Another look at the molecular mechanism of the resistance of H5N1 influenza A virus neuraminidase (NA) to oseltamivir (OTV)

In the context of a recent pandemic threat by the worldwide spread of H5N1 avian influenza, the high resistance of H5N1 virus to the most widely used commercial drug, oseltamivir (Tamiflu), is currently an important research topic. Herein, molecular bases of the mechanism of H5N1 NA resistance to oseltamivir were elucidated using a computational approach in a systematic fashion. Using the crystal structure of the complex of H5N1 NA with OTV (PDB ID: 2hu0) as the starting point, the question, how mutations at His274 by both smaller side chain (Gly, Ser, Asn, Gln) and larger side chain (Phe, Tyr) residues influence the sensitivity of N1 to oseltamivir, was addressed and correlated with the experimental data. The smaller side chain residue mutations of His274 resulted in slightly enhanced or unchanged NA sensitivity to OTV, while His274Phe and His274Tyr reduced the susceptibility of OTV to N1. In contrast to the binding free energies, the net charges of Glu276 and Arg224, making charge-charge interactions with Glu276, were established to be more sensitive to detecting subtle conformational differences induced at the key residue Glu276 by the His274X mutations. This study provides deeper insights into the possibility of developing viable drug-resistant mutants.     

Inhibition of viral group-1 and group-2 neuraminidases by oseltamivir: A comparative structural analysis by the ScrewFit algorithm

The viral surface glycoprotein neuraminidase (NA) allows the influenza virus penetration and the egress of virions. NAs are classified as A, B, and C. Type-A NAs from influenza virus are subdivided into two phylogenetically distinct families, group-1 and group-2. NA inhibition by oseltamivir represents a therapeutic approach against the avian influenza virus H5N1. Here, structural bases for oseltamivir recognition by group-1 NA1, NA8 and group-2 NA9 are highlighted by the ScrewFit algorithm for quantitative structure comparison. Oseltamivir binding to NA1 and NA8 affects the geometry of Glu119 and of regions Arg130-Ser160, Val240-Gly260, and Asp330-Glu382, leading to multiple NA conformations. Additionally, although NA1 and NA9 share almost the same oseltamivir-bound final conformation, they show some relevant differences as suggested by the ScrewFit algorithm. These results indicate that the design of new NA inhibitors should take into account these family-specific effects induced on the whole structure of NAs.     

Decreased neuraminidase activity is important for the adaptation of H5N1 influenza virus to human airway epithelium

Highly pathogenic avian H5N1 influenza viruses remain a pandemic threat. Antiviral drugs such as neuraminidase (NA) inhibitors will be crucial for disease control in the event of a pandemic. Should drug-resistant H5N1 viruses develop, all defense strategies will be compromised. To determine the likelihood and mechanisms of emergence of NA inhibitor-resistant H5N1 variants in humans, we serially passaged two H5N1 viruses, A/Hong Kong/213/03 and A/Turkey/65-1242/06, in normal human bronchial epithelial (NHBE) cells in the presence of oseltamivir, zanamivir, or peramivir. To monitor the emergence of changes associated with the adaptation of H5N1 viruses to humans, we passaged the strains in the absence of drugs. Under pressure of each NA inhibitor, A/Turkey/65-1242/06 developed mutations in the hemagglutinin (HA) (H28R and P194L/T215I) and NA (E119A) proteins that reduced virus binding to α2,3-sialyl receptor and NA activity. Oseltamivir pressure selected a variant of A/Hong Kong/213/03 virus with HA P194S mutation that decreased viral binding to α2,6 receptor. Under peramivir pressure, A/Hong Kong/213/03 virus developed a novel NA mutation, R156K, that reduced binding to all three drugs, caused about 90% loss of NA activity, and compromised replication in NHBE cells. Both strains were eliminated in NHBE cells when they were cultivated in the absence of drugs. Here, we show for the first time that decreased NA activity mediated through NA inhibitors is essential for the adaptation of pandemic H5N1 influenza virus to humans. This ability of decreased NA activity to promote H5N1 infection underlines the necessity to optimize management strategies for a plausible H5N1 pandemic.     

Computational investigation of interaction mechanisms between juglone and influenza virus surface glycoproteins

Surface glycoproteins of influenza virus [haemagglutinin (HA) and neuraminidase (NA)] are vital target proteins in current rational drug designs. Here, the molecular recognitions between juglone and A/H5N1 influenza virus membrane glycoproteins were studied through flexible docking and molecular dynamic simulations. The results revealed that juglone has the binding specificity to HA (H5) and NA (N1), especially the spatial match of 2-cyclohexene-1,4-dione ring. N1 rather than H5 protein is responsible for the binding, with the interaction energies of ? 72.48 and ? 41.91 kcal mol^? 1, respectively. The residues Arg152, Arg156, Glu276, Glu277 and Arg292 of N1 protein had important roles during the binding process. Compared with other NA inhibitors, juglone is a potential source of anti-influenza ingredients, with better interaction energy and relatively smaller size. In addition, this work also pointed out how to effectively modify the functional groups of juglone. We hope that the results will aid our understanding of recognitions involving influenza surface glycoproteins with the phenolic compounds and warrant the experimental aspects to design novel anti-influenza drugs.     

Computational studies of H5N1 influenza virus resistance to oseltamivir

Influenza A (H5N1) virus is one of the world's greatest pandemic threats. Neuraminidase (NA) inhibitors, oseltamivir and zanamivir, prevent the spread of influenza, but drug‐resistant viruses have reduced their effectiveness. Resistance depends on the binding properties of NA‐drug complexes. Key residue mutations within the active site of NA glycoproteins diminish binding, thereby resulting in drug resistance. We performed molecular simulations and calculations to characterize the mechanisms of H5N1 influenza virus resistance to oseltamivir and predict potential drug‐resistant mutations. We examined two resistant NA mutations, H274Y and N294S, and one non‐drug‐resistant mutation, E119G. Six‐nanosecond unrestrained molecular dynamic simulations with explicit solvent were performed using NA‐oseltamivir complexes containing either NA wild‐type H5N1 virus or a variant. MM_PBSA techniques were then used to rank the binding free energies of these complexes. Detailed analyses indicated that conformational change of E276 in the Pocket 1 region of NA is a key source of drug resistance in the H274Y mutant but not in the N294S mutant.     

A computational H5N1 neuraminidase model and its binding to commercial drugs

In order to understand the mechanisms of ligand binding and interaction between two commercial drugs (ligands), zanamivir and oseltamivir and H5N1 Influenza Virus Neuraminidase subtype N1, a three-dimensional model of N1-ligand (GenBank accession no. AAS654617) was initially generated by homology modeling using the 13 high-resolution X-ray structures of neuraminidase N2 and N9 as the template. With the aid of the molecular mechanics and molecular dynamics methods, the final implicit solvent refined model was obtained. It was, then, assessed by PROCHECK, PROSA and VERIFY3D. With this model, a flexible docking study was performed. The results show strong hydrogen bond interactions between the glycerol side chains of zanamivir and Arg29 of the N1. Common hydrogen bonds between the carboxyl groups and Arg279 were found for both drugs. It was also found that the Glu30, Asp62, Arg63, Arg204, Trp310, Tyr313, Glu336, Ile338, Trp348, Ala349 were observed to facilitate the enzyme-ligand non-bonding interactions as they are located within the radius of 5 Å from all atoms of both drugs. Charge distribution was evaluated using the semi-empirical AM1 method. The results show that the total net charges of the –NH side chain of zanamivir is less negative than that of oseltamivir. This is in contrast to what is observed for the amide and alkyl (ether/glycerol) side chains. In comparison of the binding free energies between the X-ray N2-ligand and N9-ligand complexes, N1-ligand binding is found to be less potent than N2 and N9 subtypes, while N2-ligand and N9-ligand are roughly comparable. In addition, it is interesting to observe that the binding free energies for all three subtypes of the zanamivir complexes are lower than those of oseltamivir.     

Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.     

Effect of an asparagine-to-serine mutation at position 294 in neuraminidase on the pathogenicity of highly pathogenic H5N1 influenza A virus

Like the histidine-to-tyrosine substitution at position 274 in neuraminidase (NA H274Y), an asparagine-to-serine mutation at position 294 in this protein (NA N294S) confers oseltamivir resistance to highly pathogenic H5N1 influenza A viruses. However, unlike viruses with the NA H274Y mutation, the properties of viruses possessing NA N294S are not well understood. Here, we assessed the effect of the NA N294S substitution on the replication and pathogenicity of human H5N1 viruses and on the efficacy of the NA inhibitors oseltamivir and zanamivir in mouse and ferret models. Although NA N294S-possessing H5N1 viruses were attenuated in mice and ferrets compared to their oseltamivir-sensitive counterparts, one of the infected ferrets died from systemic infection, demonstrating the potential lethality in ferrets of oseltamivir-resistant H5N1 viruses with the NA N294S substitution. The efficacy of oseltamivir, but not that of zanamivir, against an NA N294S-possessing virus was substantially impaired both in ferrets and in vitro. These results demonstrate the considerable pathogenicity of NA N294S substitution-possessing H5N1 viruses and underscore the importance of monitoring the emergence of the NA N294S mutation in circulating H5N1 viruses.     

Computational study on new natural polycyclic compounds of H1N1 influenza virus neuraminidase

A new strain of influenza A (H1N1) virus is a major cause of morbidity and mortality around the world. The neuraminidase of the influenza virus has been the most potential target for the anti-influenza drugs such as oseltamivir and zanamivir. However, the emergence of drug-resistant variants of these drugs makes a pressing need for the development of new neuraminidase inhibitors for controlling illness and transmission. Here a 3D structure model of H1N1 avian influenza virus neuraminidase type 1 (N1) was constructed based on the structure of the template H5N1 avian influenza virus N1. Upon application of virtual screening technique for N1 inhibitors, two novel compounds (ZINC database ID: ZINC02128091, ZINC02098378) were found as the most favorable interaction energy with N1. Docking results showed that the compounds bound not only in the active pocket, but also in a new hydrophobic cave which contains Arg368, Trp399, Ile427, Pro431 and Lys432 of N1. Our result suggested that both of the screened compounds containing the hydrophobic group bring a strong conjugation effect with Arg293, Arg368 Lys432 of N1 by pi-pi interaction. However, the control inhibitors zanamivir and oseltamivir do not have this effect. The details of N1-compound binding structure obtained will be valuable for the development of a new anti-influenza virus agent.     

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.     

Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses

An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 serotype neuraminidase (NA) has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins (HAs) have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/Shanghai/1/2013. NAs from these two viruses are distinct as the A/Shanghai/1/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance (over 100 000-fold) using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 (R294-containing) and A/Shanghai/1/2013 N9 (K294-containing). R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.     

Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses

If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.     

Understanding of known drug-target interactions in the catalytic pocket of neuraminidase subtype N1

To provide detailed information and insight into the drug-target interaction, structure, solvation, and dynamic and thermodynamic properties, the three known-neuraminidase inhibitors-oseltamivir (OTV), zanamivir (ZNV), and peramivir (PRV)-embedded in the catalytic site of neuraminidase (NA) subtype N1 were studied using molecular dynamics simulations. In terms of ligand conformation, there were major differences in the structures of the guanidinium and the bulky groups. The atoms of the guanidinium group of PRV were observed to form many more hydrogen bonds with the surrounded residues and were much less solvated by water molecules, in comparison with the other two inhibitors. Consequently, D151 lying on the 150-loop (residues 147-152) of group-1 neuraminidase (N1, N4, N5, and N8) was considerably shifted to form direct hydrogen bonds with the --OH group of the PRV, which was located rather far from the 150-loop. For the bulky group, direct hydrogen bonds were detected only between the hydrophilic side chain of ZNV and residues R224, E276, and E277 of N1 with rather weak binding, 20-70% occupation. This is not the case for OTV and PRV, in which flexibility and steric effects due to the hydrophobic side chain lead to the rearrangement of the surrounded residues, that is, the negatively charged side chain of E276 was shifted and rotated to form hydrogen bonds with the positively charged moiety of R224. Taking into account all the ligand-enzyme interaction data, the gas phase MM interaction energy of -282.2 kcal/mol as well as the binding free energy (DeltaG(binding)) of -227.4 kcal/mol for the PRV-N1 are significantly lower than those of the other inhibitors. The ordering of DeltaG(binding) of PRV < ZNV < OTV agrees well with the ordering of experimental IC(50) value.     

Structural and functional analysis of laninamivir and its octanoate prodrug reveals group specific mechanisms for influenza NA inhibition

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.     

Long time scale GPU dynamics reveal the mechanism of drug resistance of the dual mutant I223R/H275Y neuraminidase from H1N1-2009 influenza virus

Multidrug resistance of the pandemic H1N1-2009 strain of influenza has been reported due to widespread treatment using the neuraminidase (NA) inhibitors, oseltamivir (Tamiflu), and zanamivir (Relenza). From clinical data, the single I223R (IR(1)) mutant of H1N1-2009 NA reduced efficacy of oseltamivir and zanamivir by 45 and 10 times, (1) respectively. More seriously, the efficacy of these two inhibitors against the double mutant I223R/H275Y (IRHY(2)) was significantly reduced by a factor of 12?374 and 21 times, respectively, compared to the wild-type.(2) This has led to the question of why the efficacy of the NA inhibitors is reduced by the occurrence of these mutations and, specifically, why the efficacy of oseltamivir against the double mutant IRHY was significantly reduced, to the point where oseltamivir has become an ineffective treatment. In this study, 1 μs of molecular dynamics (MD) simulations was performed to answer these questions. The simulations, run using graphical processors (GPUs), were used to investigate the effect of conformational change upon binding of the NA inhibitors oseltamivir and zanamivir in the wild-type and the IR and IRHY mutant strains. These long time scale dynamics simulations demonstrated that the mechanism of resistance of IRHY to oseltamivir was due to the loss of key hydrogen bonds between the inhibitor and residues in the 150-loop. This allowed NA to transition from a closed to an open conformation. Oseltamivir binds weakly with the open conformation of NA due to poor electrostatic interactions between the inhibitor and the active site. The results suggest that the efficacy of oseltamivir is reduced significantly because of conformational changes that lead to the open form of the 150-loop. This suggests that drug resistance could be overcome by increasing hydrogen bond interactions between NA inhibitors and residues in the 150-loop, with the aim of maintaining the closed conformation, or by designing inhibitors that can form a hydrogen bond to the mutant R223 residue, thereby preventing competition between R223 and R152.     

Applications of the ArgusLab4/AScore protocol in the structure-based binding affinity prediction of various inhibitors of group-1 and group-2 influenza virus neuraminidases (NAs)

Using the crystal structures of inhibitors bound to either group-2 or group-1 neuraminidases (NAs), AScore/ShapeDock (GaDock) scoring was shown to identify the binding modes in agreement with the experiment for all inhibitors docked in their own NA/inhibitor crystal structures. To investigate the effect of small changes in protein structure on predicted binding modes, in a set of 132 docking experiments (11 inhibitors docked in 12 group-2 NA structures), AScore/ShapeDock (GaDock) identified the correct binding modes of 116 complexes. In a total of 88 docking experiments (8 inhibitors docked in 11 group-1 NA structures), AScore/ShapeDock predicted 80 binding modes correctly. Flexible AScore/ShapeDock docking, as quite reproducible, is suggested to be convenient for designing novel H5N1 inhibitors.     

Crystal structure of swine major histocompatibility complex class I SLA-1 0401 and identification of 2009 pandemic swine-origin influenza A H1N1 virus cytotoxic T lymphocyte epitope peptides

The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1 0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIV(NW9); NSDTVGWSW) or Ebola virus (Ebola(AY9); ATAAATEAY) were determined in this study. The overall peptide-SLA-1 0401 structures resemble, as expected, the general conformations of other structure-solved peptide major histocompatibility complexes (pMHC). The major distinction of SLA-1 0401 is that Arg(156) has a "one-ballot veto" function in peptide binding, due to its flexible side chain. S-OIV(NW9) and Ebola(AY9) bind SLA-1 0401 with similar conformations but employ different water molecules to stabilize their binding. The side chain of P7 residues in both peptides is exposed, indicating that the epitopes are "featured" peptides presented by this SLA. Further analyses showed that SLA-1 0401 and human leukocyte antigen (HLA) class I HLA-A 0101 can present the same peptides, but in different conformations, demonstrating cross-species epitope presentation. CTL epitope peptides derived from 2009 pandemic S-OIV were screened and evaluated by the in vitro refolding method. Three peptides were identified as potential cross-species influenza virus (IV) CTL epitopes. The binding motif of SLA-1 0401 was proposed, and thermostabilities of key peptide-SLA-1 0401 complexes were analyzed by circular dichroism spectra. Our results not only provide the structural basis of peptide presentation by SLA I but also identify some IV CTL epitope peptides. These results will benefit both vaccine development and swine organ-based xenotransplantation.     

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.     

Pseudovirus-based neuraminidase inhibition assays reveal potential H5N1 drug-resistant mutations

The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. F or a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identifi ed to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.