRUS NP 195 FIG. 6. Ability of NP mutants to support influenza virus gene expression. BHK cells were infected with recombinant vaccinia viruses expressing the three subunits of the influenza virus RNA polymerase and T7 RNA polymerase and transfected with a synthetic influenza segment containing an antisense CAT gene and plasmid encoding WT or mutant NPs, and the resulting accumulation of CAT polypeptide was quantified. (a) Activity of NP deletion mutants. Values shown are the percentage CAT accumulation (plus standard error, n 3) relative to WT NP resulting from the transfection of 1 g of each plasmid. (b) Titration of NP point mutations. CAT expression arising from transfection of the indicated amount of each plasmid is shown. tested, with 30% of the protein bound by 1.2 M GST- NP. Thus the R199 polypeptide is still capable of binding GST-NP but with lower affinity than WT NP, while the R416 mutant is more drastically impaired in its ability to self-associate. In contrast, the F479 polypeptide binds to WT NP with apparently higher affinity. Because the R199 and R416 mutations lie in regions of NP that were functionally separable in terms of mediating self-association, we tested the effects of combining the two mutations. However, although clearly defective for self-association, the double-mutant R199-R416 did not bind appreciably less than the R416 parental mutant (Fig. 5 and titration data not shown). We also tested the ability of the NP from influenza B virus to form hetero-oligomers with influenza A NP. Although binding could be detected, it was substantially weaker than homo-oligomerization between molecules of A-NP (Fig. 5). Therefore the influenza A and B NPs have diverged sufficiently to lose the capability to form high-affinity protein protein contacts with each other. Ability of the NP mutants to support virus gene expression It has been shown that NP and the three subunits of the influenza virus RNA polymerase are necessary and sufficient for the transcription and replication of a synthetic influenza virus genome segment containing an antisense CAT (flu-cat) reporter gene in transfected cells (Huang et al., 1990). We used an adaptation of this system (Digard et al., 1999) to assay the transcription competence of the NP mutants. First, to establish the regions of NP essential for influenza virus gene expression, we examined the activities of NP deletion mutants. Cells were multiply infected with vaccinia viruses expressing the three subunits of the influenza virus RNA polymerase and the bacteriophage T7 RNA polymerase, transfected with the flu-cat RNA and wild-type or mutant plasmid pkt5 containing the NP gene under control of a T7 RNA polymerase promoter, and subsequent accumulation of CAT polypeptide measured. Deletion of 11 amino acids from the N terminus of NP had no effect on the ability of the protein to support CAT expression, relative to WT NP (N12; Fig. 6a). However, further removal of N-terminal sequences essentially abolished gene expression (N105; Fig. 6a, N189 data not shown). When the effect of progressive C-terminal truncation was tested, the removal of as few as three amino-acids was found to abolish activity (C495, C475; Fig. 6, C358, C256, data not shown). Thus the ability of NP to support virus gene expression is particularly sensitive to the loss of C-

8 196 ELTON ET AL. terminal residues, but a short sequence at the N terminus of the protein is dispensable for activity. Next, we compared the activities of the NP point mutants with altered oligomerization behaviour by titrating the plasmids. When the WT plasmid was tested, CAT synthesis correlated with gene dose, with the highest activity resulting from 1 g of plasmid (Fig. 6b). The R199 mutant titrated similarly to the WT gene except that maximal synthesis was reduced compared with that of WT NP (74 12% for 1 g of plasmid, n 6). In contrast, the R416 polypeptide failed to support detectable levels of CAT synthesis irrespective of the amount of plasmid transfected ( % for 1 g, n 4). The F479 polypeptide supported influenza gene expression but titrated differently to the WT polypeptide (Fig. 6b). In contrast to the behaviour of the WT gene, maximal activity was obtained from 0.3 g of plasmid (94 24% of the activity from a similar dose of WT pkt 5, n 3), with higher doses proving inhibitory (22 16% of the activity obtained from 1 g ofwtpkt5,n 3). Thus mutations that perturb NP oligomerization either positively or negatively decrease the ability of the polypeptide to support virus gene expression. This is consistent with the importance of NP NP contacts for the formation of functional RNP structures. DISCUSSION To investigate the self-association of NP, we developed a binding assay using radiolabelled in vitro-translated NP and purified immobilised NP fusion proteins. Formation of NP NP oligomers in this system was specific because no association of NP with non-fused MBP or GST moieties was observed (Fig. 1) and a variety of mutations within NP reduced or abolished the interaction (Figs. 2 5). In addition, the assay reflected a direct protein protein contact between NP polypeptides, rather than one mediated through an RNA intermediary because it could be observed under conditions where NP would not be expected to bind nucleic acid (Fig. 1). Moreover, self-association and RNA-binding activities are mutationally separable; we have shown recently that several of the NP point mutants which oligomerise normally (Fig. 5) fail to bind RNA (e.g., R267, W330; Elton et al., 1999). Binding of the radiolabelled NP to the NP fusion protein was titratable and saturable, and this approach was used to estimate an apparent dissociation constant of M for self-association between NP molecules (Fig. 2). Because the system has the potential for the formation of higher order complexes, NP:NP and GST-NP:GST-NP interactions as well as simple NP:GST-NP binding, this estimate necessarily represents an averaged value. In comparison, NP has an 10-fold greater affinity for RNA (K d 2x10 8 M; Baudin et al., 1994; Digard et al., 1999), suggesting that NP-RNA contacts may form first during the generation of RNP structures. This is similar to the behaviour of other single-strand nucleic acid-binding proteins (SSBs) that show positive cooperativity. For instance, the bacteriophage T4 gene 32 protein binds ssdna as tightly ( 10 8 M 1 ; Kelley et al., 1976) as NP does RNA, but has an even lower affinity for itself ( 10 5 M 1 ; Carrol et al., 1972). Deletion analysis of NP oligomerization identified two nonoverlapping regions of the polypeptide capable of binding to full-length NP, comprising approximately the middle (designated NP-1) and C-terminal thirds of the protein (NP-2; Figs. 3 and 4). Point mutagenesis of residues within these regions identified R199 and R416 as important for high-affinity binding (Figs. 2 and 5). The NP-1 and NP-2 areas are distinct from the region of NP previously identified as the minimal RNA-binding domain (Fig. 4b; Kobayashi et al., 1994, Albo et al., 1995). However, further structural analysis is necessary to determine whether the three regions represent independently folding protein domains. As Ruigrok and Baudin (1995) pointed out, to multimerise, NP must contain at least two independent binding sites, and it is possible the NP-1 and NP-2 binding regions we have mapped here correspond to such contact points. However, the fact that simultaneous mutation of R199 (in the NP-1 region) and R416 (in the NP-2 region), which individually decreased self-association, did not further decrease binding argues against this interpretation. Indeed, given the highly ordered helical hairpin structure of RNPs (Pons et al., 1969; Jennings et al., 1983; Ruigrok and Baudin, 1995), it is plausible that NP monomers form additional protein protein contacts beyond those necessary for a simple string of beads structure. Here, we identify three point mutations which affect the oligomerization of NP, both positively (F479) and negatively (R199 and R416). However, it is necessary to consider whether the mutations affected other functions of NP, including perhaps the general polypeptide conformation. In respect of the latter possibility, RNA-binding activity provides a stringent test of the overall folding of NP because high-affinity binding involves N- and C- terminal regions of the protein and is sensitive to mutations throughout the length of the polypeptide (Elton et al., 1999). By this criterion, the R199 and F479 polypeptides are folded normally because they bind RNA similarly to WT NP (Elton et al., 1999, and data not shown). The case of the R416 mutation is more complex because it also renders RNA-binding activity temperature sensitive: polypeptides synthesised at 30 C bind RNA normally, but those made at 37 C show much reduced activity (Elton et al., 1999). Therefore the binding experiments with R416 employed material made at 30 C and the CAT gene expression assay was performed at 30 C, to reduce this possible confounding effect. In addition, R416 synthesised at 37 C was still capable of interacting with the influenza virus polymerase complex (Elton et al., 1999) but was no more defective than material made at

9 OLIGOMERIZATION OF INFLUENZA VIRUS NP C in the NP oligomerization assay (data not shown). Thus all three mutations that affect self-association do so without destroying other functions of the protein, arguing for a degree of specificity. The three mutations decreased the ability of the polypeptide to support transcription and replication of a model influenza segment (Fig. 6). The R199 mutation, which decreased binding affinity for low concentrations of GST-NP threefold and prevented saturation binding (Fig. 2b), had little effect on the ability of the polypeptide to support RNA synthesis except at high doses of plasmid, where its activity was reduced compared to the WT protein (Fig. 6). The R416 mutation, which had a much greater negative effect on self-association in vitro (Fig. 2b), completely abolished transcriptional activity. The F479 mutation, which increased the in vitro NP binding affinity fivefold, also decreased overall transcription in vitro and reproducibly shifted the optimum concentration of plasmid to lower doses than WT. Thus mutations that perturbed NP oligomerization in vitro also affected RNA transcription in vivo, and the magnitude of the effects broadly correlated. This is consistent with the importance of NP NP contacts for the formation of a transcriptionally active RNP structure. Somewhat unexpectedly, we found that deletion of the C-terminal 23 amino acids of NP increased oligomerization, giving a 10-fold increase in apparent binding affinity ( C475, K d from 10 7 to 10 8 M; Fig. 2). This increase in self-association was not an artifact of the in vitro assay, as a similar increase in signal was seen when the C475 mutant was tested in the yeast two-hybrid system (Fig. 1b). In addition, a recent study similarly found that removal of the C terminus of NP increased the binding of the protein to the PB2 subunit of the influenza virus RNA polymerase (Biswas et al., 1998). Thus the C-terminal amino-acids of NP may represent a general regulatory region of the protein which modulates its interaction with at least two macromolecules. Biswas and colleagues speculated that the region could function either by obscuring the interacting region(s) of NP or by affecting overall protein conformation. Our data do not help distinguish between these possibilities or the alternative hypothesis that the C terminus of NP binds a sequestering cellular factor. The fact that removal of the C terminus enhances self-association in the context of otherwise full-length NP (e.g., C475), has little apparent effect on a protein containing the NP-1 and NP-2 regions (e.g., N189), but is inhibitory in the context of the NP-2 region alone (e.g., N357) argues for a complex interplay between two or more factors, one almost certainly concerning the ability of deleted molecules to fold correctly. However, it is worth considering the function of the region in terms of its primary sequence. The study of Biswas et al., (1998) concentrated on the possible role of serine residues, but failed to identify any as important for PB2-binding. We however identified residue F479 as important for NP NP interactions, as its alteration to alanine increased self-association fivefold (Fig. 2). Nevertheless, the striking feature of the region is its overall negative charge. In contrast to full-length NP, which has a pi of 9.3, the C-terminal 30 amino acids have a pi of 3.7 (A/PR8/34 strain; Winter and Fields, 1981). Many SSBs have similar acidic C-terminal tails, ranging from the NPs of the nonsegmented negative strand viruses to the ssdna binding proteins of bacteriophage T4, Escherichia coli, and eukaryotic mitochondria, and in many cases the region is apparently involved in mediating protein protein contacts and regulating the activities of the protein (Boltrek-Krassa et al., 1991; Lohman and Ferrari, 1994; Lamb and Kolakofsky, 1996). The acidic region of NP is apparently essential for polypeptide function, as its truncation (C495) or removal (C475) abolished the ability of the protein to support virus gene expression (Fig. 6), but it is difficult to correlate this with the upregulation of oligomerization because C495 self-associated normally. Similarly, a recent study examining the effects of NP point mutations on m-, c-, and vrna synthesis found that mutation of phenylalanine 488 in the C-terminal tail specifically inhibited crna synthesis (Mena et al., 1999), but in contrast to the behaviour of our F479 mutant, the authors found no evidence that the lesion affected NP NP oligomerization. Nevertheless, further investigation of the function of the influenza NP C-terminal tail should prove worthwhile. MATERIALS AND METHODS Plasmids and protein expression Plasmids containing the native A/PR8/34 NP gene under the control of a bacteriophage T7 RNA polymerase promoter (pkt5) or fused to either Escherichia coli maltose-binding protein (pmal-np) or Schistosoma japonicum glutathione-s-transferase (pgex-np) have been described previously (Digard et al., 1999). Solution protein concentrations were determined by the method of Bradford (1976) or by absorbance at 280 nm using extinction coefficients calculated according to the formula of Gill and von Hippel (1989). The concentration of fusion proteins bound to affinity matrices was determined by densitometry of aliquots analysed by SDS PAGE and staining with Coomassie brilliant blue in parallel with standards of known concentration. Deletion mutations were created in plasmid pkt5 using convenient restriction enzyme sites and standard recombinant DNA methodology. In the interest of brevity, details of the cloning steps are not reported here but are available on request. N- terminally deleted (N) mutants are named according to the first codon of the wild-type gene present, C-terminally deleted (C) mutants according to the last. The translation of N-terminal deletion mutants exploited in frame ATG codons within the NP sequence or the ATG codon present in plasmid pkt0 (Digard et al., 1999). C-terminal

10 198 ELTON ET AL. deletions were constructed by ligating the palindromic DNA oligonucleotide dctagtctagactag (New England Biolabs) into the desired restriction enzyme site and therefore include between 1 and 4 additional noninfluenza-derived codons at the 3 end of the ORF. The major in vitro-translation products obtained from the deletion clones were of the expected sizes. Point mutations (and the C495 mutation) were introduced using oligonucleotide directed mutagenesis according to standard procedures (Kunkel, 1985). A cdna clone of the NP gene from influenza B/AA/1/66 virus was the generous gift of Dr. W. Barclay (Stevens and Barclay, 1998). Radiolabelled NP polypeptides were synthesised using a coupled in vitro transcription-translation system (Craig et al., 1992). Rabbit reticulocyte lysate (Promega) was supplemented with 0.6 Ci/ l [ 35 S]methionine, 2 U/ l T7 RNA polymerase (Gibco-BRL), 3.3 mm MgCl 2, 0.2 mm nucleoside-triphosphates and g/ml plasmid DNA before incubation at 30 or 37 C for 90 min. Maltose-binding protein (MBP), glutathione-s-transferase (GST) alone, or fused to NP (MBP-NP and GST-NP) were affinity purified from lysates of E. coli containing the appropriate plasmids as previously described (Digard et al., 1999). Protein binding assays For precipitation reactions, 1 l of in vitro-translated protein was incubated with 0.5 g of fusion protein in 100 l of IP buffer (100 mm KCl, 50 mm Tris HCl, ph 7.6, 5 mm MgCl 2, 1 mm dithiothreitol, 0.1% Nonidet-P40) for 1 h [determined from time-course experiments to be sufficient for maximal binding (data not shown)] at room temperature. 50 l of a 50% (v/v) slurry of glutathione Sepharose (Pharmacia) or amylose resin (New England Biolabs) in phosphate-buffered saline was added as appropriate and incubation continued for a further 30 min with gentle mixing. The solid phase was collected by centrifugation and washed three times with 750 l ofip buffer. Bound material was eluted by boiling in 30 l of SDS PAGE sample buffer and analysed by SDS PAGE and autoradiography. For titration experiments, in vitrotranslated protein was incubated with GST-NP already bound to glutathione Sepharose for 1 h, and the supernatant fraction was saved and an amount equivalent to the pellet fraction analysed by SDS PAGE and fluorography following impregnation of the gels with Amplify (Amersham). Bound and free protein was quantified by densitometry of preflashed X-ray film using a JADE flatbed scanner (LinoType-Hell) and NIH Image software (available by anonymous FTP from zippy.nimh.nih.gov). For yeast two-hybrid experiments, a commercial adaptation (Matchmaker system, Clontech) of the system described by Fields and Song (1989) was used. To construct gene fusions between NP and the GAL-4 DNAbinding and activation domains, the NP gene was excised from plasmid pkt5 by digestion with restriction endonucleases NcoI and SalI, and the NcoI site was end-filled with Klenow fragment DNA polymerase and then ligated into plasmids pgbt9 or pgad424 (Clontech) that had been digested with SmaI and SalI. Yeast strain SFY526 was transformed with plasmids and grown according to the manufacturer s instructions. Liquid culture assays for -galactosidase activity were also performed according to the manufacturer s instructions, measuring the release of o-nitrophenol from o-nitrophenylgalactosidase by spectrophotometry at 420 nm. Influenza virus gene expression assay For in vivo RNA transcription assays, a synthetic influenza genome segment containing an antisense chloramphenicol acetyl transferase (CAT) gene was produced by in vitro transcription of plasmid ppb2cat9 (generous gift of Dr Mark Krystal). BHK cells (in 35-mm-diameter dishes) were infected with recombinant vaccinia viruses expressing the three subunits of the influenza virus polymerase (PB1-, PB2-, and PA-VAC; Smith et al., 1987) and the bacteriophage T7 RNA polymerase (VTF-7; Fuerst et al., 1987) at a m.o.i. of 5 of each virus per cell for 2hat 37 C. The cells were washed three times with serumfree medium before transfection with 1 g of plasmid pkt5 encoding NP (or mutant derivatives) and 0.5 g of ppb2cat9 in vitro-transcribed RNA and 10 g of a cationic liposome mixture (Escort; Aldrich), according to the manufacturers instructions. The cells were incubated at 30 or 37 C for 20 h, washed three times with ice-cold PBS and solubilised in 1 ml of CAT ELISA lysis buffer (Boehringer Mannheim). The lysate was clarified by centrifugation at 14,000 g for 10 min at 4 C and CAT expression quantified relative to known standards by a commercial enzyme-linked immunosorbent assay (Boehringer Mannheim). ACKNOWLEDGMENTS We thank Drs. Wendy Barclay and Mark Stevens for the gift of an influenza B NP cdna, Dr. Geoff Smith for vaccinia recombinants, and Dr. Ian Brierley for critical appraisal of the manuscript. This work was supported by grants from the Royal Society and Wellcome Trust (048911) to P.D. P.D.is a Royal Society University Research Fellow. REFERENCES Albo, C., Valencia, A., and Portela, A. (1995). Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein. J. Virol. 69, Baudin, F., Bach, C., Cusak, S., and Ruigrok, R. W. H. (1994). Structure of influenza RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to solvent. EMBO J. 13, Beaton, A. R., and Krug, R. M. (1986). Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid proteins and the absence of a 5 capped end. Proc. Natl. Acad. Sci. USA 83, Biswas, S. K., Boutz, P. L., and Nayak, D. P. (1998). Influenza virus

11 OLIGOMERIZATION OF INFLUENZA VIRUS NP 199 nucleoprotein interacts with influenza virus polymerase proteins. J. Virol. 72, Biswas, S. K., and Nayak, D. P. (1994). Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. J. Virol. 68, Blaas, D., Patzelt, E., and Kuechler, E. (1982). Cap recognising protein of influenza virus. Virology 116, Blok, V., Cianci, C., Tibbles, K. W., Inglis, S. C., Krystal, M., and Digard, P. (1996). Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit. J. Gen. Virol. 77, Boltrek-Krassa, K., Green, L. S., and Gold, L. (1991). Protein protein interactions with the acidic COOH terminus of the single-stranded DNA-binding protein of the bacteriophage T4. Proc. Natl. Acad. Sci. USA 88, Braam, J., Ulmanen, I., and Krug, R. M. (1983). Molecular model of a eukaryotic transcription complex: Functions and movements of influenza P proteins during capped RNA-primed transcription. Cell 34, Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, Carrol, R. B., Neet, K. E., and Goldthwait, D. A. (1972). Self-association of gene-32 protein of bacteriophage T4. Proc. Natl. Acad. Sci. USA 69, Choppin, P. W., and Compans, R. W. (1975). The structure of influenza virus. In The influenza viruses and influenza (E. D. Kilbourne, Ed.), pp Academic Press, New York. Craig, D., Howell, M. T., Gibbs, C. L., Hunt, T., and Jackson, R. J. (1992). Plasmid cdna-directed protein synthesis in a coupled in vitro transcription-translation system. Nucleic Acids Res. 20, Digard, P., Elton, D., Bishop, K., Medcalf, E., Weeds, A., and Pope, B. (1999). Modulation of nuclear localization of influenza virus nucleoprotein through interaction with actin filaments. J. Virol. 73, Elton, D., Medcalf, E., Bishop, K., Harrison, D., and Digard, P. (1999). Identification of amino-acid residues of influenza virus nucleoprotein essential for RNA-binding. J. Virol., in press. Fields, S., and Song, O. (1989). A novel genetic system to detect protein protein interactions. Nature 340, Fuerst, T. R., Earl, P. L., and Moss, B. (1987). Use of a hybrid vaccinia virus-t7 RNA polymerase system for expression of target genes. Mol. Cell. Biol. 7, Gill, S. C., and von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino-acid sequence data Anal. Biochem. 182, Honda, A., Ueda, K., Nagata, K., and Ishihama, A. (1988). RNA polymerase of influenza virus: Role of NP on RNA chain elongation. J. Biochem. 104, Huang, T.-S., Palese, P., and Krystal, M. (1990). Determination of influenza virus proteins required for genome replication. J. Virol. 64, Jennings, P. A., Finch, J. T., Winter, G., and Robertson, J. S. (1983). Does the higher order structure of the influenza virus ribonucleoprotein guide the sequence rearrangements in influenza viral RNA? Cell 34, Kelley, R. C., Jensen, D. E., and von Hippel, P. H. (1976). DNA melting proteins. IV. Fluorescent measurements of binding parameters for bacteriophage T4 gene 32-protien to mono-, oligo-, and polynucleotides. J. Biol. Chem. 251, Kingsbury, D. W., Jones, I. M., and Murti, K. G. (1987). Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein. Virology 156, Kobayashi, M., Toyoda, T., Adyshev, D. M., Azuma, Y., and Ishihama, A. (1994). Molecular dissection of influenza virus nucleoprotein: Deletion mapping of the RNA binding domain. J. Virol. 68, Kunkel, T. A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, Lamb, R. A., and Kolakofsky, D. (1996). Paramyxoviridae: The viruses and their replication. In Fields Virology (B.N. Fields, D.M. Knipe, P.M. Howley et al., Eds.), pp Lippincott-Raven, Philadelphia. Lamb, R. A., and Krug, R. M. (1996). Orthomyxoviridae: The viruses and their replication. In Fields Virology (B.N. Fields, D.M. Knipe, P.M. Howley et al., Eds.), pp Lippincott-Raven, Philadelphia. Li, X., and Palese, P. (1994). Characterization of the polyadenylation signal of influenza virus RNA. J. Virol. 68, Lohman, T. M., and Ferrari, M. E. (1994). Escherichia coli singlestranded DNA-binding protein: Multiple DNA-binding modes and cooperativities. Annu. Rev. Biochem. 63, Luo, G., Luytes, W., Enami, M., and Palese, P. (1991). The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. J. Virol. 65, Mahy, B. W. J. (1983). Mutants of influenza virus. In Genetics of Influenza Virus (P. Palese and D.W. Kingsbury, Eds.), pp Springer-Verlag, New York. Mena, I., Jambrina, E., Albo, C., Perales, B., Ortin, J., Arrese, M., Vallejo, D., and Portela, A. (1999). Mutational analysis of influenza A virus nucleoprotein: Identification of mutations that affect RNA replication. J. Virol. 73, O Neill, R. E., Jaskunas, R., Blobel, G., Palese, P., and Moroianu, J. (1995). Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J. Biol. Chem. 270, O Neill, R. E., and Palese, P. (1995). NPI-1, the human homologue of SRP-1, interacts with influenza virus nucleoprotein. Virology 206, Plotch, S. J., Bouloy, M., Ulmanen, I., and Krug, R. M. (1981). A unique cap (m 7 GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, Pons, M. W., Schulze, IT., Hirst, G. K., and Hauser, R. (1969). Isolation and characterisation of the ribonucleoprotein of influenza virus. Virology 39, Pritlove, D. C., Poon, L. L. M., Fodor, E., Sharps, J., and Brownlee, G. G. (1998). Polyadenylation of influenza virus mrna trancribed in vitro from model virion RNA templates: Requirement for 5 conserved sequences. J. Virol. 72, Prokudina-Kantorovich, E. N., and Semenova, N. P. (1996). Intracellular oligomerization of influenza virus nucleoprotein. Virology 223, Romanos, M. A., and Hay, A. J. (1984). Identification of the influenza virus transcriptase by afinity-labeling with pyridoxal 5 -phosphate. Virology 132, Ruigrok, R. W. H., and Baudin, F. (1995). Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein forms structures that are indistinguishable from the intact influenza virus ribonucleoprotein particles. J. Gen. Virol. 76, Scholtissek, C., and Becht, H. (1971). Binding of ribonucleic acids to the RNP-antigen protein of influenza viruses. J.Gen. Virol. 10, Shapiro, G. I., and Krug, R. M. (1988). Influenza virus RNA replication in vitro: Synthesis of viral template RNAs and virion RNAs in the absence of an added primer. J. Virol. 62, Shi, L., Summers, D. F., Peng, Q., and Galarza, J. M. (1995). Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mrna and functions only as the trimeric enzyme. Virology 208, Smith, G. L., Levin, J. Z., Palese, P., and Moss, B. (1987). Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses. Virology 160, Stevens, M. P., and Barclay, W. S. (1998). The N-terminal extension of the influenza B virus nucleoprotein is not required for nuclear accu-