Mutational characterization of HBV reverse transcriptase gene and the genotype-phenotype correlation of antiviral resistance among Chinese chronic hepatitis B patients.

Background and Aims: The drug resistance of hepatitis B virus (HBV) originates from mutations within HBV reverse transcriptase (RT) region during the prolonged antiviral therapy. So far, the characteristics of how these mutations distribute and evolve in the process of therapy have not been clarified yet. Thus we aimed to investigate these characteristics and discuss their contributing factors. Methods: HBV RT region was direct-sequenced in 285 treatment-naive and 214 post-treatment patients. Mutational frequency and Shannon entropy were calculated to identify the specific mutations differing between genotypes or treatment status. A typical putative resistance mutation rtL229V was further studied using in-vitro susceptibility assays and molecular modeling. Results: The classical resistance mutations were rarely detected among treatment-naive individuals, while the putative resistance mutations were observed at 8 AA sites. rtV191I and rtA181T/V were the only resistance mutations identified as genotype-specific mutation. Selective pressure of drug usage not only contributed to the classical resistance mutations, but also induced the changes at a putative resistance mutation site rt229. rtL229V was the major substitution at the site of rt229. It contributed to the most potent suppression of viral replication and reduced the in-vitro drug susceptibility to entecavir (ETV) when coexisting with rtM204V, consistent with the hypothesis based on the molecular modeling and clinical data analysis. Conclusions: The analysis of mutations in RT region under the different circumstances of genotypes and therapy status might pave the way for a better understanding of resistance evolution, thus providing the basis for a rational administration of antiviral therapy.

HBV polymerase-derived peptide exerts an anti-HIV-1 effect by inhibiting the acetylation of viral integrase.

Here, we found that a 6-mer peptide, Poly6, derived from the hepatitis B virus (HBV), which overlaps with a polymerase corresponding to a preS1 deletion reported to contribute to liver disease progression, can elicit an antiviral effect against human immunodeficiency virus (HIV)-1 by inhibiting HIV-1 integrase (IN) activity of infected cells. Mechanistic studies revealed that the anti-HIV-1 effects of Poly6 occurred via the inhibition of integration, which resulted from the inhibition of acetylation of HIV-1 IN possibly by downregulation of p300 histone acetyltransferase. Our data suggest the potential therapeutic use of a 6-mer HBV-derived peptide, Poly6, as an anti-HIV-1 agent to suppress HIV-1 infection via inhibiting integrase activity.

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase.

Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain. IMPORTANCE: HBV is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the TP domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and high-resolution structure. This study mapped the TP functions according to predicted secondary structure, where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.

Different Variants in Reverse Transcriptase Domain Determined by Ultra-deep Sequencing in Treatment-naïve and Treated Indonesian Patients Infected with Hepatitis B Virus.

A nucleos(t)ide analog (NA) is the common antiviral drug available for directly treating hepatitis B virus (HBV) infection. However, its application has led to the emergence of NA-resistant mutations mostly in a conserved region of the reverse transcriptase domain of HBV polymerase. Harboring NA-resistant mutations decreases drug effectiveness and increases the frequency of end-stage liver disease. The invention of next-generation sequencing that can generate thousands of sequences from viral complex mixtures provides opportunities to detect minor changes and early viral evolution under drug stress. The present study used ultra-deep sequencing to evaluate discrepant quasispecies in the reverse transcriptase domain of HBV including NA-resistant hotspots between seven treatment-naïve Indonesian patients infected with HBV and five at the early phase of treatment. The most common sub-genotype was HBV B3 (83.34%). The substitution rate of variants determined among amino acids with a ratio of ≥ 1% changes was higher among the population in conserved regions (23.19% vs. 4.59%, P = 0.001) and in the inter-reverse transcriptase domain (23.95% vs. 2.94%, P = 0.002) in treatment naïve, than in treated patients. Nine hotspots of antiviral resistance were identified in both groups, and the mean frequency of changes in all patients was < 1%. The known rtM204I mutation was the most frequent in both groups. The lower rate of variants in HBV quasispecies in patients undergoing treatment could be associated with virus elimination and the extinction of sensitive species by NA therapy. The present findings imply that HBV quasispecies dynamically change during treatment.

Mechanism of Adefovir, Tenofovir and Entecavir Resistance: Molecular Modeling Studies of How A Novel Anti-HBV Agent (FMCA) Can Overcome the Drug Resistance.

Regardless of significant improvement in the area of anti-HBV therapy, resistance and cross-resistance against available therapeutic agents are the major consideration in drug discovery of new agents. The present study is to obtain the insight of the molecular basis of drug resistance conferred by the B and C domain mutations of HBV-polymerase on the binding affinity of four anti-HBV agents [Adefovir (ADV), Tenofovir (TNF), Entecavir (ETV) & 2'-Fluoro-6'-methylene-carbocyclic adenosine (FMCA)]. In this regard, homology modeled structure of HBV polymerase was used for minimization, conformational search and Glide XP docking followed by binding energy calculation on wild-type as well as on mutant HBV-polymerases (N236T, L180M+M204V+S202G & A194T). Our studies suggest a significant correlation between the fold resistances and the binding affinity of anti-HBV nucleosides. The domain B residue, L180 is indirectly associated with other active-site hydrophobic residues such as A87, F88 and M204, whereas the domain C residue, M204 is closely associated with sugar/pseudosugar ring positioning in the active site. These hydrophobic residues can directly influence the interaction of the incoming nucleoside triphosphates and change the binding efficacy. The carbohydrate ring part of natural substrate dATP, dGTP, FMCA and ETV, are occupied in similar passion in the grooves of HBV polymerase active site. The exocyclic double bond of Entecavir and FMCA occupies in the backside hydrophobic pocket (made by residues A87, F88, L180and M204), which enhances the overall binding affinity. Additional hydrogen bonding interaction of 2'-fluorine of FMCA with R41 residue of polymerase promotes a positive binding in wild-type as well as in ADVr, ETVr and TNFr with respect to that of entecavir.

AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

[Cell proteins that potentially interact with HBV polymerase were identified by co-immunoprecipitation-based LC-MS/MS identification and IPA].

Hepatitis B virus (HBV) is a major cause of chronic liver disease, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. HBV polymerase (Pol) is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma. Protein-protein interactions appears to be crucial for its role. The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique. The HBV Pol gene was amplified by polymerase chain reaction (PCR) and cloned into pCDNA3.1(+). The recombinant plasmid pCDNA3. 1(+)-Pol-flag was transfected into HeLa cells. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol. Eleven of these have previously been reported as proteins that interact with HBV Pol. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol. Among these proteins, four proteins may play a role in three major molecular cellular networks, and are therefore worthy of further investigation.

Large-scale production and structural and biophysical characterizations of the human hepatitis B virus polymerase.

UNLABELLED: Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies--broad-spectrum antivirals--are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of ß-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCE: The viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.

Identification and characterization of a novel bipartite nuclear localization signal in the hepatitis B virus polymerase.

AIM: To characterize the nuclear import of hepatitis B virus (HBV) polymerase (P) and its relevance for the viral life cycle. METHODS: Sequence analysis was performed to predict functional motives within P. Phosphorylation of P was analyzed by in vitro phosphorylation. Phosphorylation site and nuclear localization signal (NLS) were destroyed by site directed mutagenesis. Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding. Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV. RESULTS: We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinase II (CKII) phosphorylation site located within the terminal protein domain (TP) of the HBV polymerase. Inhibition of CKII impairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase. Binding of the import factor karyopherin-α2 to the polymerase depends on its CKII-mediated phosphorylation of the bipartite NLS. In HBV-infected primary Tupaia hepatocytes CKII inhibition in the early phase (post entry phase) of the infection process prevents the establishment of the infection. CONCLUSION: Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.

Identification and classification of feline endogenous retroviruses in the cat genome using degenerate PCR and in silico data analysis.

The purpose of this study was to identify and classify endogenous retroviruses (ERVs) in the cat genome. Pooled DNA from five domestic cats was subjected to degenerate PCR with primers specific to the conserved retroviral pro/pol region. The 59 amplified retroviral sequences were used for in silico analysis of the cat genome (Felis_catus-6.2). We identified 219 ERV γ and ß elements from cat genome contigs, which were classified into 42 ERV γ and 4 ß families and further analysed. Among them, 99 γ and 5 ß ERV elements contained the complete retroviral structure. Furthermore, we identified 757 spuma-like ERV elements based on the sequence homology to murine (Mu)ERV-L and human (H)ERV-L. To the best of our knowledge, this is the first detailed genome-scale analysis examining Felis catus endogenous retroviruses (FcERV) and providing advanced insights into their structural characteristics, localization in the genome, and diversity.

Carbonyl J acid derivatives block protein priming of hepadnaviral P protein and DNA-dependent DNA synthesis activity of hepadnaviral nucleocapsids.

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 µM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

Kinetic characterization of newly discovered inhibitors of various constructs of human T-cell leukemia virus-1 (HTLV-1) protease and their effect on HTLV-1-infected cells.

BACKGROUND: Human T-cell leukemia virus-1 (HTLV-1) was the first identified human retrovirus and was shown to be associated with diseases such as adult T-cell leukemia lymphoma and tropical spastic paraparesis/HTLV-1 associated myelopathy. Retroviral proteases (PRs) are essential for viral replication by processing viral Gag and Gag-(Pro)-Pol polyproteins during maturation. Full-length HTLV-1 PR is 125 residues long; whether the C-terminal region is required for catalytic activity is still controversial. In this study, we characterized the effect of C-terminal amino acids of HTLV-1 PR for PR activity and examined the binding of compounds identified by in silico screening. One compound showed inhibition against the virus in infected cells. METHODS: Truncated (116-, 121- and 122-residue) forms of HTLV-1 PR were prepared and proteins from expression of the genes were purified. In silico screening was performed by docking small molecules into the active site of HTLV-1 PR. The kinetic constants k(cat), K(m), k(cat)/K(m) and inhibition constants K(i) for inhibitors identified by the computational screening were determined. Western blot and ELISA analyses were used to determine the effect of the most potent PR inhibitors on HTLV-1 protein processing in infected cells. RESULTS: The constructs showed similar catalytic efficiency constants (k(cat)/K(m)); thus HTLV-1 PR C-terminal amino acids are not essential for full activity. Computational screening revealed new PR inhibitors and some were shown to be inhibitory in enzyme assays. In HTLV-1-infected cells, one of the small molecules inhibited HTLV-1 gag cleavage and decreased the amount of HTLV-1 p19 produced in the cells. CONCLUSIONS: We have identified an HTLV-1 PR inhibitor that is biologically functional. Inhibitor screening will continue to develop possible drugs for therapy of HTLV-1 infection.

Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome.

Hepatitis B virus (HBV) pregenomic RNA contains a hairpin structure (ε) located in the preCore region, essential for viral replication. ε stability is enhanced by the presence of preCore variants and ε is recognized by the HBV polymerase (Pol). Mutations in the retrotranscriptase domain (YMDD) of Pol are associated with treatment resistance. The aim of this study was to analyze the preCore region and YMDD motif by ultra-deep pyrosequencing (UDPS). To evaluate the UDPS error rate, an internal control sequence was inserted in the amplicon. A newly developed technique enabled simultaneous analysis of the preCore region and Pol in the same viral genome, as well as the conserved sequence of the internal control. Nucleotide errors in HindIII yielded a UDPS error rate <0.05%. UDPS study confirmed the possibility of simultaneous detection of preCore and YMDD mutations, and demonstrated the complexity of the HBV quasispecies and cooperation between viruses. Thermodynamic stability of the ε signal was found to be the main constraint for selecting main preCore mutations. Analysis of ε-signal variability suggested the essential nature of the ε structural motif and that certain nucleotides may be involved in ε signal functions.

PAComplex: a web server to infer peptide antigen families and binding models from TCR-pMHC complexes.

One of the most adaptive immune responses is triggered by specific T-cell receptors (TCR) binding to peptide-major histocompatibility complexes (pMHC). Despite the availability of many prediction servers to identify peptides binding to MHC, these servers are often lacking in peptide-TCR interactions and detailed atomic interacting models. PAComplex is the first web server investigating both pMHC and peptide-TCR interfaces to infer peptide antigens and homologous peptide antigens of a query. This server first identifies significantly similar TCR-pMHC templates (joint Z-value ≥ 4.0) of the query by using antibody-antigen and protein-protein interacting scoring matrices for peptide-TCR and pMHC interfaces, respectively. PAComplex then identifies the homologous peptide antigens of these hit templates from complete pathogen genome databases (≥10(8) peptide candidates from 864,628 protein sequences of 389 pathogens) and experimental peptide databases (80,057 peptides in 2287 species). Finally, the server outputs peptide antigens and homologous peptide antigens of the query and displays detailed interacting models (e.g. hydrogen bonds and steric interactions in two interfaces) of hitTCR-pMHC templates. Experimental results demonstrate that the proposed server can achieve high prediction accuracy and offer potential peptide antigens across pathogens. We believe that the server is able to provide valuable insights for the peptide vaccine and MHC restriction. The PAComplex sever is available at http://PAcomplex.life.nctu.edu.tw.

Foamy retrovirus integrase contains a Pol dimerization domain required for protease activation.

Unlike orthoretroviruses, foamy retroviruses (FV) synthesize Pol independently of Gag. The FV Pol precursor is cleaved only once between reverse transcriptase (RT) and integrase (IN) by the protease (PR), resulting in a PR-RT and an IN protein. Only the Pol precursor, not the cleaved subunits, is packaged into virions. Like orthoretroviral PRs, FV PR needs to dimerize to be active. Previously, we showed that a Pol mutant lacking IN has defects in PR activity and Pol packaging into virions. We now show that introduction of a leucine zipper (zip) dimerization motif in an IN truncation mutant can restore PR activity, leading to Pol processing in cells. However, these zip mutants neither cleave Gag nor incorporate Pol into virions. We propose that IN is required for Pol dimerization, which is necessary for the creation of a functional PR active site.

Expression and purification of a functional human hepatitis B virus polymerase.

AIM: To identify a method for efficient large-scale purification of functional hepatitis B virus polymerase (HBV-Pol) without addition of cellular factors. METHODS: Full-length HBV-Pol (843 amino acids) tagged with 5' end Polyhistidine was expressed at a high level in an Escherichia coli (E. coli) system. Sodium dodecyl sulfate lysis buffer was utilized to dissolve insoluble HBV-Pol, and Ni-NTA resin affinity chromatography was utilized for HBV-Pol purification. Most recombinant HBV-Pol was eluted with 100 mmol/L imidazole in the presence of NP-40, a weak detergent that keeps HBV-Pol in solution. A reducing agent was utilized throughout the purification steps to keep soluble HBV-Pol from redundant disulfide bond formation. RESULTS: The large-scale production of functional intact human HBV-Pol was achieved in an E. coli expression system. Purified HBV-Pol showed stable reverse transcriptase activity and DNA polymerase activity. The purified protein was of high purity and had stable reverse transcriptase activity. CONCLUSION: Large-scale production of HBV-Pol in pure form should facilitate crystallization and detailed analysis of the structure and mechanism of HBV-Pol. Ability of this purification approach to obtain human HBV-Pol in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.

[Analysis on mutation of S gene and P gene of hepatitis B virus in two counties of Sichuan Province].

OBJECTIVE: To analyze HBV S gene/P gene mutation in 2 counties (districts) of Sichuan province. METHODS: HBV DNA were extracted from sera positive both for HBsAg and HBeAg. After PCR and nucleotide sequencing, nucleotide/amino acid mutation in S and P gene were compared and analyzed. RESULTS: Of 47 serum samples from patients with chronic HBV infection, amino acid mutation in 'a' determinant occurred in 12 samples (25.53%,12/47), correlating with T126A, I126T/S, P127T, T131N, M133L, M133T and T140I; high proportion of mutation clustered in first loop of 'a' determinant (92.86%,13/14), rtV207I mutation in C domain of reverse transcription occured in one sample. CONCLUSION: Naturally occurring mutation in 'a' determinant clustered predominantly in the first loop and usually associated with altered antigenicity, posing a potential threat to successfully vaccinated individuals; Lamivudine-resistant mutant might occur in patient even without nucleotide analogue treatment.

[Resistance in hepatitis B virus]. / Resistencias en el virus de la hepatitis B.

The development hepatitis B virus (HBV) polymerase inhibitors has revolutionised the treatment of chronic HBV infection. However, the emergence of resistance mutations can compromise their clinical efficacy and it is mandatory to know the mechanisms of these resistances, its clinical implications, strategies for prevention and how to deal with the rescue. Since HBV has a high degree of replication and a high error rate, during their life cycle it will produce a large number of punctual mutations in individuals with active replication. Due to the large size of the HBV genome, all the possible changes may occur daily and should be screened before starting any antiviral therapy. Therefore, in individuals infected with HBV there is a mixture of similar viruses that evolves over time (quasispecies), some of which are carriers of resistance mutations to antivirals, which explains why they can be selected quickly after exposure to drug. Of the five drugs approved in Europe for the treatment of hepatitis B, three of them (lamivudine, adefovir and entecavir) are likely to be affected directly by these mutations, as well as other active drugs, such as telbivudine, tenofovir and the emtricitabine. The characterization of the resistance mutations is helpful for the prevention and the optimization of antiviral therapies.

Recombination analysis and structure prediction show correlation between breakpoint clusters and RNA hairpins in the pol gene of human immunodeficiency virus type 1 unique recombinant forms.

Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly throughout the genome, leading to viral recombination. Some recombination hotspots have been identified and found to correlate with RNA structure or sequence features. The aim of this study was to evaluate the presence of recombination hotspots in the pol gene of HIV-1 and to assess their correlation with the underlying RNA structure. Analysis of the recombination pattern and breakpoint distribution in a group of unique recombinant forms (URFs) detected two recombination hotspots in the pol region. Two stable and conserved hairpins were consistently predicted corresponding to the identified hotspots using six different RNA-folding algorithms on the URF parental strains. These findings suggest that such hairpins may play a role in the higher recombination rates detected at these positions.

Understanding the molecular basis of HBV drug resistance by molecular modeling.

Despite the significant successes in the area of anti-HBV agents, resistance and cross-resistance against available therapeutics are the major hurdles in drug discovery. The present investigation is to understand the molecular basis of drug resistance conferred by the B and C domain mutations of HBV-polymerase on the binding affinity of five anti-HBV agents [lamivudine (3TC, 1), adefovir (ADV, 2), entecavir (ETV, 3), telbivudine (LdT, 4) and clevudine (l-FMAU, 5)]. In this regard, homology modeled structure of HBV-polymerase was used for minimization, conformational search and induced fit docking followed by binding energy calculation on wild-type as well as on mutant HBV-polymerases (L180M, M204V, M204I, L180M+M204V, L180M-M204I). Our studies suggest a significant correlation between the fold resistances and the binding affinity of anti-HBV nucleosides. The binding mode studies reveals that the domain C residue M204 is closely associated with sugar/pseudosugar ring positioning in the active site. The positioning of oxathiolane ring of 3TC (1) is plausible due the induced fit orientation of the M204 residue in wild-type, and further mutation of M204 to V204 or I204 reduces the final binding affinity which leads to the drug resistance. The domain B residue L180 is not directly close ( approximately 6A) to the nucleoside/nucleoside analogs, but indirectly associated with other active-site hydrophobic residues such as A87, F88, P177 and M204. These five hydrophobic residues can directly affect on the incoming nucleoside analogs in terms of its association and interaction that can alter the final binding affinity. There was no sugar ring shifting observed in the case of adefovir (2) and entecavir (3), and the position of sugar ring of 2 and 3 is found similar to the sugar position of natural substrate dATP and dGTP, respectively. The exocyclic double bond of entecavir (3) occupied in the backside hydrophobic pocket (made by residues A87, F88, P177, L180 and M204), which enhances the overall binding affinity. The active site binding of LdT (4) and l-FMAU (5) showed backward shifting along with upward movement without enforcing M204 residue and this significant different binding mode makes these molecules as polymerase inhibitors, without being incorporated into the growing HBV-DNA chain. Structural results conferred by these l- and d-nucleosides, explored the molecular basis of drug resistance which can be utilized for future anti-HBV drug discovery.