For reprint orders, please contact: [email protected]
Future Microbiology
Review
Influenza virus nucleoprotein: structure, RNA binding, oligomerization and antiviral drug target Sylvie Chenavas1,2,3, Thibaut Crépin1,2,3, Bernard Delmas 4, Rob WH Ruigrok1,2,3 & Anny Slama-Schwok*4 Université Grenoble Alpes, Unit of Virus Host Cell Interactions, F-38000 Grenoble, France Centre National de la Recherche Scientifique, Unit of Virus Host Cell Interactions, F-38000 Grenoble, France 3 Unit for Virus Host–Cell Interactions, Université Grenoble Alpes–European Molecular Biology Laboratory–Centre National de la Recherche Scientifique, 6 rue Jules Horowitz, 38042 France 4 INRA UR 892, Virologie et Immunologie Moléculaires, Domaine de Vilvert, 78350, Jouy en Josas, France *Author for correspondence: Tel.: +33 134 652 615 n [email protected] 1 2
The nucleoprotein (NP) of influenza virus covers the viral RNA entirely and it is this NP–RNA complex that is the template for transcription and replication by the viral polymerase. Purified NP forms a dynamic equilibrium between monomers and small oligomers, but only the monomers can oligomerize onto RNA. Therefore, drugs that stabilize the monomers or that induce abnormal oligomerization may have an antiviral effect, as would drugs that interfere with RNA binding. Crystal structures have been produced for monomeric and dimeric mutants, and for trimers and tetramers; high-resolution electron microscopy structures have also been calculated for the viral NP–RNA complex. We explain how these structures and the dynamic oligomerization equilibrium of NP can be and have been used for anti-influenza drug development.
Influenza A viruses infect water birds as their natural hosts, but some influenza viruses infect humans, pigs and other mammals. Because influenza viruses have a segmented RNA genome, exchange of segments between different viruses can lead to viruses with totally new biological characteristics. In particular, when a human virus mixes with an animal virus, a new human virus may emerge with surface antigens that have not yet been seen in the human population. Such an occasional antigenic shift and the antigenic drift of gradual accumulation of mutations in the surface glycoproteins lead to the annual flu epidemics. This review will focus on the structure of one of the proteins of the virus, the nucleoprotein (NP), and its oligomerization and RNA binding activities. We will describe a number of drug target sites on this NP, some of which have already led to inhibitors of viral replication. Influenza viruses are negative-strand RNA viruses. This means that their genomic RNA, which is carried inside the virus particle, is in the opposite sense of mRNA and that transcription is the first viral activity after entry into the host cell. The viral RNA of all these viruses is covered by multiple copies of the viral NP and it is the NP–RNA (ribonucleoprotein [RNP]) complex that is the template for viral transcription and replication [1]. For influenza virus, the NP–RNA forms a loose helical polymer that folds back on 10.2217/FMB.13.128 © 2013 Future Medicine Ltd
itself, bringing the 3´ and 5´ ends of the RNA into close proximity Figure 1A & B), thus allowing the viral polymerase to bind to both ends [2–4]. The RNPs are of variable lengths depending on the length of the bound RNA [5] and are flexible (Figure 1) so that regular structural biology approaches, such as crystallography or helical reconstruction of the entire structure, have not led to structural models. Only very recently, two groups have analyzed the regular middle and end parts separately, and have proposed a model for the RNP structure [6,7]. They describe the double helical structure and how the NP protomers interact in a linear fashion (see below) on the RNA, and how they bind to each other in the opposite strands. They also imaged the trimeric RNA-dependent RNA polymerase at one end of the RNP and the NP-covered RNA loop at the opposite end. Oligomerization state of RNA-free NP
NP that is free from RNA can be isolated from viral RNPs [8] or in a recombinant form when expressed in Escherichia coli [9]. The RNA can be removed from the protein by very high salt or through a heparin column. Both the NP isolated from virus and the recombinant protein are found in equilibrium between monomers and oligomers. The equilibrium is very sensitive to the salt concentration and is strongly shifted to the monomeric form at low salt (10 mM NaCl) and Future Microbiol. (2013) 8(12), 1537–1545
Keywords n
antiviral n nucleoprotein RNP n viral RNA
n resistance n
part of
ISSN 1746-0913
1537
Review
A
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
B
C
HA
3
4
1 2
5
50 nm
Figure 1. Electron micrographs of ribonucleoprotein and purified nucleoprotein. (A) Ribonucleoprotein (RNP) treated at low salt after removal of the polymerase complex shows a single helical structure of nucleoprotein–RNA. (B) Double helical intact RNP complexes isolated from virus particles. Note the flexible nature of the RNPs and the variation in length that is due to the different lengths of the RNA in the RNPs. Encircled are two HA rosettes copurified with the RNP that attach end-on to the carbon support film. (C) Monomers and small oligomers of purified RNA-free nucleoprotein. Monomers, dimers, trimers, tetramers and pentamers are indicated by numbers. All micrographs are from protein negatively stained with 1% sodium silicotungstate on a carbon support film. All micrographs have the same magnification as indicated by the bar in panel (B). HA: Hemagglutinin. (A) reproduced from [2] .
to the trimeric form at high salt (300 mM) [10]. The protein isolated from viruses tends to form a larger variety of oligomers, going from dimers to circular forms with six or more protomers (Figure 1C) [8], whereas the recombinant protein mainly forms trimers and tetramers [9–11]. This difference may be due to post-translational modifications, which are probably absent in the recombinant
A
protein; influenza A viral NP is phosphorylated at positions S9/Y10, S165, S377/T378, S402/S403, S457 and T472/S473 [12]. All of these sites are differentially phosphorylated in protein isolated from viruses. In particular, the S165D mutation was found to lead to monomeric protein that did not oligomerize in the absence of RNA [13]. For the recombinant wild-type protein, the dissociation constant (Kd) for the conversion of NP3 to 3 NP is rather high, 8.5 × 10-3 M2, and very slow, with a half-life of 10 h [11]. The high Kd, very slow kinetics of oligomerization and possible phosphorylation of serine 165 all suggest that NP in the infected cell is probably monomeric when it binds to the replicating viral RNA. Apart from this, it is possible that the binding of NP to cellular proteins, such as importin-a, also helps to keep it in a monomeric state [14]. In vitro, trimeric NP binds to RNA, with a Kd between 2 and 14 nM [10,11], whereas monomeric NP binds with a slightly higher Kd of 41 nM [10]. Monomeric NP does oligomerize onto the RNA to form NP–RNA rings that resemble recombinant circular RNPs (Figure 2A) [10,13,15]. However, trimeric NP remains trimeric and does not evolve into structures that resemble viral RNP (Figure 2B). Therefore, it would appear that monomeric NP is the active form of the protein and the oligomers that form in the absence of RNA may be artifacts of the high concentrations used in biochemical or crystallization experiments (Figure 3).
B
5050nm nm Figure 2. Reconstituted complexes between nucleoprotein and an RNA oligonucleotide of 55 nucleotides. (A) Monomeric nucleoprotein was incubated with RNA and prepared for electron microscopy after 1 h; nucleoprotein–RNA rings had formed. (B) A mixture of trimer and tetramers was incubated with the RNA. The oligomers did bind the RNA, but did not evolve into nucleoprotein–RNA rings [10] . The electron microscopy was as described in Figure 1.
1538
Future Microbiol. (2013) 8(12)
future science group
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
A/WSN/33 R416A
A/WSN/33 ∆402–429
A/WSN/33 wt
Review
B/Hong Kong/2004 wt
Figure 3. Crystal structures of RNA-free nucleoprotein. (A–C) Structures of mutant monomers, mutant dimers and wild-type trimers of influenza A/WSN/33 and (D) wild-type tetramers of influenza B/HK/2004. The top row shows space filling structures with the protomers in different colors. The bottom row shows ribbon diagrams in gray, with the exchange domains in color. In (A) the exchange domain is bound to the surface of its own protein, in (B) the domain is absent because the protein is a deletion mutant. The Protein Data Bank numbers are 3ZDP for the structure in (A), 4IRY for (B), 2IQH for (C) and 3TJO for (D). Arrow indicates a wide surface that is strongly positively charged. wt: Wild-type. Please see color figure online at www.futuremedicine.com/doi/full/10.2217/FMB.13.128
Structure of RNA-free NP
The RNA-free NPs of influenza A viruses H1N1 A/WSN/33 and H5N1 A/HK/483/97 were crystallized as trimers (Figure 3C) [9,16]. The structure of the protein shows a wide surface (arrowed in Figure 3C) that is strongly positively charged. Mutations of residues making up this surface lower the affinity for RNA [16] and, therefore, it was proposed that this surface constitutes at least part of the RNA-binding platform of NPs. The protomers are attached through an exchange domain, highlighted in Figure 3C & D, consisting of residues 402–428, that insert into a groove on a neighboring protomer. One difference between the H1N1 and the H5N1 structure is that the exchange domains do not interact with the same neighbors [13]. An important interaction between the exchange domain and the neighboring groove is a salt bridge between R416 in the loop and E339 (H1N1 numbering) in the future science group
groove. Abolition of this bond by mutating either of the residues to alanine leads to the formation of monomeric proteins with a strongly lowered affinity for RNA [10,13,14,17–20]. Apart from this loop exchange, some surface residues also interact between protomers. Deletion of the exchange domain (D402–429) also leads to monomeric proteins, but with a fraction of the protein forming dimers [11]. The dimers could be crystallized and the dimer interface appeared to overlap with the trimer interface (Figure 3B & C) [11]. This interface is sufficient to keep two protomers in a dimer without the presence of the exchange domain. Crystallizing monomeric wild-type NP app eared to be impossible, as the protein always formed trimers under the very high protein concentrations and conditions necessary for crystallization. However, the monomeric R416A mutant could be crystallized [13]. Although it was previously supposed that the exchange domains www.futuremedicine.com
1539
Review
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
were exposed as in a protomer of the trimer structure, but unable to bind in the groove [21], it appeared that the loop was folded onto its own surface, taking up part of its own groove and extending into the RNA-binding platforms, leading to a lower affinity for RNA (Figure 3A) [13]. Biophysical characterization suggested that wildtype monomeric NPs may have the same conformation as the mutant. Therefore, although the monomer and dimer structures were obtained with mutant proteins, the series of structures of A/WSN/33 NP shown in Figure 3A–C show oligomeric forms observed in wild-type-purified NP preparations (Figure 1C) [9–11]. Even though tetrameric NP was observed in NP preparations (Figure 1B) [9,10,16], up to now, no tetrameric crystal form of influenza A virus NP has been described. However, the NP of influenza B/Hong Kong/2004 crystallized as a tetramer (Figure 3D) [22,23]. The main difference between influenza A and B NP, is a much larger N-terminal domain for B NP but this was not visible in the structure. Otherwise, the proteins are very similar, both having a similar RNA-binding surface and loop exchange domain that inserts into a groove on a neighboring protomer. The geometry of the tetramer structure is such that the major
Figure 4. Docking of nucleoprotein protomers into the electron microscopy reconstruction of the central section of intact ribonucleoprotein. (A) Three nucleoprotein protomers in color going from right to left and three protomers in gray going in the opposite direction. Note that the electron microscopy reconstruction shows the density for both the protein and the RNA, but here only the docked protein is shown. The distance between subsequent protomers is larger than in the RNA-free trimers and tetramers shown in Figure 3, and the details of the exchange domain inserting into the groove on the neighboring protomer are probably different. The surface contacts between protomers on opposite strands are not identical to the surface contacts observed in the dimer and trimer structures in Figure 3. (B) Suggestion for the path of the RNA bound in the ribonucleoprotein. The view of the colored strand is from the outside in order to better see the RNA. The Protein Data Bank code for the docking of the nucleoprotein crystal structure into the electron microscopy model is 4BBL. Data taken from [6] . Please see color figure online at www.futuremedicine.com/doi/full/10.2217/ FMB.13.128
1540
Future Microbiol. (2013) 8(12)
interactions between protomers is through the exchange domains, without the additional surface interactions observed for the dimers and trimers. Structure of RNP
As described above, the viral RNP consists of a helical protein–RNA filament. This single filament folds back on itself, forming a double helical structure with the 5´ and 3´ ends of the viral RNA in close opposition for binding of the polymerase (Figure 1B & C). The ascending strand of NP molecules is in contact with the descending strand. Recently, two groups used cryoelectron microscopy to analyze straight fragments of RNPs plus both ends of the RNP, the end that carries the polymerase and the end that forms a loop [6,7,9–11], and both groups combined the data into a reconstruction of the entire RNP. Because of the large depth of focus of the electron microscope, the hand of the helix cannot be derived from such images and Arranz et al. suggested a left-handed helix [6], whereas Moeller et al. suggested a right-handed helix [7], as had been suggested before from electron images of negatively stained RNPs [24]. Arranz et al. decided on the left hand because the fit of the crystal structure into the electron microscopy density was much better for the left- than the right-handed reconstruction [6]. These authors also performed cryoelectron tomography on virus particles, and the analysis of these tomograms only led to a proper reconstruction when the left-handed helix was used. Figure 4A shows three consecutive NP protomers of the descending strand in colors in contact with three protomers from the ascending strand in gray (reconstruction of [6]). The contacts between the protomers are somewhat similar to those of the tetramer structure in Figure 3D, although the distance between the protomers is significantly larger between protomers in the RNP reconstruction than in the trimer or tetramer [6]. Therefore, it is likely that the way the exchange domain is inserted from one protomer into its neighbor is different between the RNPs and the crystallized oligomers without RNA. Note that the additional interacting surfaces between NP protomers in the RNP are not the same as between protomers in the dimer and trimer, and that the biological significance of the interacting surface in the dimer and trimer is not clear. Finally, Figure 4B shows the proposed path for the RNA in the RNP. NP as an antiviral drug target
In this review, we have not described the inter actions of NP with host cell proteins. All the interaction surfaces between NP and host future science group
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
Review
proteins may be considered as antiviral targets (reviewed in [25]). In the following, we will discuss how the NP structures may be exploited for the design of molecules inhibiting NP function (reviewed in [26]). We will refer to Figure 5, which shows different areas on the surface of NP that have been used for targeting inhibitors (Figure 5A), and that shows how nucleozin ‘cross-links’ two protomers (Figure 5B). Annotations are listed in Table 1. Several approaches are listed below. Inducing the formation of higher-order oligomers to prevent nuclear transport of NP or its regular oligomerization onto RNA
High-throughput studies monitoring nuclear traffic of NP discovered that nucleozin forms higher-order oligomers that inhibited transport [27–31]. Nucleozin binds to a small hydrophobic cavity on the back of the body (Figure 5A, yellow sites). Initial modeling studies suggested that nucleozin forms a hydrogen bond with N309 and stacks onto Y289 [29]. Compound 3b derived from nucleozin was found at a NP–NP dimer interface (Figure 5B & Table 2). Each NP molecule binds two separate halves of compound 3b. Key contacts included a stacking p–p interaction between the aromatic chloronitrophenyl moiety of compound 3b and Y289, and hydrophobic interactions between the piperazine moiety and Y52, and with its own isoxazole-aryl moiety by intramolecular folding; an additional hydrogen bond is formed between the amide carbonyl of compound 3b to the S376 side chain (not shown) (F igure 5B) [27,28]. Resistance to nucleozin and some of its derivatives was reported to involve Y289H and Y52H, respectively. Interestingly, residues located in the same region as the Y52 and Y289/ N309 pockets of the nucleozin derivatives were recently shown to confer resistance to the antiviral effect of the cellular MxA proteins [32]. As mentioned above, only monomeric NP forms regular NP–RNA structures and any substance that shifts the monomer–oligomer equilibrium towards the oligomeric form of NP or that, on the contrary, stabilizes the monomeric form, will interfere with the formation of the viral RNP. Inhibiting NP oligomerization by interfering with the insertion of the exchange domain into the groove on the neighboring protomer
The exchange domain-binding groove of the NP may be a viable target for small molecules that mimic the key structural features of the exchange domain of NP (Figure 5A, blue) and therefore may interfere with NP oligomerization [33]. The proof of future science group
Figure 5. Localization of antiviral-binding sites on nucleoproteins mapped onto the trimeric nucleoprotein structure (2IQH) deduced from x-ray crystallography or molecular dynamics simulation supported by experimental data. (A) Depicts the binding sites of the antiviral compounds detailed in Tables 1 & 2 with the inset corresponding to the potential interaction site between naproxen and nucleoprotein. (B) Explains how the nucleozin at the interface of two monomers induces the formation of higher-order nucleoprotein oligomers. Protein Data Bank code for the structure of nucleoprotein plus nucleozin is 3TG6 (Table 1) . Data taken from [9,28] . Please see color figure online at www.futuremedicine.com/doi/full/10.2217/ FMB.13.128
principle was first given by expressing an exchangedomain peptide (residues 402–428) fused to the EGFP in HEK293T cells; evidence for binding to FLAG-tagged NP was given by immuno precipitation assays [20]. Binding of this peptide inhibited NP oligomerization in the presence of RNA shown by analytical ultracentrifugation and resulted in antiviral effects in the mM concentration range. Virtual screening selected compounds that had the ability to inhibit NP oligomerization with antiviral effects in the low µM range. The best candidate was docked in a hydrophobic pocket, formed by F304, W330, A336, I347 and A387, which is originally occupied by F412 from the exchange domain [20]. The aromatic ring of the inhibitor formed p–p interactions with F304 and W330, and a cation–p interaction with R389. The nitrogen atoms of the thiazole–carboxamide of compound 3b (shown in Table 2) mimicked the exchange domain R416 and interacted with the carboxylate of E339. In addition, the morpholino and propyl moieties of compound 3b could be docked into the pocket that was occupied by I408 and P419 of the exchange domain. Inhibiting RNA binding by targeting the RNA binding groove of NP
One of the main roles of NP is to bind the viral genome. Compounds able to inhibit this www.futuremedicine.com
1541
Review
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
Table 1. Annotations for Figure 5. Color
NP residues involved in Antiviral/target antiviral binding
Critical residues†
Ref.
Blue
402–428
Peptides, small molecules
A337, E339, Q405, F412, R416
[20]
Yellow
2 sites: Y52, Y313 and Y289, N309
Nucleozin derivatives
–
Purple
Y148, R361, R355, F489
Naproxen
Y148, R150, R152, R361, F489
[27–30] [35]
Data taken from [34]. NP: Nucleoprotein. †
process are expected to exhibit antiviral properties. By virtual screening of small molecules binding to NP, we identified within the putative RNA-binding groove a small hydrophobic
site defined by two aromatic residues, Y148 and F489, with flanking basic residues: R361, R355 on one side and R150 and R152 on the other side (Figure 5A , purple). All these residues
Table 2. Comparison of the efficacy and toxicity of antiviral compounds directed against nucleoprotein. Color Blue
Chemical structure H N
Cl
O
N S
Cl
CC50 (µM)
IC50
Ref.
35.5
2.7 µM (WSN)
>250
69 nM (WSN) 160 nM (H3N2) 330 nM (H5N1)
>100
0.68 µM (WSN) 0.52 µM (H1N1 2009) 2 µM H3N2 0.69 µM (H5N1)
1605 ± 328
16 ± 5 µM (WSN) 12 ± 5 µM (H3N2)
[20]
N
N H
O
Compound 3 Yellow
Cl O NO2
N
N
[27–30]
R
R =
X
N O
Nucleozin, X = H R1 R5 R6
R4 O
A N N
R7
N
N
(
B
R2
) n
R3 N
Compound 3b , n = 1, R1 = Cl, R2 = NO2, R3 = OCH3, R4, R5, R6, R7 = H Purple
O
O
Na+
CH3
O CH3
Naproxen CC50: 50% cytotoxic concentration.
1542
Future Microbiol. (2013) 8(12)
future science group
[35]
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
present in the RNA-binding groove are critical, since engineered substitutions at these positions do not allow virus rescue [34]. R150 was shown to be essential to the interaction with the PB2 subunit of the influenza virus RNA polymerase [22,23]. Naproxen was identified by virtual screening from a chemical library. Naproxen, a known anti-inflammatory drug, interacted with NP by p–p stacking of its naphatalene core onto Y148, hydrophobic interactions between the methyl of its methoxy group with Q149 and a strong salt bridge between its carboxylate group and R361 (Figure 5A , purple area and inset) [35]. A combination of biophysical experiments with wild-type NP and selected mutants (Y148A, R152A, R355A and R361A) confirmed the binding of naproxen at this site, as anticipated from molecular modeling studies. The naproxen binding site is located close to S165 and, interestingly, naproxen introduces negative charges mimicking phosphorylation of S165 that may interfere with NP polymerization.
Review
Naproxen protected MDCK cells against a viral challenge with H1N1 and H3N2 influenza virus, and had antiviral effects in a mouse model of viral infection. The efficacy and toxicity of the antiviral compounds directed to NP are briefly summarized in Table 2 . The nucleozin derivatives are the most potent antivirals against the WSN strain, IC50 = 70 nM. Due to the high sequence conservation of NP among viral strains, it would be expected that these compounds inhibit influenza A viruses H1N1, H3N2 and H5N1. In the case of naproxen, whose binding site is only composed of critical residues, similar IC50 values for H1N1 and H3N2 were observed, and no escape mutants could be produced. Similarly, drugs that target the exchange domain produced minimal resistance [30]. In conclusion, the structural and functional work on NP and RNP has defined three functional structures for the protein; a monomer, a trimer/tetramer and NP bound to RNA in the
Executive summary Nucleoprotein of influenza virus Nucleoprotein (NP) of influenza virus entirely covers the viral RNA and this NP–RNA complex is the template for transcription and replication. Oligomerization state of RNA-free NP Isolated NP free from RNA is in a dynamic equilibrium between monomers and trimers/tetramers. The equilibrium is slow and depends on protein and salt concentration. Only monomeric NP can oligomerize onto RNA. Structure of RNA-free NP In the crystallized trimeric form of NP, the exchange domain of one protomer reaches over to a groove on the surface of a neighboring protomer in order to oligomerize. In the monomeric form, the same domain wraps around its own monomer in an autoinhibited state, with loops reaching into the RNA-binding platform. Structure of ribonucleoprotein In the viral ribonucleoprotein, the NP protomers are further apart than the protomers in the RNA-free trimer or tetramer structure. This suggests that the exchange domain is probably not inserted in the neighboring protomer in the same manner as in the RNA-free trimer/tetramer structures. The double helical structure reveals a contact surface that is absent in the trimer/tetramer structures. NP as antiviral drug target; the various NP & RNP structures suggest the following ways of inhibiting NP function & oligomerization Inducing the formation of higher-order oligomers to prevent nuclear transport of NP or its regular oligomerization onto RNA. Inhibiting NP oligomerization by interfering with the insertion of the exchange domain into the groove on the neighboring protomer or by stabilizing the monomeric form. Inhibiting RNA binding by targeting the RNA binding site of NP. Conclusion The structural and functional work on NP and RNP has defined three functional structures for the protein; a monomer, a trimer/tetramer and NP bound to RNA in the RNP. Interfering with the monomer–oligomer equilibrium by inducing natural or non-natural oligomerization, or by preventing oligomerization interferes with the normal NP function. Targeting the RNA binding site that contains a significant number of conserved residues also seems a promising avenue for the development of anti-influenza virus drugs.
future science group
www.futuremedicine.com
1543
Review
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
RNP. Interfering with the monomer–oligomer equilibrium by inducing natural or non-natural oligomerization, or by preventing oligomerization interferes with the normal NP function. Targeting the RNA-binding site that contains a significant number of conserved residues also seems a promising avenue for the development of anti-influenza virus drugs. Future perspective
The influenza virus NP has several conformations and activities, such as RNA binding and self-oligomerization, which can all be inhibited. It is likely that this protein, which is relatively well conserved between all influenza A viruses and has no cellular equivalent, will be the target of commercial drugs to come in the next 10 years.
1.
Ruigrok RWH, Crepin T, Kolakofsky D. Nucleoproteins and nucleocapsids of negativestrand RNA viruses. Curr. Opin. Microbiol. 14(4), 504–510 (2011).
2.
Klumpp K, Ruigrok RW, Baudin F. Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO J. 16(6), 1248–1257 (1997).
3.
4.
5.
6.
nn
7.
Fodor E, Pritlove DC, Brownlee GG. The influenza virus panhandle is involved in the initiation of transcription. J. Virol. 68(6), 4092–4096 (1994). Hagen M, Chung TD, Butcher JA, Krystal M. Recombinant influenza virus polymerase: requirement of both 5´ and 3´ viral ends for endonuclease activity. J. Virol. 68(3), 1509–1515 (1994). Compans RW, Content J, Duesberg PH. Structure of the ribonucleoprotein of influenza virus. J. Virol. 10(4), 795–800 (1972). Arranz R, Coloma R, Chichon FJ et al. The structure of native influenza virion ribonucleoproteins. Science 338(6114), 1634–1637 (2012). Electron microscopy model of intact viral ribonucleoprotein (RNP) showing the contacts between nucleoprotein (NP) protomers along the RNA and between ascending and descending strands, as well as a tomogram of RNP inside the virus. Moeller A, Kirchdoerfer RN, Potter CS, Carragher B, Wilson IA. Organization of the
1544
This work was supported by the French agency for res earch (A N R ; Fl unu cl e ovir; A N R-2 010 Blanc-1307-1301 to A Slama-Schwok and RWH Ruigrok), MEDICEN and Lyonbiopole, the European Commission under grant agreement no. 259751 (Flupharm: New drugs targeting influenza virus polymerase, 11/2010-2005/2014 to RWH Ruigrok) and by a grant of the Programme de Recherche Influenza A (H1N1), coordinated by the Institut de Microbiologie et Maladies Infectieuses of the Institut National de la Santé et de la Recherche Médicale, INSERM, France (to B Delmas). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
influenza virus replication machinery. Science 338(6114), 1631–1634 (2012).
References Papers of special note have been highlighted as: n of interest nn of considerable interest
Financial & competing interests disclosure
nn
8.
9.
Electron microscopy model of recombinant and replicating RNP. Ruigrok RW, Baudin F. 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(Pt 4), 1009–1014 (1995). Ye Q, Krug RM, Tao YJ. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444(7122), 1078–1082 (2006).
10. Tarus B, Bakowiez O, Chenavas S et al.
Oligomerization paths of the nucleoprotein of influenza A virus. Biochimie 94(3), 776–785 (2012). n
Biochemistry paper describing the monomer–oligomer equilibrium of isolated NP and the polymerization of the monomeric form onto RNA.
11. Ye Q, Guu TS, Mata DA et al. Biochemical
and structural evidence in support of a coherent model for the formation of the double-helical influenza A virus ribonucleoprotein. MBio 4(1), e00467-12 (2012). n
Describes the kinetics of RNA-free NP polymerization, as well as the dimer structure of the deletion mutant of NP missing the exchange domain.
12. Hutchinson EC, Denham EM, Thomas B
et al. Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry. PLoS Pathog. 8(11), e1002993 (2012).
Future Microbiol. (2013) 8(12)
13. Chenavas S, Estrozi LF, Slama-Schwok A
et al. Monomeric nucleoprotein of influenza A virus. PLoS Pathog. 9(3), e1003275 (2013). n
Describes the structure of the R416A monomeric mutant and the role of phosphorylation of serine 165 in keeping NP monomeric.
14. Boulo S, Akarsu H, Lotteau V, Muller CW,
Ruigrok RWH, Baudin F. Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein. Virology 409(1), 84–90 (2011). 15. Coloma R, Valpuesta JM, Arranz R,
Carrascosa JL, Ortin J, Martin-Benito J. The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog. 5(6), e1000491 (2009). 16. Ng AK-L, Zhang H, Tan K et al. Structure
of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. FASEB J. 22(10), 3638–3647 (2008). 17. Chan W-H, Ng AK-L, Robb NC et al.
Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities. J. Virol. 84(14), 7337–7345 (2010). 18. Elton D, Medcalf E, Bishop K, Digard P.
Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements. Virology 260(1), 190–200 (1999). 19. Elton D, Medcalf L, Bishop K, Harrison D,
Digard P. Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding. J. Virol. 73(9), 7357–7367 (1999).
future science group
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
20. Shen Y-F, Chen Y-H, Chu S-Y et al.
E339...R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors. Proc. Natl. Acad. Sci. USA 108(40), 16515–16520 (2011).
antiviral agents. Trends Pharmacol. Sci. 33(2), 89–99 (2012). 26. Cianci C, Gerritz SW, Deminie C, Krystal
Vreede FT. The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes. Nat. Commun. 4, 1591 (2013).
27. Cheng H, Wan J, Lin M-I et al. Design,
synthesis, and in vitro biological evaluation of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents targeting virus nucleoprotein. J. Med. Chem. 55(5), 2144–2153 (2012).
22. Ng AK-L, Chan W-H, Choi S-T et al.
Influenza polymerase activity correlates with the strength of interaction between nucleoprotein and PB2 through the hostspecific residue K/E627. PLoS ONE 7(5), e36415 (2012).
24. Jennings PA, Finch JT, Winter G, Robertson
JS. Does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral RNA? Cell 34(2), 619–627 (1983). 25. Muller KH, Kakkola L, Nagaraj AS,
Cheltsov AV, Anastasina M, Kainov DE. Emerging cellular targets for influenza
future science group
31. Amorim MJ, Kao RY, Digard P. Nucleozin
targets cytoplasmic trafficking of viral ribonucleoprotein–Rab11 complexes in influenza A virus infection. J. Virol. 87(8), 4694–4703 (2013). 32. Zimmermann P, Manz B, Haller O,
Schwemmle M, Kochs G. The viral nucleoprotein determines Mx sensitivity of influenza A viruses. J. Virol. 85(16), 8133–8140 (2011). 33. Das K, Aramini JM, Ma L-C, Krug RM,
28. Gerritz SW, Cianci C, Kim S et al. Inhibition
23. Ng AK-L, Lam MK-H, Zhang H et al.
Structural basis for RNA binding and homo-oligomer formation by influenza B virus nucleoprotein. J. Virol. 86(12), 6758–6767 (2012).
polymerase activity. Proc. Natl. Acad. Sci. USA 107(45), 19151–19156 (2010).
M. Influenza nucleoprotein: promising target for antiviral chemotherapy. Antiviral Chem. Chemother. doi:10.3851/IMP2235 (2012) (Epub ahead of print).
21. Turrell L, Lyall JW, Tiley LS, Fodor E,
nn
Review
Arnold E. Structures of influenza A proteins and insights into antiviral drug targets. Nat. Struct. Mol. Biol. 17(5), 530–538 (2010).
of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers. Proc. Natl. Acad. Sci. USA 108(37), 15366–15371 (2011).
34. Li Z, Watanabe T, Hatta M et al. Mutational
Describes the function of nucleozin and shows the structure of NP trimers ‘cross-linked’ by the drug.
35. Lejal N, Tarus B, Bouguyon E et al.
analysis of conserved amino acids in the influenza A virus nucleoprotein. J. Virol. 83(9), 4153–4162 (2009). Structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza A virus. Antimicrob. Agents Chemother. 57(5), 2231–2242 (2013).
29. Kao RY, Yang D, Lau L-S et al. Identification
of influenza A nucleoprotein as an antiviral target. Nat. Biotechnol. 28(6), 600–605 (2010). 30. Su C-Y, Cheng T-JR, Lin M-I et al. High-
throughput identification of compounds targeting influenza RNA-dependent RNA
www.futuremedicine.com
n
Describes the binding site of naproxen to the RNA-binding platform of NP and its antiviral effect.
1545
Future Microbiology
Review
Influenza virus nucleoprotein: structure, RNA binding, oligomerization and antiviral drug target Sylvie Chenavas1,2,3, Thibaut Crépin1,2,3, Bernard Delmas 4, Rob WH Ruigrok1,2,3 & Anny Slama-Schwok*4 Université Grenoble Alpes, Unit of Virus Host Cell Interactions, F-38000 Grenoble, France Centre National de la Recherche Scientifique, Unit of Virus Host Cell Interactions, F-38000 Grenoble, France 3 Unit for Virus Host–Cell Interactions, Université Grenoble Alpes–European Molecular Biology Laboratory–Centre National de la Recherche Scientifique, 6 rue Jules Horowitz, 38042 France 4 INRA UR 892, Virologie et Immunologie Moléculaires, Domaine de Vilvert, 78350, Jouy en Josas, France *Author for correspondence: Tel.: +33 134 652 615 n [email protected] 1 2
The nucleoprotein (NP) of influenza virus covers the viral RNA entirely and it is this NP–RNA complex that is the template for transcription and replication by the viral polymerase. Purified NP forms a dynamic equilibrium between monomers and small oligomers, but only the monomers can oligomerize onto RNA. Therefore, drugs that stabilize the monomers or that induce abnormal oligomerization may have an antiviral effect, as would drugs that interfere with RNA binding. Crystal structures have been produced for monomeric and dimeric mutants, and for trimers and tetramers; high-resolution electron microscopy structures have also been calculated for the viral NP–RNA complex. We explain how these structures and the dynamic oligomerization equilibrium of NP can be and have been used for anti-influenza drug development.
Influenza A viruses infect water birds as their natural hosts, but some influenza viruses infect humans, pigs and other mammals. Because influenza viruses have a segmented RNA genome, exchange of segments between different viruses can lead to viruses with totally new biological characteristics. In particular, when a human virus mixes with an animal virus, a new human virus may emerge with surface antigens that have not yet been seen in the human population. Such an occasional antigenic shift and the antigenic drift of gradual accumulation of mutations in the surface glycoproteins lead to the annual flu epidemics. This review will focus on the structure of one of the proteins of the virus, the nucleoprotein (NP), and its oligomerization and RNA binding activities. We will describe a number of drug target sites on this NP, some of which have already led to inhibitors of viral replication. Influenza viruses are negative-strand RNA viruses. This means that their genomic RNA, which is carried inside the virus particle, is in the opposite sense of mRNA and that transcription is the first viral activity after entry into the host cell. The viral RNA of all these viruses is covered by multiple copies of the viral NP and it is the NP–RNA (ribonucleoprotein [RNP]) complex that is the template for viral transcription and replication [1]. For influenza virus, the NP–RNA forms a loose helical polymer that folds back on 10.2217/FMB.13.128 © 2013 Future Medicine Ltd
itself, bringing the 3´ and 5´ ends of the RNA into close proximity Figure 1A & B), thus allowing the viral polymerase to bind to both ends [2–4]. The RNPs are of variable lengths depending on the length of the bound RNA [5] and are flexible (Figure 1) so that regular structural biology approaches, such as crystallography or helical reconstruction of the entire structure, have not led to structural models. Only very recently, two groups have analyzed the regular middle and end parts separately, and have proposed a model for the RNP structure [6,7]. They describe the double helical structure and how the NP protomers interact in a linear fashion (see below) on the RNA, and how they bind to each other in the opposite strands. They also imaged the trimeric RNA-dependent RNA polymerase at one end of the RNP and the NP-covered RNA loop at the opposite end. Oligomerization state of RNA-free NP
NP that is free from RNA can be isolated from viral RNPs [8] or in a recombinant form when expressed in Escherichia coli [9]. The RNA can be removed from the protein by very high salt or through a heparin column. Both the NP isolated from virus and the recombinant protein are found in equilibrium between monomers and oligomers. The equilibrium is very sensitive to the salt concentration and is strongly shifted to the monomeric form at low salt (10 mM NaCl) and Future Microbiol. (2013) 8(12), 1537–1545
Keywords n
antiviral n nucleoprotein RNP n viral RNA
n resistance n
part of
ISSN 1746-0913
1537
Review
A
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
B
C
HA
3
4
1 2
5
50 nm
Figure 1. Electron micrographs of ribonucleoprotein and purified nucleoprotein. (A) Ribonucleoprotein (RNP) treated at low salt after removal of the polymerase complex shows a single helical structure of nucleoprotein–RNA. (B) Double helical intact RNP complexes isolated from virus particles. Note the flexible nature of the RNPs and the variation in length that is due to the different lengths of the RNA in the RNPs. Encircled are two HA rosettes copurified with the RNP that attach end-on to the carbon support film. (C) Monomers and small oligomers of purified RNA-free nucleoprotein. Monomers, dimers, trimers, tetramers and pentamers are indicated by numbers. All micrographs are from protein negatively stained with 1% sodium silicotungstate on a carbon support film. All micrographs have the same magnification as indicated by the bar in panel (B). HA: Hemagglutinin. (A) reproduced from [2] .
to the trimeric form at high salt (300 mM) [10]. The protein isolated from viruses tends to form a larger variety of oligomers, going from dimers to circular forms with six or more protomers (Figure 1C) [8], whereas the recombinant protein mainly forms trimers and tetramers [9–11]. This difference may be due to post-translational modifications, which are probably absent in the recombinant
A
protein; influenza A viral NP is phosphorylated at positions S9/Y10, S165, S377/T378, S402/S403, S457 and T472/S473 [12]. All of these sites are differentially phosphorylated in protein isolated from viruses. In particular, the S165D mutation was found to lead to monomeric protein that did not oligomerize in the absence of RNA [13]. For the recombinant wild-type protein, the dissociation constant (Kd) for the conversion of NP3 to 3 NP is rather high, 8.5 × 10-3 M2, and very slow, with a half-life of 10 h [11]. The high Kd, very slow kinetics of oligomerization and possible phosphorylation of serine 165 all suggest that NP in the infected cell is probably monomeric when it binds to the replicating viral RNA. Apart from this, it is possible that the binding of NP to cellular proteins, such as importin-a, also helps to keep it in a monomeric state [14]. In vitro, trimeric NP binds to RNA, with a Kd between 2 and 14 nM [10,11], whereas monomeric NP binds with a slightly higher Kd of 41 nM [10]. Monomeric NP does oligomerize onto the RNA to form NP–RNA rings that resemble recombinant circular RNPs (Figure 2A) [10,13,15]. However, trimeric NP remains trimeric and does not evolve into structures that resemble viral RNP (Figure 2B). Therefore, it would appear that monomeric NP is the active form of the protein and the oligomers that form in the absence of RNA may be artifacts of the high concentrations used in biochemical or crystallization experiments (Figure 3).
B
5050nm nm Figure 2. Reconstituted complexes between nucleoprotein and an RNA oligonucleotide of 55 nucleotides. (A) Monomeric nucleoprotein was incubated with RNA and prepared for electron microscopy after 1 h; nucleoprotein–RNA rings had formed. (B) A mixture of trimer and tetramers was incubated with the RNA. The oligomers did bind the RNA, but did not evolve into nucleoprotein–RNA rings [10] . The electron microscopy was as described in Figure 1.
1538
Future Microbiol. (2013) 8(12)
future science group
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
A/WSN/33 R416A
A/WSN/33 ∆402–429
A/WSN/33 wt
Review
B/Hong Kong/2004 wt
Figure 3. Crystal structures of RNA-free nucleoprotein. (A–C) Structures of mutant monomers, mutant dimers and wild-type trimers of influenza A/WSN/33 and (D) wild-type tetramers of influenza B/HK/2004. The top row shows space filling structures with the protomers in different colors. The bottom row shows ribbon diagrams in gray, with the exchange domains in color. In (A) the exchange domain is bound to the surface of its own protein, in (B) the domain is absent because the protein is a deletion mutant. The Protein Data Bank numbers are 3ZDP for the structure in (A), 4IRY for (B), 2IQH for (C) and 3TJO for (D). Arrow indicates a wide surface that is strongly positively charged. wt: Wild-type. Please see color figure online at www.futuremedicine.com/doi/full/10.2217/FMB.13.128
Structure of RNA-free NP
The RNA-free NPs of influenza A viruses H1N1 A/WSN/33 and H5N1 A/HK/483/97 were crystallized as trimers (Figure 3C) [9,16]. The structure of the protein shows a wide surface (arrowed in Figure 3C) that is strongly positively charged. Mutations of residues making up this surface lower the affinity for RNA [16] and, therefore, it was proposed that this surface constitutes at least part of the RNA-binding platform of NPs. The protomers are attached through an exchange domain, highlighted in Figure 3C & D, consisting of residues 402–428, that insert into a groove on a neighboring protomer. One difference between the H1N1 and the H5N1 structure is that the exchange domains do not interact with the same neighbors [13]. An important interaction between the exchange domain and the neighboring groove is a salt bridge between R416 in the loop and E339 (H1N1 numbering) in the future science group
groove. Abolition of this bond by mutating either of the residues to alanine leads to the formation of monomeric proteins with a strongly lowered affinity for RNA [10,13,14,17–20]. Apart from this loop exchange, some surface residues also interact between protomers. Deletion of the exchange domain (D402–429) also leads to monomeric proteins, but with a fraction of the protein forming dimers [11]. The dimers could be crystallized and the dimer interface appeared to overlap with the trimer interface (Figure 3B & C) [11]. This interface is sufficient to keep two protomers in a dimer without the presence of the exchange domain. Crystallizing monomeric wild-type NP app eared to be impossible, as the protein always formed trimers under the very high protein concentrations and conditions necessary for crystallization. However, the monomeric R416A mutant could be crystallized [13]. Although it was previously supposed that the exchange domains www.futuremedicine.com
1539
Review
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
were exposed as in a protomer of the trimer structure, but unable to bind in the groove [21], it appeared that the loop was folded onto its own surface, taking up part of its own groove and extending into the RNA-binding platforms, leading to a lower affinity for RNA (Figure 3A) [13]. Biophysical characterization suggested that wildtype monomeric NPs may have the same conformation as the mutant. Therefore, although the monomer and dimer structures were obtained with mutant proteins, the series of structures of A/WSN/33 NP shown in Figure 3A–C show oligomeric forms observed in wild-type-purified NP preparations (Figure 1C) [9–11]. Even though tetrameric NP was observed in NP preparations (Figure 1B) [9,10,16], up to now, no tetrameric crystal form of influenza A virus NP has been described. However, the NP of influenza B/Hong Kong/2004 crystallized as a tetramer (Figure 3D) [22,23]. The main difference between influenza A and B NP, is a much larger N-terminal domain for B NP but this was not visible in the structure. Otherwise, the proteins are very similar, both having a similar RNA-binding surface and loop exchange domain that inserts into a groove on a neighboring protomer. The geometry of the tetramer structure is such that the major
Figure 4. Docking of nucleoprotein protomers into the electron microscopy reconstruction of the central section of intact ribonucleoprotein. (A) Three nucleoprotein protomers in color going from right to left and three protomers in gray going in the opposite direction. Note that the electron microscopy reconstruction shows the density for both the protein and the RNA, but here only the docked protein is shown. The distance between subsequent protomers is larger than in the RNA-free trimers and tetramers shown in Figure 3, and the details of the exchange domain inserting into the groove on the neighboring protomer are probably different. The surface contacts between protomers on opposite strands are not identical to the surface contacts observed in the dimer and trimer structures in Figure 3. (B) Suggestion for the path of the RNA bound in the ribonucleoprotein. The view of the colored strand is from the outside in order to better see the RNA. The Protein Data Bank code for the docking of the nucleoprotein crystal structure into the electron microscopy model is 4BBL. Data taken from [6] . Please see color figure online at www.futuremedicine.com/doi/full/10.2217/ FMB.13.128
1540
Future Microbiol. (2013) 8(12)
interactions between protomers is through the exchange domains, without the additional surface interactions observed for the dimers and trimers. Structure of RNP
As described above, the viral RNP consists of a helical protein–RNA filament. This single filament folds back on itself, forming a double helical structure with the 5´ and 3´ ends of the viral RNA in close opposition for binding of the polymerase (Figure 1B & C). The ascending strand of NP molecules is in contact with the descending strand. Recently, two groups used cryoelectron microscopy to analyze straight fragments of RNPs plus both ends of the RNP, the end that carries the polymerase and the end that forms a loop [6,7,9–11], and both groups combined the data into a reconstruction of the entire RNP. Because of the large depth of focus of the electron microscope, the hand of the helix cannot be derived from such images and Arranz et al. suggested a left-handed helix [6], whereas Moeller et al. suggested a right-handed helix [7], as had been suggested before from electron images of negatively stained RNPs [24]. Arranz et al. decided on the left hand because the fit of the crystal structure into the electron microscopy density was much better for the left- than the right-handed reconstruction [6]. These authors also performed cryoelectron tomography on virus particles, and the analysis of these tomograms only led to a proper reconstruction when the left-handed helix was used. Figure 4A shows three consecutive NP protomers of the descending strand in colors in contact with three protomers from the ascending strand in gray (reconstruction of [6]). The contacts between the protomers are somewhat similar to those of the tetramer structure in Figure 3D, although the distance between the protomers is significantly larger between protomers in the RNP reconstruction than in the trimer or tetramer [6]. Therefore, it is likely that the way the exchange domain is inserted from one protomer into its neighbor is different between the RNPs and the crystallized oligomers without RNA. Note that the additional interacting surfaces between NP protomers in the RNP are not the same as between protomers in the dimer and trimer, and that the biological significance of the interacting surface in the dimer and trimer is not clear. Finally, Figure 4B shows the proposed path for the RNA in the RNP. NP as an antiviral drug target
In this review, we have not described the inter actions of NP with host cell proteins. All the interaction surfaces between NP and host future science group
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
Review
proteins may be considered as antiviral targets (reviewed in [25]). In the following, we will discuss how the NP structures may be exploited for the design of molecules inhibiting NP function (reviewed in [26]). We will refer to Figure 5, which shows different areas on the surface of NP that have been used for targeting inhibitors (Figure 5A), and that shows how nucleozin ‘cross-links’ two protomers (Figure 5B). Annotations are listed in Table 1. Several approaches are listed below. Inducing the formation of higher-order oligomers to prevent nuclear transport of NP or its regular oligomerization onto RNA
High-throughput studies monitoring nuclear traffic of NP discovered that nucleozin forms higher-order oligomers that inhibited transport [27–31]. Nucleozin binds to a small hydrophobic cavity on the back of the body (Figure 5A, yellow sites). Initial modeling studies suggested that nucleozin forms a hydrogen bond with N309 and stacks onto Y289 [29]. Compound 3b derived from nucleozin was found at a NP–NP dimer interface (Figure 5B & Table 2). Each NP molecule binds two separate halves of compound 3b. Key contacts included a stacking p–p interaction between the aromatic chloronitrophenyl moiety of compound 3b and Y289, and hydrophobic interactions between the piperazine moiety and Y52, and with its own isoxazole-aryl moiety by intramolecular folding; an additional hydrogen bond is formed between the amide carbonyl of compound 3b to the S376 side chain (not shown) (F igure 5B) [27,28]. Resistance to nucleozin and some of its derivatives was reported to involve Y289H and Y52H, respectively. Interestingly, residues located in the same region as the Y52 and Y289/ N309 pockets of the nucleozin derivatives were recently shown to confer resistance to the antiviral effect of the cellular MxA proteins [32]. As mentioned above, only monomeric NP forms regular NP–RNA structures and any substance that shifts the monomer–oligomer equilibrium towards the oligomeric form of NP or that, on the contrary, stabilizes the monomeric form, will interfere with the formation of the viral RNP. Inhibiting NP oligomerization by interfering with the insertion of the exchange domain into the groove on the neighboring protomer
The exchange domain-binding groove of the NP may be a viable target for small molecules that mimic the key structural features of the exchange domain of NP (Figure 5A, blue) and therefore may interfere with NP oligomerization [33]. The proof of future science group
Figure 5. Localization of antiviral-binding sites on nucleoproteins mapped onto the trimeric nucleoprotein structure (2IQH) deduced from x-ray crystallography or molecular dynamics simulation supported by experimental data. (A) Depicts the binding sites of the antiviral compounds detailed in Tables 1 & 2 with the inset corresponding to the potential interaction site between naproxen and nucleoprotein. (B) Explains how the nucleozin at the interface of two monomers induces the formation of higher-order nucleoprotein oligomers. Protein Data Bank code for the structure of nucleoprotein plus nucleozin is 3TG6 (Table 1) . Data taken from [9,28] . Please see color figure online at www.futuremedicine.com/doi/full/10.2217/ FMB.13.128
principle was first given by expressing an exchangedomain peptide (residues 402–428) fused to the EGFP in HEK293T cells; evidence for binding to FLAG-tagged NP was given by immuno precipitation assays [20]. Binding of this peptide inhibited NP oligomerization in the presence of RNA shown by analytical ultracentrifugation and resulted in antiviral effects in the mM concentration range. Virtual screening selected compounds that had the ability to inhibit NP oligomerization with antiviral effects in the low µM range. The best candidate was docked in a hydrophobic pocket, formed by F304, W330, A336, I347 and A387, which is originally occupied by F412 from the exchange domain [20]. The aromatic ring of the inhibitor formed p–p interactions with F304 and W330, and a cation–p interaction with R389. The nitrogen atoms of the thiazole–carboxamide of compound 3b (shown in Table 2) mimicked the exchange domain R416 and interacted with the carboxylate of E339. In addition, the morpholino and propyl moieties of compound 3b could be docked into the pocket that was occupied by I408 and P419 of the exchange domain. Inhibiting RNA binding by targeting the RNA binding groove of NP
One of the main roles of NP is to bind the viral genome. Compounds able to inhibit this www.futuremedicine.com
1541
Review
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
Table 1. Annotations for Figure 5. Color
NP residues involved in Antiviral/target antiviral binding
Critical residues†
Ref.
Blue
402–428
Peptides, small molecules
A337, E339, Q405, F412, R416
[20]
Yellow
2 sites: Y52, Y313 and Y289, N309
Nucleozin derivatives
–
Purple
Y148, R361, R355, F489
Naproxen
Y148, R150, R152, R361, F489
[27–30] [35]
Data taken from [34]. NP: Nucleoprotein. †
process are expected to exhibit antiviral properties. By virtual screening of small molecules binding to NP, we identified within the putative RNA-binding groove a small hydrophobic
site defined by two aromatic residues, Y148 and F489, with flanking basic residues: R361, R355 on one side and R150 and R152 on the other side (Figure 5A , purple). All these residues
Table 2. Comparison of the efficacy and toxicity of antiviral compounds directed against nucleoprotein. Color Blue
Chemical structure H N
Cl
O
N S
Cl
CC50 (µM)
IC50
Ref.
35.5
2.7 µM (WSN)
>250
69 nM (WSN) 160 nM (H3N2) 330 nM (H5N1)
>100
0.68 µM (WSN) 0.52 µM (H1N1 2009) 2 µM H3N2 0.69 µM (H5N1)
1605 ± 328
16 ± 5 µM (WSN) 12 ± 5 µM (H3N2)
[20]
N
N H
O
Compound 3 Yellow
Cl O NO2
N
N
[27–30]
R
R =
X
N O
Nucleozin, X = H R1 R5 R6
R4 O
A N N
R7
N
N
(
B
R2
) n
R3 N
Compound 3b , n = 1, R1 = Cl, R2 = NO2, R3 = OCH3, R4, R5, R6, R7 = H Purple
O
O
Na+
CH3
O CH3
Naproxen CC50: 50% cytotoxic concentration.
1542
Future Microbiol. (2013) 8(12)
future science group
[35]
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
present in the RNA-binding groove are critical, since engineered substitutions at these positions do not allow virus rescue [34]. R150 was shown to be essential to the interaction with the PB2 subunit of the influenza virus RNA polymerase [22,23]. Naproxen was identified by virtual screening from a chemical library. Naproxen, a known anti-inflammatory drug, interacted with NP by p–p stacking of its naphatalene core onto Y148, hydrophobic interactions between the methyl of its methoxy group with Q149 and a strong salt bridge between its carboxylate group and R361 (Figure 5A , purple area and inset) [35]. A combination of biophysical experiments with wild-type NP and selected mutants (Y148A, R152A, R355A and R361A) confirmed the binding of naproxen at this site, as anticipated from molecular modeling studies. The naproxen binding site is located close to S165 and, interestingly, naproxen introduces negative charges mimicking phosphorylation of S165 that may interfere with NP polymerization.
Review
Naproxen protected MDCK cells against a viral challenge with H1N1 and H3N2 influenza virus, and had antiviral effects in a mouse model of viral infection. The efficacy and toxicity of the antiviral compounds directed to NP are briefly summarized in Table 2 . The nucleozin derivatives are the most potent antivirals against the WSN strain, IC50 = 70 nM. Due to the high sequence conservation of NP among viral strains, it would be expected that these compounds inhibit influenza A viruses H1N1, H3N2 and H5N1. In the case of naproxen, whose binding site is only composed of critical residues, similar IC50 values for H1N1 and H3N2 were observed, and no escape mutants could be produced. Similarly, drugs that target the exchange domain produced minimal resistance [30]. In conclusion, the structural and functional work on NP and RNP has defined three functional structures for the protein; a monomer, a trimer/tetramer and NP bound to RNA in the
Executive summary Nucleoprotein of influenza virus Nucleoprotein (NP) of influenza virus entirely covers the viral RNA and this NP–RNA complex is the template for transcription and replication. Oligomerization state of RNA-free NP Isolated NP free from RNA is in a dynamic equilibrium between monomers and trimers/tetramers. The equilibrium is slow and depends on protein and salt concentration. Only monomeric NP can oligomerize onto RNA. Structure of RNA-free NP In the crystallized trimeric form of NP, the exchange domain of one protomer reaches over to a groove on the surface of a neighboring protomer in order to oligomerize. In the monomeric form, the same domain wraps around its own monomer in an autoinhibited state, with loops reaching into the RNA-binding platform. Structure of ribonucleoprotein In the viral ribonucleoprotein, the NP protomers are further apart than the protomers in the RNA-free trimer or tetramer structure. This suggests that the exchange domain is probably not inserted in the neighboring protomer in the same manner as in the RNA-free trimer/tetramer structures. The double helical structure reveals a contact surface that is absent in the trimer/tetramer structures. NP as antiviral drug target; the various NP & RNP structures suggest the following ways of inhibiting NP function & oligomerization Inducing the formation of higher-order oligomers to prevent nuclear transport of NP or its regular oligomerization onto RNA. Inhibiting NP oligomerization by interfering with the insertion of the exchange domain into the groove on the neighboring protomer or by stabilizing the monomeric form. Inhibiting RNA binding by targeting the RNA binding site of NP. Conclusion The structural and functional work on NP and RNP has defined three functional structures for the protein; a monomer, a trimer/tetramer and NP bound to RNA in the RNP. Interfering with the monomer–oligomer equilibrium by inducing natural or non-natural oligomerization, or by preventing oligomerization interferes with the normal NP function. Targeting the RNA binding site that contains a significant number of conserved residues also seems a promising avenue for the development of anti-influenza virus drugs.
future science group
www.futuremedicine.com
1543
Review
Chenavas, Crépin, Delmas, Ruigrok & Slama-Schwok
RNP. Interfering with the monomer–oligomer equilibrium by inducing natural or non-natural oligomerization, or by preventing oligomerization interferes with the normal NP function. Targeting the RNA-binding site that contains a significant number of conserved residues also seems a promising avenue for the development of anti-influenza virus drugs. Future perspective
The influenza virus NP has several conformations and activities, such as RNA binding and self-oligomerization, which can all be inhibited. It is likely that this protein, which is relatively well conserved between all influenza A viruses and has no cellular equivalent, will be the target of commercial drugs to come in the next 10 years.
1.
Ruigrok RWH, Crepin T, Kolakofsky D. Nucleoproteins and nucleocapsids of negativestrand RNA viruses. Curr. Opin. Microbiol. 14(4), 504–510 (2011).
2.
Klumpp K, Ruigrok RW, Baudin F. Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO J. 16(6), 1248–1257 (1997).
3.
4.
5.
6.
nn
7.
Fodor E, Pritlove DC, Brownlee GG. The influenza virus panhandle is involved in the initiation of transcription. J. Virol. 68(6), 4092–4096 (1994). Hagen M, Chung TD, Butcher JA, Krystal M. Recombinant influenza virus polymerase: requirement of both 5´ and 3´ viral ends for endonuclease activity. J. Virol. 68(3), 1509–1515 (1994). Compans RW, Content J, Duesberg PH. Structure of the ribonucleoprotein of influenza virus. J. Virol. 10(4), 795–800 (1972). Arranz R, Coloma R, Chichon FJ et al. The structure of native influenza virion ribonucleoproteins. Science 338(6114), 1634–1637 (2012). Electron microscopy model of intact viral ribonucleoprotein (RNP) showing the contacts between nucleoprotein (NP) protomers along the RNA and between ascending and descending strands, as well as a tomogram of RNP inside the virus. Moeller A, Kirchdoerfer RN, Potter CS, Carragher B, Wilson IA. Organization of the
1544
This work was supported by the French agency for res earch (A N R ; Fl unu cl e ovir; A N R-2 010 Blanc-1307-1301 to A Slama-Schwok and RWH Ruigrok), MEDICEN and Lyonbiopole, the European Commission under grant agreement no. 259751 (Flupharm: New drugs targeting influenza virus polymerase, 11/2010-2005/2014 to RWH Ruigrok) and by a grant of the Programme de Recherche Influenza A (H1N1), coordinated by the Institut de Microbiologie et Maladies Infectieuses of the Institut National de la Santé et de la Recherche Médicale, INSERM, France (to B Delmas). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
influenza virus replication machinery. Science 338(6114), 1631–1634 (2012).
References Papers of special note have been highlighted as: n of interest nn of considerable interest
Financial & competing interests disclosure
nn
8.
9.
Electron microscopy model of recombinant and replicating RNP. Ruigrok RW, Baudin F. 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(Pt 4), 1009–1014 (1995). Ye Q, Krug RM, Tao YJ. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444(7122), 1078–1082 (2006).
10. Tarus B, Bakowiez O, Chenavas S et al.
Oligomerization paths of the nucleoprotein of influenza A virus. Biochimie 94(3), 776–785 (2012). n
Biochemistry paper describing the monomer–oligomer equilibrium of isolated NP and the polymerization of the monomeric form onto RNA.
11. Ye Q, Guu TS, Mata DA et al. Biochemical
and structural evidence in support of a coherent model for the formation of the double-helical influenza A virus ribonucleoprotein. MBio 4(1), e00467-12 (2012). n
Describes the kinetics of RNA-free NP polymerization, as well as the dimer structure of the deletion mutant of NP missing the exchange domain.
12. Hutchinson EC, Denham EM, Thomas B
et al. Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry. PLoS Pathog. 8(11), e1002993 (2012).
Future Microbiol. (2013) 8(12)
13. Chenavas S, Estrozi LF, Slama-Schwok A
et al. Monomeric nucleoprotein of influenza A virus. PLoS Pathog. 9(3), e1003275 (2013). n
Describes the structure of the R416A monomeric mutant and the role of phosphorylation of serine 165 in keeping NP monomeric.
14. Boulo S, Akarsu H, Lotteau V, Muller CW,
Ruigrok RWH, Baudin F. Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein. Virology 409(1), 84–90 (2011). 15. Coloma R, Valpuesta JM, Arranz R,
Carrascosa JL, Ortin J, Martin-Benito J. The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog. 5(6), e1000491 (2009). 16. Ng AK-L, Zhang H, Tan K et al. Structure
of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. FASEB J. 22(10), 3638–3647 (2008). 17. Chan W-H, Ng AK-L, Robb NC et al.
Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities. J. Virol. 84(14), 7337–7345 (2010). 18. Elton D, Medcalf E, Bishop K, Digard P.
Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements. Virology 260(1), 190–200 (1999). 19. Elton D, Medcalf L, Bishop K, Harrison D,
Digard P. Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding. J. Virol. 73(9), 7357–7367 (1999).
future science group
Influenza virus nucleoprotein: structure, RNA binding, oligomerization & antiviral drug target
20. Shen Y-F, Chen Y-H, Chu S-Y et al.
E339...R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors. Proc. Natl. Acad. Sci. USA 108(40), 16515–16520 (2011).
antiviral agents. Trends Pharmacol. Sci. 33(2), 89–99 (2012). 26. Cianci C, Gerritz SW, Deminie C, Krystal
Vreede FT. The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes. Nat. Commun. 4, 1591 (2013).
27. Cheng H, Wan J, Lin M-I et al. Design,
synthesis, and in vitro biological evaluation of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents targeting virus nucleoprotein. J. Med. Chem. 55(5), 2144–2153 (2012).
22. Ng AK-L, Chan W-H, Choi S-T et al.
Influenza polymerase activity correlates with the strength of interaction between nucleoprotein and PB2 through the hostspecific residue K/E627. PLoS ONE 7(5), e36415 (2012).
24. Jennings PA, Finch JT, Winter G, Robertson
JS. Does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral RNA? Cell 34(2), 619–627 (1983). 25. Muller KH, Kakkola L, Nagaraj AS,
Cheltsov AV, Anastasina M, Kainov DE. Emerging cellular targets for influenza
future science group
31. Amorim MJ, Kao RY, Digard P. Nucleozin
targets cytoplasmic trafficking of viral ribonucleoprotein–Rab11 complexes in influenza A virus infection. J. Virol. 87(8), 4694–4703 (2013). 32. Zimmermann P, Manz B, Haller O,
Schwemmle M, Kochs G. The viral nucleoprotein determines Mx sensitivity of influenza A viruses. J. Virol. 85(16), 8133–8140 (2011). 33. Das K, Aramini JM, Ma L-C, Krug RM,
28. Gerritz SW, Cianci C, Kim S et al. Inhibition
23. Ng AK-L, Lam MK-H, Zhang H et al.
Structural basis for RNA binding and homo-oligomer formation by influenza B virus nucleoprotein. J. Virol. 86(12), 6758–6767 (2012).
polymerase activity. Proc. Natl. Acad. Sci. USA 107(45), 19151–19156 (2010).
M. Influenza nucleoprotein: promising target for antiviral chemotherapy. Antiviral Chem. Chemother. doi:10.3851/IMP2235 (2012) (Epub ahead of print).
21. Turrell L, Lyall JW, Tiley LS, Fodor E,
nn
Review
Arnold E. Structures of influenza A proteins and insights into antiviral drug targets. Nat. Struct. Mol. Biol. 17(5), 530–538 (2010).
of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers. Proc. Natl. Acad. Sci. USA 108(37), 15366–15371 (2011).
34. Li Z, Watanabe T, Hatta M et al. Mutational
Describes the function of nucleozin and shows the structure of NP trimers ‘cross-linked’ by the drug.
35. Lejal N, Tarus B, Bouguyon E et al.
analysis of conserved amino acids in the influenza A virus nucleoprotein. J. Virol. 83(9), 4153–4162 (2009). Structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza A virus. Antimicrob. Agents Chemother. 57(5), 2231–2242 (2013).
29. Kao RY, Yang D, Lau L-S et al. Identification
of influenza A nucleoprotein as an antiviral target. Nat. Biotechnol. 28(6), 600–605 (2010). 30. Su C-Y, Cheng T-JR, Lin M-I et al. High-
throughput identification of compounds targeting influenza RNA-dependent RNA
www.futuremedicine.com
n
Describes the binding site of naproxen to the RNA-binding platform of NP and its antiviral effect.
1545
