Katsumi Omagari

Possibility of Mutation Prediction of Influenza Hemagglutinin by Combination of Hemadsorption Experiment and Quantum Chemical Calculation for Antibody Binding

We have performed a quantum-chemical MP2/6-31G* calculation for the hemagglutinin (HA) antigen-antibody system of the H3N2 influenza virus with the fragment molecular orbital method, which provides one of the worlds largest ab initio electron-correlated calculations for biomolecular systems. On the basis of the calculated interfragment interaction energies (IFIEs) representing the molecular interactions between the amino acid residues in the antigen-antibody complex, we have identified those residues in the antigenic region E of HA protein that are significantly recognized by the Fab fragment of antibody with strongly attractive interactions. Combining these IFIE results with those of hemadsorption experiments by which the mutation-prohibited sites are specified has enabled us to explain most of the historical mutation data (five of six residues), which would thus provide a promising method for predicting the HA residues that have a high probability of forthcoming mutation.

Characterization of novel entecavir resistance mutations

BACKGROUND & AIMS Entecavir (ETV) is approved for the treatment of chronic hepatitis B virus (HBV) infections, but the virus can acquire resistance to the drug. This requires lamivudine resistance mutations (LAMr) and at least one additional mutation. Here, we characterized two novel mutations, rtI163V and rtA186T, associated with viral breakthrough (VBT) in an ETV-refractory patient. METHODS HBV from an ETV-refractory patient was sequenced, and newly identified mutations were inserted into a replication-competent clone by mutagenesis. Clones were analyzed for replication efficacy and susceptibility to ETV in vitro. Chimeric mice with human hepatocytes were inoculated with the patients serum at VBT, and monitored for viral mutation pattern using a next-generation sequencing approach. RESULTS RtI163V and rtA186T mutations were detected together with LAMr (rtL180M and rtM204V) at VBT. RtA186T plus LAMr reduced susceptibility to ETV more than 111.1-fold compared with the wild-type clone, while rtI163V plus LAMr resulted in a 20.4-fold reduction. RtA186T significantly reduced viral replication efficacy, while the rtI163V mutation rescued it. Interestingly, the viral mutation pattern in the chimeric mice indicated dominant (or selective) proliferation of a clone containing rtI163V and rtA186T mutations plus LAMr under ETV treatment. Three-dimensional docking simulation indicated that rtA186T reduced the binding affinity of the HBV polymerase to ETV. CONCLUSIONS VBT in this ETV-refractory patient is attributable to the novel ETV resistance mutations rtI163V and rtA186T. RtA186T was apparently responsible for ETV resistance but the selection of a clone with the double mutation plus LAMr suggests that rtI163V is required to sustain viral fitness.

A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins

Introduction of direct‐acting antivirals against hepatitis C virus (HCV) has provided a revolutionary improvement in the treatment outcome. In contrast to HCV, however, the strategy for developing new antiviral agents against hepatitis B virus (HBV), especially viral‐targeting compounds, is limited because HBV requires only four viral genes for its efficient replication/infection. Here, we identify an oligomeric flavonoid, proanthocyanidin (PAC) and its analogs, which inhibit HBV entry into host cells by targeting the HBV large surface protein (LHBs). Through cell‐based chemical screening, PAC was identified to inhibit HBV infection with little cytotoxic effect. PAC prevented the attachment of the preS1 region in the LHBs to its cellular receptor, sodium taurocholate cotransporting polypeptide (NTCP). PAC was shown to target HBV particles and impair their infectivity, whereas it did not affect the NTCP‐mediated bile acid transport activity. Chemical biological techniques demonstrated that PAC directly interacted with the region essential for receptor binding in the preS1 region in the LHBs protein. Importantly, PAC had a pan‐genotypic anti‐HBV activity and was also effective against a clinically relevant nucleoside analog‐resistant HBV isolate. We further showed that PAC augmented the ability of a nucleoside analog, tenofovir, to interrupt HBV spread over time in primary human hepatocytes by cotreatment. Moreover, derivative analysis could identify small molecules that demonstrated more‐potent anti‐HBV activity over PAC. Conclusion: PAC and its analogs represent a new class of anti‐HBV agents that directly target the preS1 region of the HBV large surface protein. These agents could contribute to the development of a potent, well‐tolerated, and broadly active inhibitor of HBV infection. (Hepatology 2017;65:1104‐1116).

Sialic Acid Recognition of the Pandemic Influenza 2009 H1N1 Virus: Binding Mechanism Between Human Receptor and Influenza Hemagglutinin

Quantum mechanical fragment molecular orbital calculations have been performed for receptor binding of the hemagglutinin protein of the recently pandemic influenza 2009 H1N1, A/swine/Iowa/1930, and A/Puerto Rico/8/1934 viruses to α2-6 linked sialyloligosaccharides, as analogs of human receptors. The strongest receptor binding affinity was observed for the 2009/H1N1pdm. The inter-fragment interaction energy analysis revealed that the amino acid mutation of 2009/H1N1pdm, Ser145Lys, was a major cause of such strong binding affinity. Strong ionic pair interaction between the sialic acid and Lys145 was observed only in the 2009/H1N1pdm, in addition to the hydrogen bond between the sialic acid and Gln226 observed in all the HAs. Therefore, pandemic 2009/H1N1pdm has been found to recognize the α2-6 receptor much stronger than the 1930-swine and 1934-human.

Effect of Hepatitis B Virus Reverse Transcriptase Variations on Entecavir Treatment Response

BACKGROUND Entecavir therapy often reduces hepatitis B virus (HBV) DNA to an undetectable level, but HBV DNA remain detectable in some patients. We investigated whether baseline HBV reverse transcriptase (rt) polymorphism and quasispecies complexity and diversity were associated with treatment response. METHODS Pretreatment HBV DNA levels, HBV rt sequence, serology, and quasispecies complexity and diversity from 305 entecavir-treated patients were determined. These data were tested for their association with year 1 virological outcome, defined by optimal response (undetectable HBV DNA; lower limit of detection, ≤12 IU/mL) or partial response (detectable HBV DNA). RESULTS Four rt variants were more frequently detected in the 64 partial responders than in the 241 optimal responders (all P < .05). Multivariate analysis revealed that high baseline HBV DNA level (P < .0001; odds ratio [OR], 2.32), HBV e antigen (HBeAg) positivity (P < .001; OR, 3.70), and rt124N (P = .002; OR, 3.06) were associated with a partial entecavir response. Compared with the optimal responders, the partial responders had a lower quasispecies complexity and diversity. CONCLUSIONS Apart from the known factors (high baseline HBV DNA level and HBeAg positivity), a novel single nucleotide polymorphism (rt124N) and lower quasispecies complexity and diversity were associated with partial entecavir response at year 1.

ΔG‐based prediction and experimental confirmation of SYCRP1‐binding sites on the Synechocystis genome

DNA‐binding sites for SYCRP1, which is a regulatory protein of the cyanobacterium Synechocystis sp. PCC6803, were predicted for the whole genome sequence by estimating changes in the binding free energy () for SYCRP1 for those sites. The values were calculated by summing ΔΔG values derived from systematic single base‐pair substitution experiments (symmetrical and cooperative binding model). Of the calculated binding sites, 23 sites with a value < 3.9 kcal·mol−1 located upstream or between the ORFs were selected as putative binding sites for SYCRP1. In order to confirm whether SYCRP1 actually binds to these binding sites or not, 11 sites with the lowest values were tested experimentally, and we confirmed that SYCRP1 binds to ten of the 11 sites with a ΔΔGtotal value < 3.9 kcal·mol−1. The best correlation coefficient between and the observed ΔΔGtotal for binding of SYCRP1 to those sites was 0.78. These results suggest that the ΔΔG values derived from systematic single base‐pair experiments may be used to screen for potential binding sites of a regulatory protein in the genome sequence.

Reactivity of human convalescent sera with influenza virus hemagglutinin protein mutants at antigenic site A.

How the antibodies of individual convalescent human sera bind to each amino acid residue at the antigenic sites of hemagglutinin (HA) of influenza viruses, and how the antigenic drift strains of influenza viruses are selected by human sera, is not well understood. In our previous study, it was found by a binding assay with a chimeric HA between A/Kamata/14/91 (Ka/91) and A/Aichi/2/68 that convalescent human sera, following Ka/91 like (H3N2) virus infection, bind to antigenic site A of Ka/91 HA. Here using chimeric HAs possessing single amino acid substitutions at site A, it was determined how those human sera recognize each amino acid residue at antigenic site A. It was found that the capacity of human sera to recognize amino acid substitutions at site A differs from one person to another and that some amino acid substitutions result in all convalescent human sera losing their binding capacity. Among these amino acid substitutions, certain ones might be selected by chance, thus creating successive antigenic drift. Phylogenetic analysis of the drift strains of Ka/91 showed amino acid substitutions at positions 133, 135 and 145 were on the main stream of the phylogenetic tree. Indeed, all of the investigated convalescent sera failed to recognize one of them.

Quantification of the HIV transcriptional activator complex in live cells by image-based protein-protein interaction analysis.

The virus‐encoded Tat protein is essential for HIV transcription in infected cells. The interaction of Tat with the cellular transcription elongation factor P‐TEFb (positive transcriptional elongation factor b) containing cyclin T1 (CycT1) and cyclin‐dependent kinase 9 (CDK9) is critical for its activity. In this study, we use the Fluoppi (fluorescent‐based technology detecting protein–protein interaction) system, which enables the quantification of interactions between biomolecules, such as proteins, in live cells. Quantitative measurement of the molecular interactions among Tat, CycT1 and CDK9 has showed that any third molecule enhances the binding between the other two molecules. These findings suggest that each component of the Tat:P‐TEFb complex stabilizes the overall complex, thereby supporting the efficient transcriptional elongation during viral RNA synthesis. These interactions may serve as appropriate targets for novel anti‐HIV therapy.

Effect of hepatitis B virus subgenotype on antiviral response in nucleoside‐treated hepatitis B envelope antigen‐positive patients

Previous studies have reported that hepatitis B virus (HBV) genotype is not a predictor of treatment response with nucleos(t)ide analog therapy. However, the impact of subgenotype on treatment response is unknown. The aim of this study is to identify the effect of HBV subgenotype on treatment response.