The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus.

Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.

2-Arylthio-5-iodo pyrimidine derivatives as non-nucleoside HBV polymerase inhibitors.

In this study, a series of 2-arylthio-5-iodo pyrimidine derivatives, as non-nucleoside hepatitis B virus inhibitors, were evaluated and firstly reported as potential anti-HBV agents. To probe the mechanism of active agents, DHBV polymerase was isolated and a non-radioisotopic assay was established for measuring HBV polymerase. The biological results demonstrated that 2-arylthio-5-iodo pyrimidine derivatives targeted HBV polymerase. In addition, pharmacophore models were constructed for future optimization of lead compounds. Further study will be performed for the development of non-nucleoside anti-HBV agents.

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance.

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.

Antiviral therapy of chronic hepatitis B.

Since the licensing of lamivudine in 1999, the treatment of chronic hepatitis B has been revolutionized by the introduction of oral nucleoside and nucleotide analogues (NAs), which act as inhibitors of the HBV polymerase. The effectiveness of the first of these substances was limited by incomplete response and resistance development in many patients, but today, highly potent substances are available that make a reliable and durable suppression of HBV replication, a reduction of necroinflammatory activity in the liver, and even a reversion of liver fibrosis achievable for almost all patients. Beyond that, NA treatment can prevent the development of hepatocellular carcinoma in many patients. HBeAg seroconversion appears in approximately 50% of all HBeAg-positive patients during NA treatment. However, the ideal treatment endpoint, the serologic loss of HBsAg, remains a rare event almost exclusively achievable for HBeAg-positive patients. After cessation of the treatment, HBV replication tends to relapse in most patients, which is why the duration of NA treatment is indefinite. Future treatment strategies should aim at tailoring individual NA treatment regimens to increase HBs loss rates and optimize treatment duration.

Effect of HBV polymerase inhibitors on renal function in patients with chronic hepatitis B.

BACKGROUND & AIMS: Therapy of chronic hepatitis B with HBV-polymerase inhibitors, in particular tenofovir or adefovir, may affect renal function. To assess renal function more accurately in the normal range, we used the recently validated, Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula to calculate the estimated glomerular filtration rate (eGFR). METHODS: Patient subgroups included: patients with HBV-monoinfection treated with lamivudine (n=36), adefovir (n=32), entecavir (n=32), or tenofovir (n=37). HBsAg-positive untreated patients (n=60) served as control. For comparison HIV-monoinfected patients treated with tenofovir (n=120) or zidovudine (n=52) based antiretroviral therapy and antiretroviral naive patients (n=109) were assessed. CKD-EPI equation was used to calculate eGFR. In a more sensitive approach, we modeled the individual change in eGFR over time with linear mixed effects models (LME). RESULTS: Yearly predicted median changes in individual eGFR according to the LME model were: HBV untreated -2.05 ml/min, HBV lamivudine -0.92 ml/min, HBV adefovir -1.02 ml/min, HBV entecavir -1.00 ml/min, and HBV tenofovir -0.92 ml/min (p<0.01 for HBV untreated vs. HBV treated). In HIV-monoinfected patients: HIV untreated -0.62 ml/min, HIV treated with tenofovir -2.64 ml/min, HIV treated with zidovudine -1.0 ml/min (p=0.017 for tenofovir vs. no treatment, p<0.001 for tenofovir vs. zidovudine). CONCLUSIONS: Therapy of HBV infection irrespective of medication seems to result in a milder decrease of renal function. In contrast tenofovir as part of HIV combination therapy seems to impair renal function in this Caucasian population.

Screening and Evaluation of Novel Compounds against Hepatitis B Virus Polymerase Using Highly Purified Reverse Transcriptase Domain.

Hepatitis B virus (HBV) polymerase seems to be very hard to express and purify sufficiently, which has long hampered the generation of anti-HBV drugs based on the nature of the polymerase. To date, there has been no useful system developed for drug screening against HBV polymerase. In this study, we successfully obtained a highly purified reverse transcriptase (RT) domain of the polymerase, which has a template/primer and substrate binding activity, and established a novel high-throughput screening (HTS) system using purified RT protein for finding novel polymerase inhibitors. To examine whether the assay system provides reliable results, we tested the small scale screening using pharmacologically active compounds. As a result, the pilot screening identified already-known anti-viral polymerase agents. Then, we screened 20,000 chemical compounds and newly identified four hits. Several of these compounds inhibited not only the HBV RT substrate and/ template/primer binding activity, but also Moloney murine leukemia virus RT activity, which has an elongation activity. Finally, these candidates did show to be effective even in the cell-based assay. Our screening system provides a useful tool for searching candidate inhibitors against HBV.

Therapy of chronic hepatitis B: new goals and new treatments.

Treatment of chronic hepatitis B has shown a rapid development in the last years leading to a shift of treatment strategies from interferon to hepatitis B virus (HBV)-polymerase inhibitors. In particular, treatment with HBV-polymerase inhibitors has changed the indication on how to treat a patient and when to stop therapy. Long-term treatment with HBV-polymerase inhibitors may often be required, even if it raises the possibility of resistance and subsequent treatment failure. This review provides a strategy on how to manage HBV therapy with the currently available treatment options.

Rosmarinic acid is a novel inhibitor for Hepatitis B virus replication targeting viral epsilon RNA-polymerase interaction.

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the "two phenolic hydroxyl groups at both ends" and the "caffeic acid-like structure" of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.

[Human monoclonal antibody inhibiting reverse transcriptase activity of hepatitis B virus polymerase protein].

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1 x 10(10), human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1 micro g of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15 x 10(-8) M to 1.75 x 10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

Effects of the combined administration of entecavir and adefovir dipivoxil to improve hepatic fibrosis in hepatitis B patients with interferon resistance.

BACKGROUND: To observe the effect of the combined administration of entecavir and adefovir dipivoxil to improve hepatic fibrosis in hepatitis B patients with interferon resistance. METHODS: This study comprised 90 hepatitis B patients with hepatic fibrosis and interferon (IFN) resistance who were admitted in the hospital's department of infectious disease for diagnosis and treatment between January 2013 and September 2015. They were randomly divided into two groups in accordance with the random number table: the combination treatment group (N.=45) and the entecavir group (N.=45). They were observed for any variations in the indexes of liver function and fibrosis, as well as the Model for end-stage liver disease (MELD) scores, before and after treatment. RESULTS: After treatment, the levels of the indexes in both groups (the combination treatment group vs. the entecavir group) were as follows: bilirubin (67.5±7.7 vs. 82.4±13.5 µmol/L); International Normalized Ratio (INR) (1.21±0.8 vs. 1.14±0.7); creatinine (147.3±12.4 vs. 287.4±21.6 mg/dL); GGT (67.4±23.2 vs. 88.4±23.7 U/L); and ALT (63.4±40.8 vs. 96.5±23.5 U/L). In comparison of the indexes of hepatic fibrosis between the two groups, we found the following differences: PCIII (67.5±7.7 vs. 82.4±13.5 µg/L); IV-C (61.3±18.7 vs. 74.5±17.9 µg/L); HA (147.3±12.4 vs. 87.4±31.6 µg/L); and LN (88.7±13.2 vs 102.5±23.4 µg/L). The results showed that the differences in comparison of the indexes before and after the treatment were statistically significant (P<0.05). After treatment, the MELD score of patients in the combination treatment group was significantly lower than that in the entecavir group (18.7±3.2 vs. 22.5±3.4), with a statistically significant difference (P<0.05). CONCLUSIONS: In the chronic hepatitis B patients with interferon resistance, the combined administration of entecavir and adefovir dipivoxil can significantly improve liver function, hepatic fibrosis and MELD scores. The results highlight the need to promote the benefits of this drug combination in helping chronic hepatitis B patients with interferon resistance, and to promote its application in clinical practices.

The packaging and maturation of the HIV-1 Pol proteins.

The Pol protein of human immunodeficiency virus type 1 (HIV-1) harbours the viral enzymes critical for viral replication; protease (PR), reverse transcriptase (RT), and integrase (IN). PR, RT and IN are not functional in their monomeric forms and must come together as either dimers (PR), heterodimers (RT) or tetramers (IN) to be catalytically active. Our knowledge of the tertiary structures of the functional enzymes is well advanced, and substantial progress has recently been made towards understanding the precise steps leading from Pol protein synthesis through viral assembly to the release of active viral enzymes. This review will summarise our current understanding of how the Pol proteins, which are initially expressed as a Gag-Pol fusion product, are packaged into the assembling virion and discuss the maturation process that results in the release of the viral enzymes in their active forms. Our discussion will focus on the relationship between structure and function for each of the viral enzymes. This review will also provide an overview of the current status of inhibitors against the HIV-1 Pol proteins. Effective inhibitors of PR and RT are well established and we will discuss the next generation inhibitors of these enzymes as well recent investigations that have highlighted the potential of IN and RNase H as antiretroviral targets.

esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity.

Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.

Antiviral immunity. Dual attack by RIG-I.

RIG-I induces a type III interferon response during hepatitis B virus infection and also directly interferes with viral replication.

RIG-I works double duty.

The pathogen sensor RIG-I recognizes viral RNA and signals to induce an antiviral response. In this issue of Cell Host & Microbe, Weber et al. (2015), along with recent work by Sato et al. (2015), demonstrate that RIG-I directly inhibits viral replication independent of antiviral signaling.

HBV carrying drug-resistance mutations in chronically infected treatment-naive patients.

BACKGROUND: Nucleoside/nucleotide analogue (NA) treatment causes selection pressure for HBV strains carrying mutations conferring NA resistance. Drug-resistance mutations occur in the reverse transcriptase (RT) region of the HBV polymerase gene and spontaneously arise during viral replication. These mutations can also alter the hepatitis B surface (HBs) protein and in some cases reduce binding to HBs antibodies. The spread of NA-resistant HBV may impact the efficacy of antiviral treatment and hepatitis B immunization programmes. In this study, we used direct sequencing to assess the occurrence of HBV carrying known mutations that confer NA resistance in the largest cohort of treatment-naive patients with chronic hepatitis B (CHB) to date. METHODS: HBV DNA samples isolated from 702 patients were sequenced and the RT region subjected to mutational analysis. RESULTS: There was high genetic variability among the HBV samples analysed: A1 (63.7%), D3 (14.5%), A2 (3.3%), A3 (0.1%), B1 (0.1%), B2 (0.1%), C2 (0.9%), D1 (0.9%), D2 (4.6%), D4 (5.1%), D unclassified subgenotype (0.7%), E (0.6%), F2a (4.6%), F4 (0.4%) and G (0.4%). HBV strains harbouring mutations conferring NA resistance alone or combined with compensatory mutations were identified in 1.6% (11/702) of the patients. CONCLUSIONS: HBV strains harbouring resistance mutations can comprise the major population of HBV quasispecies in treatment-naive patients. In Brazil, there is a very low frequency of untreated patients who are infected with these strains. These findings suggest that the spread and natural selection of drug-resistant HBV is an uncommon event and/or most of these strains remain unstable in the absence of NA selective pressure.

Mechanism of antiviral activities of 3'-substituted L-nucleosides against 3TC-resistant HBV polymerase: a molecular modelling approach.

Comparison of the active sites of the human HIV-1 reverse transcriptase (RT) and the homology-modelled hepatitis B virus (HBV) polymerase shows that the active sites of both enzymes are open to L-nucleosides, but the position where the 3'-substituent of the L-ribose projects in HBV polymerase is wider and deeper than HIV-1 RT, which enables the HBV polymerase to accommodate various 3'-substituted L-nucleosides. However, the space is not sufficient to accommodate a bulky 3'-substituent such as the 3'-azido group of L-3'-azido-3'-deoxythymidine. Analysis of the minimized structure of rtM204V HBV polymerase/3TCTP complex shows that, instead of the steric stress produced by rtV204, a loss of the van der Waals contact around the oxathiolane sugar moiety of 3TCTP caused by the mutation results in the disruption of the active site. Therefore, nucleosides, which are stabilized by additional specific interaction with the enzyme residues, can have more opportunities to circumvent the destabilization by the loss of hydrophobic interaction conferred by mutation. Specifically, the substitution at the 3'-position would be beneficial as the HBV polymerase has wide open space composed of the highly conserved motif (YMDD) where the 3'-substituents of the L-nucleosides project. As an example, our study shows that the 3'-fluorine atom contributes to the antiviral activity of L-3'-Fd4CTP against rtM204V HBV polymerase by readily compensating for the loss of the van der Waals interaction around the 2',3'-double bond through a formation of a hydrogen bond to the amide backbone of rtD205.

Resistance of hepatitis B virus to antiviral drugs: current aspects and directions for future investigation.

Despite the existence of vaccines, chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Interferon therapy successfully controls infection in only a small percentage of chronically infected individuals. The recent approval of the nucleoside analogue lamivudine for the treatment of chronic HBV infection has ushered in a new era of antiviral therapy. While lamivudine is highly effective at controlling viral infection short-term, prolonged therapy has been associated with an increasing incidence of viral resistance. Thus, it appears that lamivudine alone will not be sufficient to control chronic viral infection in the majority of individuals. In addition to lamivudine, several new nucleoside and nucleotide analogues that show promising antihepadnaviral activity are in various stages of development. Lamivudine resistance has been found to confer cross-resistance to some of these compounds and it is likely that resistance to newer antivirals may also develop during prolonged use. Drug resistance therefore poses a major threat to nucleoside analogue-based therapies for chronic HBV infection. Fortunately, combination chemotherapy (antiviral therapy with two or more agents) can minimize the chance that resistance will develop and can be expected to achieve sustained reductions in viral load, provided that suitable combinations of agents are chosen. Here we review the basis of drug resistance in HBV, with emphasis on aspects that are likely to affect drug choice in future.

The molecular biology of HIV. Insights into pathogenesis and targets for therapy.

In the past 10 years, a large number of investigators have produced an enormous amount of information concerning the molecular biology of HIV. These studies at the most basic biological level have provided essential insights into the pathogenesis of the disease. They have supplied the information necessary for the creation of the antiviral therapies now available and have indicated the direction for the development of new therapies now in clinical trials and under investigation. Although the relatively ineffective therapies currently available serve as a constant source of disappointment for those practitioners who care for HIV-infected patients, there is some comfort to be gained from the rapid pace of investigation into the basic biology of the virus and the certainty that any more effective therapy must build upon the basic biological knowledge already obtained. A detailed study of some of the unique features observed during pediatric and perinatal HIV infection, particularly the relatively shortened time from infection to symptoms and the relative importance of CNS disease, may suggest new therapeutic approaches that will benefit both adult and pediatric patients. Finally, a comprehensive knowledge of HIV biology is an essential requirement for therapeutic maneuvers designed to interrupt the transmission of HIV from mother to child.

The newly synthetized pyridobenzoxazocynone inhibits HIV-1 reverse transcriptase activity.

We synthetized pyridobenzoxazocynone that differs in the enlarged eight-membered heterocyclic system from the basic structure of pyridobenzoxazepinones a known class of non-nucleoside HIV-1 reverse transcriptase inhibitors. Pyridobenzoxazocynone hydrochloride was found to inhibit HIV-1 reverse transcriptase activity. At concentration 0.35 microM the enzyme activity decreased by 64 +/- 14%. Higher concentrations of pyridobenzoxazocynone hydrochloride completely abolished the enzyme activity expressed as radioactivity of acid insoluble products. These results suggest that pyridobenzoxazocynones may represent a new class of HIV-1 reverse transcriptase inhibitors.