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ABSTRACT: Conserved tryptophan-187 facilitates homodimerization of the influenza A virus NS1 effector domain. We generated a mutant influenza virus expressing NS1-W187R to destabilize this self-interaction. NS1-W187R protein exhibited lower dsRNA-binding activity, showed a temporal redistribution during infection, and was minimally compromised for interferon-antagonism. The mutant virus replicated similarly to wild-type in vitro, but was slightly attenuated for replication in mice, causing notably reduced morbidity/mortality. These data suggest biological relevance for the W187-mediated homotypic interaction of NS1.
Journal of Virology 09/2012; · 5.40 Impact Factor
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ABSTRACT: Neuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D+I222L, E119V+I222L, D198N+I222L, and H274Y+I222L (N2 numbering). Among the viruses produced, the E119D+I222L mutant virus was not able to grow without bacterial NA complementation and the D198N+I222L mutant and H274Y+I222L mutant were not stable after passages in MDCK cells. The E119V+I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V+I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V+I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.
Antimicrobial Agents and Chemotherapy 03/2011; 55(6):2942-52. · 4.84 Impact Factor
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ABSTRACT: We have utilized glycan microarray technology to determine the receptor binding properties of early isolates from the recent 2009 H1N1 human pandemic (pdmH1N1), and compared them to North American swine influenza isolates from the same year, as well as past seasonal H1N1 human isolates. We showed that the pdmH1N1 strains, as well as the swine influenza isolates examined, bound almost exclusively to glycans with α2,6-linked sialic acid with little binding detected for α2,3-linked species. This is highlighted by pair-wise comparisons between compounds with identical glycan backbones, differing only in the chemistry of their terminal linkages. The overall similarities in receptor binding profiles displayed by pdmH1N1 strains and swine isolates indicate that little or no adaptation appeared to be necessary in the binding component of HA for transmission from pig to human, and subsequent human to human spread.
Virology 02/2011; 413(2):169-82. · 3.35 Impact Factor
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ABSTRACT: Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins.
PLoS ONE 01/2011; 6(3):e17946. · 4.09 Impact Factor
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ABSTRACT: Seasonal epidemics and periodic worldwide pandemics caused by influenza A viruses are of continuous concern. The viral nonstructural (NS1) protein is a multifunctional virulence factor that antagonizes several host innate immune defenses during infection. NS1 also directly stimulates class IA phosphoinositide 3-kinase (PI3K) signaling, an essential cell survival pathway commonly mutated in human cancers. Here, we present a 2.3-A resolution crystal structure of the NS1 effector domain in complex with the inter-SH2 (coiled-coil) domain of p85beta, a regulatory subunit of PI3K. Our data emphasize the remarkable isoform specificity of this interaction, and provide insights into the mechanism by which NS1 activates the PI3K (p85beta:p110) holoenzyme. A model of the NS1:PI3K heterotrimeric complex reveals that NS1 uses the coiled-coil as a structural tether to sterically prevent normal inhibitory contacts between the N-terminal SH2 domain of p85beta and the p110 catalytic subunit. Furthermore, in this model, NS1 makes extensive contacts with the C2/kinase domains of p110, and a small acidic alpha-helix of NS1 sits adjacent to the highly basic activation loop of the enzyme. During infection, a recombinant influenza A virus expressing NS1 with charge-disruption mutations in this acidic alpha-helix is unable to stimulate the production of phosphatidylinositol 3,4,5-trisphosphate or the phosphorylation of Akt. Despite this, the charge-disruption mutations in NS1 do not affect its ability to interact with the p85beta inter-SH2 domain in vitro. Overall, these data suggest that both direct binding of NS1 to p85beta (resulting in repositioning of the N-terminal SH2 domain) and possible NS1:p110 contacts contribute to PI3K activation.
Proceedings of the National Academy of Sciences 02/2010; 107(5):1954-9. · 9.68 Impact Factor
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ABSTRACT: The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the "Asian Influenza" of 1957. We compare it with the 1918 "Spanish Influenza" hemagglutinin, H1, and the 1968 "Hong Kong Influenza" hemagglutinin, H3, and show that despite its close overall structural similarity to H1, and its more distant relationship to H3, the H2 receptor binding site is closely related to that of H3 hemagglutinin. By analyzing hemagglutinins of potential H2 avian precursors of the pandemic virus, we show that the human receptor can be bound by avian hemagglutinins that lack the human-specific mutations of H2 and H3 pandemic viruses, Gln-226Leu, and Gly-228Ser. We show how Gln-226 in the avian H2 receptor binding site, together with Asn-186, form hydrogen bond networks through bound water molecules to mediate binding to human receptor. We show that the human receptor adopts a very similar conformation in both human and avian hemagglutinin-receptor complexes. We also show that Leu-226 in the receptor binding site of human virus hemagglutinins creates a hydrophobic environment near the Sia-1-Gal-2 glycosidic linkage that favors binding of the human receptor and is unfavorable for avian receptor binding. We consider the significance for the development of pandemics, of the existence of avian viruses that can bind to both avian and human receptors.
Proceedings of the National Academy of Sciences 10/2009; 106(40):17175-80. · 9.68 Impact Factor
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ABSTRACT: A panel of influenza A viruses encoding mutant NS1 proteins was created in which a number of NS1 functions, including interactions with dsRNA, PI3K, CPSF30 and PKR, were inhibited. Surprisingly, given previous reports that NS1 activates PI3K to prevent apoptosis, the mutant viruses rUd-Y89F and rUd-P164/7A that fail to activate PI3K did not induce any more apoptosis than wild-type virus in MRC-5 and A549 cells, even though these cells are highly sensitive to inducers of apoptosis. Induction of cell death by the apoptogenic rUd-184-8(P) virus could not be prevented by serum-mediated activation of PI3K/Akt. Neither infection of MRC-5 or A549 cells with wild-type virus nor constitutive expression of NS1 prevented cell death caused by apoptosis inducers, suggesting that NS1 is not directly anti-apoptotic. Our data suggest that the loss of a functionally intact NS1 protein promotes apoptosis, but this is not due to an inability to activate PI3K.
Virology 10/2009; 396(1):94-105. · 3.35 Impact Factor
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ABSTRACT: A panel of eight single amino acid deletion mutants was generated within the first 24 residues of the fusion peptide domain of the of the hemagglutinin (HA) of A/Aichi/2/68 influenza A virus (H3N2 subtype). The mutant HAs were analyzed for folding, cell surface transport, cleavage activation, capacity to undergo acid-induced conformational changes, and membrane fusion activity. We found that the mutant DeltaF24, at the C-terminal end of the fusion peptide, was expressed in a non-native conformation, whereas all other deletion mutants were transported to the cell surface and could be cleaved into HA1 and HA2 to activate membrane fusion potential. Furthermore, upon acidification these cleaved HAs were able to undergo the characteristic structural rearrangements that are required for fusion. Despite this, all mutants were inhibited for fusion activity based on two separate assays. The results indicate that the mutant fusion peptide domains associate with target membranes in a non-functional fashion, and suggest that structural features along the length of the fusion peptide are likely to be relevant for optimal membrane fusion activity.
Virology 09/2009; 394(2):321-30. · 3.35 Impact Factor
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ABSTRACT: Fusion of the influenza virus envelope with the endosomal membrane of host cells is mediated by the hemagglutinin glycoprotein (HA). The most conserved region of HA is at the N-terminus of the HA2 subunit, a relatively hydrophobic sequence of amino acids referred to as the fusion peptide. This domain is critical both for setting the trigger for fusion and for destabilizing target membranes during the fusion process. The “trigger” is set by cleavage of the HA precursor polypeptide, when the newly-generated HA2 N-terminal fusion peptide positions itself into the trimer interior and makes contacts with ionizable residues to generate a fusion competent neutral pH structure. This essentially “primes” the HA such that subsequent acidification of the endosomal environment can induce the irreversible conformational changes that result in membrane fusion. A key component of these acid-induced structural rearrangements involves the extrusion of the fusion peptide from its buried position and its relocation to interact with the target membrane. The role of the fusion peptide for both priming the neutral pH structure and interacting with cellular membranes during the fusion process is discussed.
Protein and Peptide Letters 06/2009; 16(7):766-778. · 1.94 Impact Factor
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ABSTRACT: Fusion of the influenza virus envelope with the endosomal membrane of host cells is mediated by the hemagglutinin glycoprotein (HA). The most conserved region of HA is at the N-terminus of the HA2 subunit, a relatively hydrophobic sequence of amino acids referred to as the fusion peptide. This domain is critical both for setting the trigger for fusion and for destabilizing target membranes during the fusion process. The "trigger" is set by cleavage of the HA precursor polypeptide, when the newly-generated HA2 N-terminal fusion peptide positions itself into the trimer interior and makes contacts with ionizable residues to generate a fusion competent neutral pH structure. This essentially "primes" the HA such that subsequent acidification of the endosomal environment can induce the irreversible conformational changes that result in membrane fusion. A key component of these acid-induced structural rearrangements involves the extrusion of the fusion peptide from its buried position and its relocation to interact with the target membrane. The role of the fusion peptide for both priming the neutral pH structure and interacting with cellular membranes during the fusion process is discussed.
Protein and Peptide Letters 02/2009; 16(7):766-78. · 1.94 Impact Factor
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ABSTRACT: The influenza surface glycoprotein hemagglutinin (HA) is a potential target for antiviral drugs because of its key roles in the initial stages of infection: receptor binding and the fusion of virus and cell membranes. The structure of HA in complex with a known inhibitor of membrane fusion and virus infectivity, tert-butyl hydroquinone (TBHQ), shows that the inhibitor binds in a hydrophobic pocket formed at an interface between HA monomers. Occupation of this site by TBHQ stabilizes the neutral pH structure through intersubunit and intrasubunit interactions that presumably inhibit the conformational rearrangements required for membrane fusion. The nature of the binding site suggests routes for the chemical modification of TBHQ that could lead to the development of more potent inhibitors of membrane fusion and potential anti-influenza drugs.
Proceedings of the National Academy of Sciences 12/2008; 105(46):17736-41. · 9.68 Impact Factor
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ABSTRACT: The antiviral drug arbidol (ARB), which is licensed in Russia for use against influenza, is known to inhibit early membrane fusion events in influenza A and B virus replication. To investigate in more detail the target and mechanism of ARB action we generated and studied the characteristics of ARB-resistant influenza virus mutants. Observations of the ARB susceptibility of reassortants between A/Singapore/1/57(H2N2) and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of mutants of the latter virus identified the virus haemagglutinin (HA) as the major determinant of ARB sensitivity. ARB-resistant mutants, selected from the most sensitive reassortant, possessed single amino acid substitutions in the HA2 subunit which caused an increase in the pH of fusion and the associated conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2 pH unit reduction in the pH of the transition to the low pH form, which was specifically abrogated by the resistance mutations. Some of the resistance mutations, which reduce acid stability and would disrupt ARB-HA interactions, are located in the vicinity of a potential ARB binding site identified using the docking programme Gold. Together, the results of these investigations indicate that ARB falls within a class of inhibitor which interacts with HA to stabilize it against the low pH transition to its fusogenic state and consequently inhibit HA-mediated membrane fusion during influenza virus infection.
Antiviral research 12/2008; 81(2):132-40. · 3.61 Impact Factor
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ABSTRACT: During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of fusion is currently unknown. Here, we examine mutant HAs and influenza viruses containing such HAs to determine whether these peptide linkers are subject to specific length requirements for the proper folding of native HA and for membrane fusion function. Using pairwise deletions and insertions, we show that the region flanking the fusion peptide appears to be important for the folding of the native HA structure but that mutant proteins with small insertions can be expressed on the cell surface and are functional for membrane fusion. HA mutants with deletions of up to 10 residues and insertions of as many as 12 amino acids were generated for the peptide linker to the viral transmembrane domain, and all folded properly and were expressed on the cell surface. For these mutants, it was possible to designate length restrictions for efficient membrane fusion, as functional activity was observed only for mutants containing linkers with insertions or deletions of eight residues or less. The linker peptide mutants are discussed with respect to requirements for the folding of native HAs and length restrictions for membrane fusion activity.
Journal of Virology 08/2008; 82(13):6337-48. · 5.40 Impact Factor
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ABSTRACT: Influenza A virus NS1 protein is a multifunctional virulence factor. Here, we report a crystal structure for the NS1 effector domain of avian influenza virus A/Duck/Albany/76. Comparison of this structure with that reported for a human strain shows both proteins share a common monomer conformation, albeit with subtle differences. Strikingly, our data reveal a novel helix-helix dimeric interface between monomers of the avian NS1 protein, which is also found in the human NS1 crystal lattice. We re-evaluate the current model of NS1 dimeric assembly, and provide biochemical evidence to show tryptophan-187 (a residue located at the helix-helix interface) is essential for dimerization of this effector domain.
Virology 07/2008; 378(1):1-5. · 3.35 Impact Factor
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ABSTRACT: The potential impact of pandemic influenza makes effective measures to limit the spread and morbidity of virus infection a public health priority. Antiviral drugs are seen as essential requirements for control of initial influenza outbreaks caused by a new virus, and in pre-pandemic plans there is a heavy reliance on drug stockpiles. The principal target for these drugs is a virus surface glycoprotein, neuraminidase, which facilitates the release of nascent virus and thus the spread of infection. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two currently used neuraminidase inhibitors that were developed using knowledge of the enzyme structure. It has been proposed that the closer such inhibitors resemble the natural substrate, the less likely they are to select drug-resistant mutant viruses that retain viability. However, there have been reports of drug-resistant mutant selection in vitro and from infected humans. We report here the enzymatic properties and crystal structures of neuraminidase mutants from H5N1-infected patients that explain the molecular basis of resistance. Our results show that these mutants are resistant to oseltamivir but still strongly inhibited by zanamivir owing to an altered hydrophobic pocket in the active site of the enzyme required for oseltamivir binding. Together with recent reports of the viability and pathogenesis of H5N1 (ref. 7) and H1N1 (ref. 8) viruses with neuraminidases carrying these mutations, our results indicate that it would be prudent for pandemic stockpiles of oseltamivir to be augmented by additional antiviral drugs, including zanamivir.
Nature 07/2008; 453(7199):1258-61. · 36.28 Impact Factor
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Jeffrey Meisner,
Kristy J Szretter,
Konrad C Bradley,
William A Langley,
Zhu-Nan Li,
Byeong-Jae Lee,
Sudha Thoennes,
Javier Martin,
John J Skehel, Rupert J Russell,
Jacqueline M Katz,
David A Steinhauer
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ABSTRACT: The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.
Journal of Virology 06/2008; 82(10):5079-83. · 5.40 Impact Factor
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ABSTRACT: Sialic acids (Sias) are regarded as receptors for influenza viruses and are usually bound to galactose (Gal) in an alpha2-3 or alpha2-6 configuration. The detection of these Sia configurations in tissues has commonly been through the use of plant lectins that are able to identify which cells contain Siaalpha2-3- and Siaalpha2-6-linked glycans, although other techniques for receptor distribution have been used. Initial experiments indicated that avian versus human influenza virus binding was determined by either Siaalpha2-6 or Siaalpha2-3 expression. In this review, we suggest that the distribution and detection of these terminal Siaalpha2-3- and Siaalpha2-6-linked receptors within the respiratory tract might not be as clear cut as has been reported. We will also review how other viral and receptor components might act as determinants for successful viral replication and transmission. Understanding these additional components is important in comprehending the infection and the transmission of both existing human influenza viruses and newly emerging avian influenza viruses.
Trends in Microbiology 05/2008; 16(4):149-57. · 7.91 Impact Factor
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ABSTRACT: Influenza virus entry occurs in endosomes, where acidification triggers irreversible conformational changes of the hemagglutinin glycoprotein (HA) that are required for membrane fusion. The acid-induced HA structural rearrangements have been well documented, and several models have been proposed to relate these to the process of membrane fusion. However, details regarding the role of specific residues in the initiation of structural rearrangements and membrane fusion are lacking. Here we report the results of studies on the HA of A/Aichi/2/68 virus (H3 subtype), in which mutants with changes at several ionizable residues in the vicinity of the "fusion peptide" were analyzed for their effects on the pH at which conformational changes and membrane fusion occur. A variety of phenotypes was obtained, including examples of substitutions that lead to an increase in HA stability at reduced pH. Of particular note was the observation that a histidine to tyrosine substitution at HA1 position 17 resulted in a decrease in pH at which HA structural changes and membrane fusion take place by 0.3 relative to WT. The results are discussed in relation to possible mechanisms by which HA structural rearrangements are initiated at low pH and clade-specific differences near the fusion peptide.
Virology 02/2008; 370(2):403-14. · 3.35 Impact Factor
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Shinya Yamada,
Yasuo Suzuki,
Takashi Suzuki,
Mai Q Le,
Chairul A Nidom,
Yuko Sakai-Tagawa,
Yukiko Muramoto,
Mutsumi Ito,
Maki Kiso,
Taisuke Horimoto, [......],
Taiichi Usui,
Takeomi Murata,
Yipu Lin,
Alan Hay,
Lesley F Haire,
David J Stevens, Rupert J Russell,
Steven J Gamblin,
John J Skehel,
Yoshihiro Kawaoka
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ABSTRACT: H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.
Nature 12/2006; 444(7117):378-82. · 36.28 Impact Factor
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ABSTRACT: The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.
Nature 10/2006; 443(7107):45-9. · 36.28 Impact Factor
