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Trends Pharmacol Sci. Author manuscript; available in PMC Oct 27, 2010.
Published in final edited form as:
PMCID: PMC2964876
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Emerging antiviral targets for influenza A virus

Abstract

The potential threat of a pandemic caused by H5N1 influenza A viruses has stimulated increased research on developing new antivirals against influenza A viruses. Current antivirals are directed against the M2 protein (named adamantanes) and the neuraminidase (named zanamivir and oseltamivir). However, both seasonal and H5N1 influenza A viruses have developed resistance to adamantanes and oseltamivir. Accordingly, new antivirals directed at the M2 and neuraminidase proteins, and against the hemagglutinin protein, are being developed. In addition, elucidation of the structural basis for several crucial functions of other viral proteins (specifically the non-structural NS1A protein, the nucleoprotein and the viral polymerase) has identified novel targets for the development of new antivirals. Here, we describe how functional and structural studies led to the discovery of these novel targets and also how structural information is facilitating the rational design of new drugs against previously identified targets.

Introduction

Influenza A and B viruses are enveloped RNA viruses, of which the genomes are in the form of eight segments [1] (Box 1). These viruses cause a highly contagious respiratory disease in humans, resulting in ~36 000 deaths and >100 000 hospitalizations in the USA annually (www.cdc.gov/flu). Influenza A viruses, but not influenza B viruses, are responsible for the periodic widespread epidemics, or pandemics, that have caused high mortality rates [2]. The most devastating pandemic occurred in 1918, which caused an estimated 40 million deaths worldwide [3]. Less devastating pandemics occurred in 1957 and 1968. H5N1 influenza A viruses (commonly known as ‘avian flu’), which are highly pathogenic in humans, are candidates for the next pandemic influenza virus [4,5] (Box 2). H5N1 viruses were first transmitted from chickens to humans in 1997 in Hong Kong, killing 6 out of 18 infected people [6,7]. After all the chickens in the poultry markets in Hong Kong were culled, H5N1 viruses continued to circulate in poultry markets in China, but transmission to humans was not documented until 2003 when fatal transmissions to humans resumed [8]. The human mortality rate has been high (~65%), resulting in >240 deaths. H5N1 viruses in avian species have now spread from Asia to Europe and Africa (www.who.int/csr/disease/avian_influenza/en/index.html). Fortunately, H5N1 viruses have not yet acquired the ability for efficient human-to-human transmission.

Box 1Influenza virus genome and proteins

Influenza A and B viruses are enveloped viruses that contain negative-stranded RNA genomes comprising eight RNA segments [1]. Because the viral genomes are of negative-strand polarity, the genome RNA segments must be transcribed by a virion-associated RNA-dependent RNA polymerase to produce the plus-strand viral mRNAs needed to initiate virus replication. Figure I shows a schematic representation of influenza A virus and its genome. The three largest genome segments encode the three subunits of the polymerase: PB1, PB2 and PA. The segment encoding PB1 also encodes a small nonstructural protein, PB1-F2, which has apoptotic functions. The intermediate-sized segments encode the hemagglutinin (HA), the neuraminidase (NA) and the nucleocapsid (NP) proteins. HA, the major surface protein of the virus, binds to sialic-acid-containing receptors on host cells, and is the protein against which neutralizing antibodies are produced. The NA viral surface protein removes sialic acid from glycoproteins. One of its major functions is to remove sialic acid during virus budding from the cell surface and from the HA and NA of the newly assembled virions, thereby eliminating aggregation of budding virions on the cell surface. NP protein molecules are bound at regular intervals along the entire length of each of the genomic RNAs to form viral ribonucleoproteins (vRNPs) and also have essential functions in viral RNA replication. The seventh genomic RNA segment encodes two proteins: M1 (matrix protein) and M2. The M1 protein underlies the viral lipid membrane and is thought to interact with the genomic vRNPs and with the inner (cytoplasmic) tails of HA and NA. The M2 protein is an ion channel protein that is essential for the uncoating of the virus. The smallest segment encodes two proteins, NS1A and NS2. The NS2 protein, also called NEP, mediates the export of newly synthesized viral RNPs from the nucleus to the cytoplasm. The NS1A protein, which is not incorporated into virions, is a multi-functional protein that has an integral role in suppressing host antiviral responses. We denote the NS1 protein of influenza A virus as the NS1A protein to distinguish it from the NS1 protein of influenza B virus, which can then be denoted as the NS1B protein. The sizes of both the genome segments and their encoded proteins differ between A and B viruses [1]. There are also other differences; for example, influenza B viruses do not have a M2 protein, and the NS1B protein does not bind the host proteins that bind to the NS1A protein (see main text).

Figure I
Schematic representation of influenza A virus.

Box 2Influenza virus strains

Influenza A and B viruses are enveloped RNA viruses in the Orthomyxoviridae family [1]. Influenza A viruses infect a wide number of avian and mammalian species, whereas influenza B viruses infect only humans. Influenza A viruses are organized into subtypes based on their surface hemagglutinin (HA) and neuraminidase (NA). Sixteen HA (H1 to H16) and nine NA (N1 to N9) subtypes are currently identified. The H3N2 and H1N1 influenza A subtypes (along with influenza B viruses) are responsible for recent seasonal influenza epidemics. The pathogenic influenza A virus that has been transmitted from avian species to humans since 1997 (commonly known as ‘avian flu’) is a H5N1 strain. A specific influenza strain is named according to a standard convention in the following order: virus type (the source organism of avian strains is also often specified), geographic origin, strain number, year and virus subtype; for example, influenza A/Udorn/307/1972 (H3N2) virus.

The primary means for controlling influenza virus epidemics has been vaccination with inactivated or live-attenuated virus [2]. The hemagglutinin (HA) of the virus in the vaccine elicits an immune response that neutralizes virus infectivity. The antigenic structures of the HAs of both influenza A and B viruses undergo antigenic drift, which results from the selection of mutant viruses that evade antibodies directed against the predominant antigenic type of the HA circulating in the human population. Mutant viruses are readily generated because the viral RNA polymerase has no proof-reading function. Because of antigenic drift, the vaccine has to be reformulated each year.

Pandemic influenza A viruses can apparently arise by two different mechanisms. In one mechanism, influenza A viruses undergo a process called antigenic shift, the reassortment of genomic RNA segments between human and avian influenza A virus strains, resulting in a new (potentially pandemic) virus encoding a novel avian-type HA that is immunologically distinct [2]. The influenza A viruses containing the H2 and H3 HA subtypes that caused pandemics in 1957 and 1968, respectively, resulted from the reassortment of avian and human genomic RNA segments. Influenza B viruses cannot undergo antigenic shift because they infect only humans. A second mechanism for generating pandemic influenza A viruses has been proposed based on the finding that all eight genomic RNAs of the 1918 pandemic virus contain avian-like sequences [9]. This finding suggested that all eight genomic RNAs were derived from an avian virus and that such a progenitor virus then underwent multiple mutations in the process of adapting to the mammalian host. H5N1 viruses, which contain only avian genes, seem to be undergoing this route for acquiring pandemic capability in humans [4,5]. As is the case for other avian HAs, humans have no immunological protection against the H5 HA subtype.

Antivirals have been used for both prophylactic and therapeutic treatments during seasonal epidemics [10]. For example, prophylactic treatment has been used for individuals at high risk who have not been vaccinated, such as the elderly in nursing homes and hospital personnel who could spread infection to patients. The most effective therapeutic treatment of patients requires early diagnosis because antivirals are most effective in alleviating symptoms if given within the first 30 hours after infection. If a H5N1 pandemic occurs, effective control of such a pandemic will require the use of antiviral drugs because it is not likely that sufficient amounts of an effective vaccine will be available, particularly in the early phase of a fast-spreading pandemic [1113]. Antivirals can be stockpiled and, if used appropriately, should limit the spread of pandemic influenza virus. Importantly, the strategies that have been proposed for the use of antivirals to stem a H5N1 pandemic [1113] could lead to more common and effective uses of antivirals during annual epidemics.

Current antivirals are directed against the M2 protein (these antivirals are known as adamantanes) and neuraminidase (NA; antivirals known as zanamivir and oseltamivir) [1418]. However, both seasonal and H5N1 influenza A viruses have developed resistance to adamantanes and oseltamivir [1926]. Consequently, new antivirals directed at these two viral proteins, and against HA, are being developed. In addition, novel targets for the development of new antivirals have been identified in the non-structural NS1A protein, the nucleoprotein (NP) and the viral polymerase. Here, we describe how functional and structural studies led to the discovery of these novel targets, and also how structural information is facilitating the rational design of new drugs against previously identified targets.

Current antiviral targets

M2 protein

Adamantanes (amantadine and rimantadine, marketed under the trade names Symmetrel and Flumadine, respectively) are directed against the M2 protein, a 97-amino-acid protein that is assembled into a tetramer that spans the viral membrane [14,15] (Figure 1). The tetramer possesses low pH-activated (H+)-ion-channel activity that is crucial for uncoating of influenza A virions, which enter cells via endosomes [14,15]. The M2 protein channel promotes the influx of H+ ions into the interior of the virions, thereby disrupting protein–protein interactions, specifically including those between the M1 protein and viral ribonucleoproteins (vRNPs). Two key residues (His37 and Trp41) within this tetrameric helical channel act as a pH sensor and a proton (H+ ion) gate, respectively. After the vRNPs are released from the endosomes into the cytoplasm, they are transported to the nucleus where they catalyze the initial transcription of the viral genome. Adamantanes inhibit M2-ion-channel activity and, as a result, the M1 protein remains associated with the released vRNPs, thereby inhibiting their nuclear import.

Figure 1
Current structures of the influenza A virus M2 protein channel in complex with either amantadine or rimantadine. (a) X-ray structure (3.5 Å resolution) of the TM domain of M2 (residues 22–46) tetramer in complex (1:1) with amantadine in ...

Virus mutants resistant to adamantanes readily emerge and are as fit as the wild-type virus [19,20]. In the 2005–2006 influenza season, 90.5% of the H3N2 viruses and 15.5% of the H1N1 viruses analyzed were resistant to adamantanes [21]; many human isolates of H5N1 viruses are also resistant [22]. A single substitution of any one of five amino acids in the M2 transmembrane (TM) domain can confer resistance [19,20]. In the 2005–2006 influenza season, the substitution of Asn for Ser at position 31 in the M2 protein was the predominant change that conferred resistance [21].

Several recent 3D structures of the M2 channel in the presence of one of the adamantane drugs have been determined using X-ray crystallography and NMR spectroscopy [2729] (Figure 1). The X-ray structure [27] and one NMR structure [29] indicate that the M2 ion channel TM domain binds to a single amantadine molecule plugging the pore in the vicinity of Ser31 (Figure 1a,c). The position of amantadine in the pore suggested that new drugs that target different regions of the pore would be less susceptible to the generation of resistant mutants. In contrast, another NMR structure [28] of the closed state of the M2 ion channel suggested a totally different, allosteric mechanism for inhibition by rimantadine. In this model, the drug binds at four equivalent sites in the vicinity of the Trp41 proton gate, but on the outside of the TM helices, thereby stabilizing the closed state of the channel and inhibiting its opening (Figure 1b). Subsequent functional results have favored the first model [30]. The key question is whether these new structures taken together will enable investigators to develop new antiviral drugs against the M2 protein that are less susceptible to the development of resistant virus mutants. To this end, researchers are screening for chemicals that bind to mutant forms of M2 that are not inhibited by adamantanes.

NA

The first antivirals directed against NA were rationally designed on the basis of the X-ray crystal structure of N2-type NAs (i.e. N2 and N9) [16,31,32]. NA is a tetramer, and each monomer has a sialic-acid-binding pocket lined by amino acids that are conserved in all influenza A and B virus strains. A sialic acid derivative in which the 4-hydroxyl group of sialic acid is replaced with a guanidinyl group binds to this pocket tighter than sialic acid itself. The resulting compound, zanamivir (commercially called Relenza), effectively inhibits the NA of influenza A and B viruses and is orders of magnitude less active against non-influenza neuraminidases. Zanamivir must be delivered by an oral inhaler.

Subsequently, a different compound containing a lipophilic side chain was synthesized (oseltamivir, commercially called Tamiflu), which is administered orally as the prodrug for oseltamivir carboxylate, the actual NA inhibitor [17,18]. Pathogenic H5N1 viruses resistant to oseltamivir were isolated from patients undergoing treatment with this drug [2325]. In addition, oseltamivir-resistant H1N1 viruses emerged globally in the 2007–2009 influenza seasons in the absence of exposure of patients to the drug [26]. All these resistant viruses had the same amino acid substitution, His274Tyr, in their NA protein, which had no apparent effect on the fitness of the virus. The H5N1 and H1N1 viruses with the His274Tyr mutant NA remained susceptible to zanamivir, demonstrating that even if such mutant viruses became predominant at least one NA inhibitor would still be potent. In addition, a recent structural analysis has shown that type 1 NAs (i.e. N1, N4 and N8), unlike the N2 NA that was used to design oseltamivir and zanamivir, have a large cavity adjacent to the sialic-acid-binding site [33]. This cavity can potentially be exploited for the design of new antivirals directed at type 1 NAs by adding additional chemical groups to existing NA inhibitors. Such antivirals would be expected to be effective both against currently circulating human H1N1 viruses and pathogenic H5N1 viruses.

HA

The HA of influenza A viruses is a homotrimer that binds to sialic receptors on cells [1,34]. The low pH in the endosomes causes irreversible conformational changes of the HA [35] that lead to the fusion of the viral and cellular membranes and hence uncoating of the virus. Chemicals that inhibit these conformational changes inhibit virus replication [3638]. Insight into ways to improve the activity of such inhibitors was provided by the crystal structures of two HAs in complex with one such inhibitor, tert-butyl hydroquinone (TBHQ) [39]. This compound binds in a hydrophobic pocket at the interface between HA monomers, thereby stabilizing the neutral pH conformation of HA. The way that TBHQ fits into this pocket suggests that specific modifications of this chemical would improve binding affinity, potentially leading to an effective antiviral drug. The HAs used for this crystal structure fall into the phylogenetic group of HAs that includes the H3 HA of viruses currently circulating in humans. However, the TBHQ-binding pocket is absent from the HAs of the second phylogenetic HA group that includes both the H1 HA of viruses currently circulating in humans and the H5 HA of pathogenic H5N1 viruses. Other chemicals inhibit membrane fusion by the HAs of this second phylogenetic group [38], and crystal structures of these HAs in complex with their inhibitory compounds might lead to the design of antiviral drugs against viruses containing these HAs.

Emerging antiviral targets

NS1A protein

The NS1 protein from influenza A (NS1A) is a small (230–237 residue), dimeric, multi-functional protein that has a crucial role in the ability of the virus to evade the antiviral response of the infected cell [40,41]. The protein comprises two domains: (i) the N-terminal (residues 1–73) RNA-binding domain (RBD), and (ii) a C-terminal (residues 74–230/237) effector domain (ED) [40,41]. The NS1A RBD binds to double-stranded RNA (dsRNA) in a non-sequence-specific manner [4245]. The primary, if not only, role of NS1A dsRNA-binding activity is the inhibition of the 2′–5′ oligo A synthetase/RNase L pathway that is induced by interferon-α/β (IFN-α/β) [46]. The C-terminal ED of NS1A interacts with several host cellular proteins, including: (i) the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), thereby inhibiting the 3′ end processing of cellular pre-mRNAs and, hence, the production of all cellular mRNAs including IFN-β mRNA; (ii) the p85β subunit of phosphatidylinositol-3 (PI3) kinase, resulting in the activation of PI3 kinase signaling; and (iii) protein kinase R (PKR), resulting in the inhibition of PKR activation that would otherwise inhibit virus replication [4752].

The dsRNA-binding surface of the RBD is an attractive target for antiviral drug development. Solution NMR and X-ray crystal structures of the isolated RBD of the NS1A protein of influenza A/Udorn/307//72 (Ud) virus (H3N2) revealed a unique six-helical symmetric head-to-tail homodimer structure [53,54] (Figure 2a). Only one amino acid, Arg38/Arg38′, is absolutely required for dsRNA binding [44]. A mutant Ud virus expressing a NS1A protein in which Arg at position 38 was replaced with an Ala residue is highly attenuated [46], demonstrating that dsRNA-binding activity is required for efficient virus replication. Several basic residues including Arg38/Arg38′, are located in symmetry-related, antiparallel helices (α2 and α2′) on one face of the protein [53,54]. An analogous six-helical homodimer motif was observed in the X-ray crystal structure of the RBD of the NS1B protein of an influenza B virus [55]. The dsRNA-binding surfaces in both NS1A and NS1B are defined by tracks of highly conserved basic and hydrophilic residues that are complementary to the phosphate backbone of canonical A-form dsRNA, as supported by NMR chemical shift perturbation data for the Ud NS1A(1–73):dsRNA complex [55] (Figure 2b). This binding mode was confirmed by an X-ray crystal structure of the complex of a NS1A RBD and 19-base-pair dsRNA [56] (Figure 2c). This structure shows that the NS1A RBD interacts with backbone atoms and 2′-OH moieties along the major groove of the RNA duplex. Numerous hydrophilic or charged residues on the binding face of NS1A contribute to dsRNA binding, particularly Arg38/Arg38′, which penetrates into the helix, forming hydrogen bonds with phosphates on both strands. An important feature of the NS1A dsRNA-binding face is a pocket consisting of numerous hydrophilic and hydrophobic residues. Chemicals that specifically bind to this pocket and interfere with the interaction of NS1A residues with dsRNA would inhibit virus replication, making this pocket an inviting target for antiviral drug design.

Figure 2
Potential antiviral target in the NS1A dsRNA-binding domain. (a) Ribbon representation of the solution NMR structure of Ud NS1A(1–73) [53]. NS1A and NS1A′ subunits are shown in green and cyan, respectively, and the side chains of Arg38 ...

The CPSF30-binding pocket in the ED is also an attractive target for antiviral drug development. Several lines of evidence established the importance of this binding site for virus replication. One set of experiments utilized the CPSF30 domain comprising its second and third zinc finger (F2F3) that binds efficiently to the NS1A ED [57]. In cells that constitutively express F2F3, replication of influenza A viruses was inhibited coupled with increased production of IFN-β mRNA, presumably as a result of F2F3 occupying the CPSF30 binding site on the NS1A protein and, hence, blocking the binding of endogenous CPSF30 to this site. These results show that the CPSF30-binding site of the NS1A protein can be blocked without any apparent effect on the ability of CPSF30 to carry out its required functions in the processing of cellular pre-mRNAs. The X-ray crystal structure of Ud NS1A(85–215) in complex with the F2F3 fragment revealed that the ED undergoes a dramatic structural rearrangement from its dimer structure [5860] to form a tetrameric complex comprising two F2F3 units wrapped around two NS1A EDs arranged in a head-to-head manner [61] (Figure 3, inset). The CPSF30-binding pocket identified in this crystal structure is largely hydrophobic and contains several highly conserved residues that interact with aromatic side chains from the F3 zinc finger [61] (Figure 3). A recombinant Ud virus expressing a NS1A mutant protein that does not bind to F2F3 of CPSF30 is attenuated and does not inhibit IFN-β pre-mRNA processing [61]. Hence, disruption of the NS1A:CPSF30 complex, which suppresses this crucial host antiviral response and is required for efficient virus replication, makes this conserved binding pocket in NS1A ED an attractive target for antiviral drug development.

Figure 3
Potential antiviral target in the NS1A effector domain. Expanded view of the F2F3-binding pocket of the Ud NS1A ED [61]. NS1A residues labeled red interact with aromatic side chains in the F3 zinc finger. Inset: ribbon representation of the Ud NS1A(85–215)–F2F3 ...

A recent study showed that several chemicals that bind to undetermined regions of the NS1A protein inhibit virus replication [62], verifying that the NS1A protein is a viable target for the generation of antivirals against influenza A viruses. Other investigators are specifically targeting the dsRNA- and CPSF30-binding sites for the development of antivirals.

NP

The viral NP, which has RNA-binding activity with no apparent specificity, has several crucial functions during infection. It binds along the entire length of the viral RNA segments to form the vRNPs that are packaged into the virus [63] and is required for the switch from viral mRNA synthesis to viral RNA replication [64]. Viral mRNA synthesis is initiated by capped RNA fragments of 10–13 bases that are excised from cellular pre-mRNAs by the intrinsic endonuclease of the viral polymerase (a process called cap-snatching), whereas viral RNA replication is initiated without a primer [64]. A recent study has shown how NP mediates this switch: it interacts with one or more subunits of the viral polymerase, resulting in modification of the polymerase in favor of unprimed initiation [65].

The X-ray crystal structures of NP from both H1N1 and H5N1 viruses identified two potential targets for antiviral development [66,67]. NP (which forms trimers in the crystal) folds into a crescent shape with a head and body domain (Figure 4a). Each NP monomer has a tail loop at the back of the molecule that mediates oligomerization via its insertion into a pocket in the body domain of a neighboring molecule. The tail loop interactions between subunits are almost identical in the H1N1 and H5N1 NP structures. Single amino acid substitutions in the tail loop eliminate oligomerization, and the resulting monomeric NP is not active in viral RNA replication. Consequently, the tail-loop-binding pocket is a potential target for the development of antivirals. Another possible antiviral target in NP is its RNA-binding site [66] (Figure 4b). The RNA-binding groove is found between the head and body domains at the exterior surface of the trimer. It contains a large number of basic residues, and some of the residues required for RNA-binding have been identified.

Figure 4
Potential antiviral targets in NP. Crystal structure of the NP from HINI influenza A/WSN/33 [66]. (a) Interactions between subunit A (green) and the tail loop of subunit B (red) from the trimeric structure of NP shown on the left. For each denoted amino ...

The viral RNA-dependent RNA polymerase

The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a large (250 kDa) heterotrimer complex comprising three subunits: PB1, PB2 and PA. All three subunits are required for both capped RNA-primed viral mRNA synthesis and for unprimed viral RNA replication [65]. Proper formation of the trimeric polymerase requires a specific interaction between the C-terminal domain of PA (PAC) and the N terminus of PB1 (PB1N). The 3D structure of this binding surface was revealed in crystal structures of PAC in complex with PB1N, using both H5N1 and H1N1 viral proteins. PAC forms a clamp around PB1N [68,69] (Figure 5). Binding is mediated by hydrophobic interactions and hydrogen bonds. The PA and PB1 residues involved in this interaction are conserved across all influenza A virus strains. Mutation of key residues in the PB1-binding site of PAC and in PB1N lead to a loss of polymerase activity and a reduction in virus replication. Because the PB1N peptide has been shown to inhibit viral replication, presumably by blocking trimer assembly [70], the PAC–PB1N binding surface provides a potential target for anti-influenza drug development.

Figure 5
Potential antiviral target in the viral polymerase. Structure of the PAC–PB1N complex. Surface image showing the hydrophobic contacts between PA (green) and the N terminus of PB1 (orange) in the crystal structure of PA(239–716)–PB1(1–81) ...

The crystal structures of the two functional domains of the cap-dependent endonuclease, its cap-binding site and endonuclease active site have recently been determined [7173]. These two polymerase domains might also be suitable targets for influenza antiviral drug development.

Conclusions

The potential threat of a pandemic caused by H5N1 influenza A viruses has stimulated increased research on developing new antivirals directed against several viral proteins. An additional incentive has been the emergence of influenza A viruses, both seasonal and pathogenic H5N1 viruses, that are resistant to some of the current antivirals that are directed against the M2 and NA proteins. Accordingly, new antivirals against these two viral proteins are being developed relying largely on structure-based optimization of drug candidates. In addition, recent structural results have provided a more informed approach to the development of antivirals against a well-known target, HA. Furthermore, functional and structural studies have identified novel targets in other viral proteins, specifically the NS1A protein, NP and viral polymerase proteins. The new targets in these proteins are conserved structures that mediate crucial virus functions. Because many amino acid changes in most of these conserved functional sites are deleterious [44,46,61,6669], it is anticipated that it will be difficult for influenza virus to develop resistance to compounds targeting these sites. However, this prediction will have to be verified, particularly considering that a similar prediction was made for NA antivirals. The development of antivirals targeted against several influenza proteins would make it possible to use combinations of antivirals to minimize the selection of resistant viruses, in light of the report that combined use of amantadine and oseltamivir reduced the emergence of drug-resistant influenza A viruses [74].

Acknowledgments

Research in our laboratories is supported by grants from the National Institutes of Health USA (www.nih.gov).

Footnotes

Disclosure statement

The authors are funded by a grant from the National Institutes of Health USA to develop antivirals against the dsRNA- and CPSF30-binding surfaces in the NS1A protein. Applications for patents directed at targeting these two NS1A surfaces for the development of antivirals have been submitted.

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