ABSTRACT
DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.
Subject(s)
DNA, Viral/analysis , Human papillomavirus 16/isolation & purification , Phosphates/chemistry , SARS-CoV-2/isolation & purification , Selenium/chemistry , DNA, Viral/biosynthesis , Human papillomavirus 16/metabolism , Humans , Nucleic Acid Amplification Techniques , Polymerization , SARS-CoV-2/metabolism , TemperatureABSTRACT
Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Herpes Simplex/drug therapy , Herpesvirus 1, Human/drug effects , Herpesvirus 2, Human/drug effects , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Virus Replication/drug effects , Acyclovir/pharmacology , Animals , Cell Line, Tumor , Chlorocebus aethiops , DNA Replication/drug effects , DNA, Viral/biosynthesis , Dihydroorotate Dehydrogenase , Drug Synergism , Drug Therapy, Combination , Gene Expression Regulation, Viral/drug effects , Herpes Simplex/virology , Humans , Pyrimidines/biosynthesis , Vero CellsABSTRACT
BACKGROUND: Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro. METHODS: We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase. RESULTS: We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 µM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs. CONCLUSIONS: We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.
Subject(s)
DNA, Viral/biosynthesis , Hepatitis B virus/enzymology , RNA-Directed DNA Polymerase/biosynthesis , Reverse Transcriptase Inhibitors/pharmacology , Virus Replication/drug effects , Antiviral Agents , Drug Evaluation, Preclinical/methods , Drug Resistance, Viral , Guanine/analogs & derivatives , Hep G2 Cells , Humans , In Vitro Techniques , Inhibitory Concentration 50 , Lamivudine , Protein Biosynthesis/drug effects , Recombinant Proteins , Stilbenes/pharmacologyABSTRACT
The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.
Subject(s)
Hepatitis B virus/growth & development , Hepatocytes/physiology , Hepatocytes/virology , Primary Cell Culture/methods , Virus Cultivation/methods , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , DNA, Circular/biosynthesis , DNA, Circular/isolation & purification , DNA, Viral/biosynthesis , DNA, Viral/isolation & purification , Drug Evaluation, Preclinical , Hepatitis B virus/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Transcriptome , Virion/drug effects , Virion/growth & developmentABSTRACT
APOBEC3G is a member of the human cytidine deaminase family that restricts Vif-deficient viruses by being packaged with progeny virions and inducing the G to A mutation during the synthesis of HIV-1 viral DNA when the progeny virus infects new cells. HIV-1 Vif protein resists the activity of A3G by mediating A3G degradation. Phorbol esters are plant-derived organic compounds belonging to the tigliane family of diterpenes and could activate the PKC pathway. In this study, we identified an inhibitor 12-O-tricosanoylphorbol-20-acetate (hop-8), a novel ester of phorbol which was isolated from Ostodes katharinae of the family Euphorbiaceae, that inhibited the replication of wild-type HIV-1 and HIV-2 strains and drug-resistant strains broadly both in C8166 cells and PBMCs with low cytotoxicity and the EC50 values ranged from 0.106 µM to 7.987 µM. One of the main mechanisms of hop-8 is to stimulate A3G expressing in HIV-1 producing cells and upregulate the A3G level in progeny virions, which results in reducing the infectivity of the progeny virus. This novel mechanism of hop-8 inhibition of HIV replication might represents a promising approach for developing new therapeutics for HIV infection.
Subject(s)
Anti-HIV Agents/pharmacology , Euphorbiaceae/chemistry , HIV-1/drug effects , Host-Pathogen Interactions , Phorbol Esters/pharmacology , Virion/drug effects , Virus Replication/drug effects , APOBEC-3G Deaminase/genetics , APOBEC-3G Deaminase/metabolism , Anti-HIV Agents/chemistry , Anti-HIV Agents/isolation & purification , Cell Line , DNA, Viral/antagonists & inhibitors , DNA, Viral/biosynthesis , Gene Expression Regulation , HIV-1/genetics , HIV-1/metabolism , HIV-2/drug effects , HIV-2/genetics , HIV-2/metabolism , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Mutation , Phorbol Esters/chemistry , Phorbol Esters/isolation & purification , Plant Extracts/chemistry , Primary Cell Culture , Protein Kinase C/genetics , Protein Kinase C/metabolism , Signal Transduction , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Virion/genetics , Virion/metabolism , vif Gene Products, Human Immunodeficiency Virus/deficiency , vif Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA. METHODS: Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI). RESULTS: We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system. CONCLUSION: Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery. LAY SUMMARY: To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.
Subject(s)
DNA, Circular/genetics , DNA, Viral/genetics , Genetic Techniques , Hepatitis B virus/genetics , Adaptive Immunity , Animals , Cell Line , DNA, Circular/biosynthesis , DNA, Circular/immunology , DNA, Viral/biosynthesis , DNA, Viral/immunology , Drug Discovery , Drug Evaluation, Preclinical , Genes, Viral , Genetic Engineering , Hep G2 Cells , Hepatitis B/drug therapy , Hepatitis B/virology , Hepatitis B virus/immunology , Hepatitis B virus/physiology , Hepatocytes/virology , Humans , Male , Mice , Mice, Inbred C3H , Models, Genetic , Transcription, Genetic , Transfection , Virus Replication/geneticsABSTRACT
Betulinic acid is a naturally occurring pentacyclic triterpenoid, which has antiretroviral, antimalarial, and anti-inflammatory properties. The purpose of this study is to investigate the HBV DNA replication inhibition in the mouse model with betulinic acid. Hydrodynamic injection method via the tail vein with the Paywl. 3 plasmid was used to establish the animal mode (n = 15), and the mice were randomly divided into the PBS control group (n = 5), Betulinic acid treatment group (n = 5) and lamivudine control group (n = 5). The day after successful modeling , the mice would have taken Betulinic acid (100 mg x kg(-1)), lamivudine (50 mg x kg(-1)), PBS drugs orally, once daily for 7 days, blood samples were acquired from the orbital venous blood at 3, 5, 7 days after the administering, HBsAg and HBeAg in serum concentration were measured by ELISA and the mice were sacrificed after 7 days, HBV DNA southern detections were used with part of mice livers. The results showed that betulinic acid significantly inhibited the expression of HbsAg in the mice model at the fifth day compared with the control group, and there was no significant differences between the effects of lamivudine and the PBS control group; both the betulinic acid and lamivudine groups had no significant inhibition for the HBeAg expression; the HBV DNA expressions of the liver tissue from the betulinic acid and lamivudine groups were inhibited compared with the control group. Taken together, these results reveal betulinic acid can inhibit the HBsAg expression and replication of the liver HBV DNA in the mouse model.
Subject(s)
Antiviral Agents/pharmacology , DNA, Viral/biosynthesis , Hepatitis B virus/genetics , Hepatitis B virus/physiology , Hepatitis B/virology , Plasmids/genetics , Triterpenes/pharmacology , Acute Disease , Animals , DNA Replication/drug effects , Hepatitis B/blood , Hepatitis B Surface Antigens/blood , Hepatitis B virus/drug effects , Hepatitis B virus/immunology , Male , Mice , Pentacyclic Triterpenes , Virus Replication/drug effects , Betulinic AcidABSTRACT
Hepatitis B virus (HBV)-associated chronic liver diseases are treated with nucleoside analogs that target the virus polymerase. While these analogs are potent, drugs are needed to target other virus-encoded gene products to better block the virus replication cycle and chronic liver disease. This work further characterized GLS4 and compared it to the related BAY 41-4109, both of which trigger aberrant HBV core particle assembly, where the virus replication cycle occurs. This was done in HepAD38 cells, which replicate HBV to high levels. In vitro, GLS4 was significantly less toxic for primary human hepatocytes (P < 0.01 up to 100 µM), inhibited virus accumulation in the supernantant of HepAD38 cells (P < 0.02 up to 100 nM), inhibited HBV replicative forms in the liver with a significantly lower 50% effective concentration (EC50) (P < 0.02), and more strongly inhibited core gene expression (P < 0.001 at 100 to 200 nM) compared to BAY 41-4109. In vivo characterization was performed in nude mice inoculated with HepAD38 cells, which grew out as tumors, resulting in viremia. Treatment of mice with GLS4 and BAY 41-4109 showed strong and sustained suppression of virus DNA to about the same extents both during and after treatment. Both drugs reduced the levels of intracellular core antigen in the tumors. Alanine aminotransferase levels were normal. Tumor and total body weights were not affected by treatment. Thus, GLS4 was as potent as the prototype, BAY 41-4109, and was superior to lamivudine, in that there was little virus relapse after the end of treatment and no indication of toxicity.
Subject(s)
Antiviral Agents/pharmacology , DNA, Viral/antagonists & inhibitors , Hepatitis B virus/drug effects , Hepatitis B, Chronic/drug therapy , Pyrimidines/pharmacology , Thiazoles/pharmacology , Viremia/drug therapy , Virion/drug effects , Animals , Antiviral Agents/chemical synthesis , Cell Line , DNA, Viral/biosynthesis , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Hepatitis B virus/growth & development , Hepatitis B virus/ultrastructure , Hepatitis B, Chronic/virology , Hepatocytes/drug effects , Hepatocytes/pathology , Hepatocytes/virology , Humans , Lamivudine/pharmacology , Male , Mice , Mice, Nude , Microbial Sensitivity Tests , Primary Cell Culture , Pyridines/pharmacology , Pyrimidines/chemical synthesis , Thiazoles/chemical synthesis , Viral Core Proteins/antagonists & inhibitors , Viral Core Proteins/metabolism , Viremia/virology , Virion/ultrastructure , Virus Replication/drug effectsABSTRACT
Tomato leaf curl New Delhi virus (ToLCNDV) (Geminiviridae) is an important pathogen that severely affects tomato production. An extensive survey was carried out during 2003-2010 to study the diversity of begomoviruses found in tomato, potato, and cucurbits that showed symptoms of leaf puckering, distortion, curling, vein clearing, and yellow mosaic in various fields in different regions of India. Ten begomovirus isolates were cloned from infected samples and identified as belonging to the species ToLCNDV. A total of 44 % of the samples showed association of betasatellites, with CLCuMuB and LuLDB being the most frequent. The ToLCNDV cloned component DNA A and DNA B were agroinoculated on Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with or without betasatellites, CLCuMuB or LuLDB. The viral genome levels were then monitored by real-time polymerase chain reaction at different time points of disease development. Plants co-inoculated with betasatellites showed enhanced symptom severity in both N. benthamiana and tomato, as well as increases in helper viral DNA A and DNA B levels. The DNA B and betasatellites acted antagonistically to each other, so that the level of DNA B was 16-fold greater in the presence of betasatellites, while accumulation of betasatellites, CLCuMuB and LuLDB, were reduced by 60 % in the presence of DNA B. DNA B-mediated symptoms predominated in CLCuMuB-inoculated plants, whereas betasatellite-mediated leaf abnormalities were prominent in LuLDB-co-inoculated plants. Inoculation with the cloned components will be a good biotechnological tool in resistance breeding program.
Subject(s)
Begomovirus/growth & development , Begomovirus/genetics , DNA, Satellite/genetics , Helper Viruses/growth & development , Plant Diseases/virology , Viral Interference , Begomovirus/isolation & purification , Cucurbita/virology , DNA, Viral/biosynthesis , Genetic Variation , Genome, Viral , Solanum lycopersicum/virology , Plant Leaves/virology , Real-Time Polymerase Chain Reaction , Solanum tuberosum/virology , Nicotiana/virologyABSTRACT
Since currently available therapies against HIV/AIDS still show important drawbacks, the development of novel anti-HIV treatments is a key issue. We recently characterized methylated oligoribonucleotides (mONs) that extensively inhibit HIV-1 replication in primary T cells at nanomolar concentrations. The mONs were shown to target both HIV-1 reverse transcriptase (RT) and the nucleocapsid protein (NC), which is an essential partner of RT during viral DNA synthesis. To further understand the mechanism of such mONs, we studied by isothermal titration calorimetry and fluorescence-based techniques their NC binding properties and ability to inhibit the nucleic acid chaperone properties of NC. Notably, we investigated the ability of mONs to inhibit the NC-induced destabilization of the HIV-1 cTAR (complementary DNA sequence to TAR [transactivation response element]) stem-loop and the NC-promoted cTAR annealing to its complementary sequence, required at the early stage of HIV-1 viral DNA synthesis. Moreover, we compared the activity of the mONs to that of a number of modified and nonmodified oligonucleotides. Results show that the mONs inhibit NC by a competitive mechanism whereby the mONs tightly bind the NC peptide, mainly through nonelectrostatic interactions with the hydrophobic platform at the top of the NC zinc fingers. Taken together, these results favor the notion that the mONs impair the process of the RT-directed viral DNA synthesis by sequestering NC molecules, thus preventing the chaperoning of viral DNA synthesis by NC. These findings contribute to the understanding of the molecular basis for NC inhibition by mONs, which could be used for the rational design of antiretroviral compounds targeting HIV-1 NC protein.
Subject(s)
HIV-1/metabolism , Molecular Chaperones/antagonists & inhibitors , Nucleocapsid Proteins/antagonists & inhibitors , Oligoribonucleotides/pharmacology , Amino Acid Sequence , Base Sequence , DNA, Viral/biosynthesis , HIV Reverse Transcriptase/antagonists & inhibitors , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , Oligoribonucleotides/chemistry , Oligoribonucleotides/metabolismABSTRACT
Beet curly top virus (BCTV) limits sugarbeet production considerably. Previous studies have shown that infections are associated with the generation of defective DNAs (D-DNA) which may attenuate symptoms. Transgenic sugarbeet lines were established carrying a partial direct repeat construct of D-DNA in order to examine whether they are useful as a means of generating tolerance against BCTV. Thirty four independent transgenic lines were challenged. Viral full-length and D-DNAs were monitored by polymerase chain reaction (PCR) or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP). The differential accumulation of both DNA species was compared with symptom severity during the course of infection. RCA/RFLP allowed the discrimination of two D-DNA classes which were either derived from the transgenic construct (D(0)) or had been generated de novo (D(n)). The statistical analysis of the results showed that the presence of D(0)-DNA correlated with increased symptom severity, whereas D(n)-DNAs correlated with attenuated symptoms.
Subject(s)
Beta vulgaris/immunology , DNA, Viral/biosynthesis , Defective Viruses/genetics , Defective Viruses/immunology , Geminiviridae/immunology , Geminiviridae/pathogenicity , Plant Diseases/prevention & control , Beta vulgaris/virology , DNA, Viral/genetics , DNA, Viral/isolation & purification , Defective Viruses/isolation & purification , Geminiviridae/isolation & purification , Genotype , Nucleic Acid Amplification Techniques , Plant Diseases/virology , Plants, Genetically Modified/immunology , Plants, Genetically Modified/virology , Polymorphism, Restriction Fragment LengthABSTRACT
In this study, 1,2,4,6-tetra-O-galloyl-ß-D-glucose (1246TGG), a polyphenolic compound isolated from traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae), was found to inhibit herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infection at different magnitudes of activity in vitro. Further studies revealed that 1246TGG directly inactivated HSV-1 particles, leading to the failure of early infection, including viral attachment and penetration. 1246TGG also suppressed the intracellular growth of HSV-1 within a long period post-infection (from 0 h p.i. to 12 h p.i.), while it might exert an antiviral effect mainly before 3 h p.i. It inhibited HSV-1 E and L gene expressions as well as viral DNA replication but did not affect the RNA synthesis of IE gene in our study. Also, in the presence of 1246TGG, the synthesis of viral protein was reduced. Taken together, it was suggested that 1246TGG might exert anti-HSV activity both by inactivating extracellular viral particles and by inhibiting viral biosynthesis in host cells. These results warrant further studies on the antiviral mechanisms of 1246TGG and suggest that it might be a candidate for HSV therapy.
Subject(s)
Antiviral Agents/pharmacology , Gallic Acid/analogs & derivatives , Herpesvirus 1, Human/drug effects , Herpesvirus 2, Human/drug effects , Monosaccharides/pharmacology , Animals , Antiviral Agents/isolation & purification , Chlorocebus aethiops , DNA, Viral/biosynthesis , Gallic Acid/isolation & purification , Gallic Acid/pharmacology , Herpesvirus 1, Human/physiology , Herpesvirus 2, Human/physiology , Monosaccharides/isolation & purification , Phyllanthus emblica/chemistry , Vero Cells , Viral Proteins/biosynthesis , Virus Replication/drug effectsABSTRACT
Previous studies showed that the root extract of Boehmeria nivea (BNE) can significantly suppress the production of hepatitis B virus (HBV) in vitro and in vivo. In this study, viral core and large-surface proteins accompanied with their encapsidated viral DNA were observed to accumulate within the cells. Notably, 78-kDa glucose-regulated protein (GRP78) was found to be suppressed by BNE, and stimulation of the GRP78 expression by thapsigargin could rescue virus production initially inhibited by BNE. The antiviral effect of BNE was reversible, which also coincided with the level of GRP78. Furthermore, we synthesized the GRP78 siRNA to knockdown the expression of GRP78 protein, and the production of supernatant HBV DNA was reduced simultaneously. Moreover, combined treatment of BNE and 3TC exhibited an additive anti-hepatitis B virus effect. In conclusion, the inhibitory effect of BNE on blocking assembled virion secretion might be via the reduction of GRP78.
Subject(s)
Antiviral Agents/pharmacology , Boehmeria/chemistry , Heat-Shock Proteins/metabolism , Hepatitis B virus/drug effects , Molecular Chaperones/metabolism , Plant Extracts/pharmacology , Virus Replication/drug effects , Cell Line , DNA, Viral/biosynthesis , Endoplasmic Reticulum Chaperone BiP , Gene Knockdown Techniques , Hepatitis B virus/physiology , Hepatocytes/virology , HumansABSTRACT
INTRODUCTION: The treatment of chronic hepatitis B virus (HBV) infection has been revolutionized in the past decade by the increased availability of effective antiviral agents. Telbivudine is an L-nucleoside that is structurally related to lamivudine and has recently been approved for use in patients with chronic HBV infection. Telbivudine is highly selective for HBV DNA and inhibits viral DNA synthesis with no effect on human DNA or other viruses. This article reviews the pharmacology, pharmacokinetics, therapeutic efficacy and safety of telbivudine, and discusses its place in the current armamentarium against HBV. METHODS: Relevant publications were identified from searches of Medline and PubMed between 2000 and 2008, using the search terms "hepatitis B/HBV," "telbivudine/LdT," "beta-L-thymidine," "pharmacokinetics," "safety," "adverse events," and "resistance." The reference lists of retrieved articles were searched for relevant studies. RESULTS: Phase 3 clinical studies demonstrate that telbivudine is superior to lamivudine over a 2-year period in hepatitis B e-antigen (HBeAg)-positive and HBeAg-negative patients. Telbivudine was associated with a statistically significantly greater reduction in HBV DNA, greater proportion of alanine aminotransferase normalization, and greater histological response than lamivudine. Furthermore, telbivudine use resulted in fewer cases of treatment failure and less virological resistance than lamivudine. However, after 2 years of therapy, telbivudine resistance was appreciable (25%) and considerably higher than that seen with other new antivirals such as tenofovir and entecavir. Overall, telbivudine was found to be safe, although grade 3 or 4 adverse events, including elevations in creatine kinase, were more commonly found in patients receiving telbivudine than lamivudine. Telbivudine is not active against lamivudine-resistant HBV. CONCLUSIONS: Telbivudine is a new antiviral agent joining the armamentarium against HBV. It is superior to lamivudine in terms of therapeutic response and resistance profile. However, concerns about resistance with long-term use, along with inferior cost-effective analyses, have relegated telbivudine to a second-line agent in the management of chronic HBV infection.
Subject(s)
Hepatitis B, Chronic/drug therapy , Nucleosides/therapeutic use , Pyrimidinones/therapeutic use , Alanine Transaminase/blood , Alanine Transaminase/drug effects , Clinical Trials, Phase II as Topic , Clinical Trials, Phase III as Topic , DNA, Viral/biosynthesis , DNA, Viral/drug effects , Drug Evaluation, Preclinical , Drug Interactions , Drug Resistance, Viral/drug effects , Hepatitis B virus/drug effects , Hepatitis B, Chronic/metabolism , Hepatitis B, Chronic/virology , Humans , Nucleosides/chemistry , Nucleosides/pharmacology , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Safety , Telbivudine , Thymidine/analogs & derivatives , Treatment Outcome , Virus Replication/drug effectsABSTRACT
The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich 'structurally poor' RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5-100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using 'structurally poor' RNA domains in regulating biological process.
Subject(s)
DNA, Complementary/biosynthesis , DNA, Viral/biosynthesis , Genes, pol , HIV-1/genetics , RNA, Viral/chemistry , Regulatory Sequences, Ribonucleic Acid , Reverse Transcription , Adenine/analysis , Base Sequence , Cell Line , Codon , Dimerization , HIV-1/physiology , Humans , Nucleic Acid Conformation , Viral Proteins/metabolism , Virion/metabolism , Virus Internalization , Virus ReplicationABSTRACT
Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs.
Subject(s)
DNA, Viral/biosynthesis , HIV-1/genetics , Reverse Transcription , gag Gene Products, Human Immunodeficiency Virus/chemistry , Amino Acid Sequence , Cell Line , Conserved Sequence , DNA, Complementary/analysis , DNA, Complementary/biosynthesis , HIV-1/growth & development , HIV-1/physiology , Humans , Molecular Sequence Data , Point Mutation , Protein Structure, Tertiary , Virion/chemistry , Virus Replication , Zinc Fingers , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Natural products provide a large reservoir of potentially active agents with anti-hepatitis B virus (HBV) activity. We examined the effect of the polyphenolic extract from Geranium carolinianum L. (PPGC) on HBV replication both in vitro and in vivo. In the human HBV-transfected liver cell line HepG(2) 2.2.15, PPGC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC(50) values of 46.85 microg/ml for HBsAg and 65.60 microg/ml for HBeAg at day 9. Consistent with the HBV antigen reduction, PPGC (100 microg/ml) also reduced HBV DNA level by 35.9%. In the duck hepatitis B virus (DHBV) infected ducks, after PPGC was dosed intragastricly (i.g.) once a day for 10 days, the plasma DHBV DNA level was reduced, with an ED(50) value of 47.54 mg/kg. In addition, Southern blot analysis confirmed the in vivo anti-HBV effect of PPGC in ducks and PPGC also reduced the plasma and the liver DHBV DNA level in a dose-dependent manner. Furthermore, significant improvement of the liver was observed after PPGC treatment, as evaluated by the histopathological analysis.
Subject(s)
Antiviral Agents/pharmacology , Geranium/chemistry , Hepatitis B virus/drug effects , Plant Extracts/pharmacology , Virus Replication , Animals , Antigens, Viral/biosynthesis , Antiviral Agents/administration & dosage , Antiviral Agents/therapeutic use , Cell Line , DNA, Viral/biosynthesis , DNA, Viral/blood , Dose-Response Relationship, Drug , Ducks , Hepadnaviridae Infections/drug therapy , Hepatitis B Virus, Duck/drug effects , Hepatitis, Viral, Animal/drug therapy , Hepatocytes/virology , Humans , Inhibitory Concentration 50 , Liver/pathology , Liver/virology , Plant Extracts/administration & dosage , Plant Extracts/therapeutic use , Viral LoadABSTRACT
Human cytomegalovirus (CMV), an agent of infection (CMVI), lethally dangerous for immune deficient neonates and adults was investigated in vitro as a target for a therapeutic effect of new membrane-active polyanionic compounds (MPC). Previous studies on the alicycle- and sulfate-modified carboxy-MPCs revealed a well-defined tendency of the anti-CMV activity amplification in parallel with increasing of the content of sulfate groups, enhancing the negative charge of the macromolecule. The dominating role of the electrostatic factor was confirmed by the highest activity of AS-688, compound with maximum sulfation among the tested MPCs. Its selectivity index (SI) of the CMVI inhibition in human diploid fibroblast cells reached 5450, 7500, 250 and 4286 in the microbicidal, viricidal, prophylactic and therapeutic schemes of the experiment respectively. The antiviral activity at the first, second and third schemes was explained by the polyanion-typical potential of electrostatic neutralization of the countercharged virions and prevention of the virus adsorption on the cell membranes (in competition with heparin sulfate, a cellular receptor of CMV), whereas the therapeutic effect required the ability of MPC to influence the intracellular stages of the CMV life cycle. The PCR and immunochemical assays revealed an inhibitory action of AS-688 on replication of the viral DNA and the following synthesis of the late viral protein gB with efficiency similar to that of gancyclovir (GCV). However, in contrast to GCV, acting as inhibitor of enzyme (viral RNA-polymerase) factor of the biosynthesis, the therapeutic activity of MPC could be interpreted by competition with viral RNA/DNA due to the specific character of the MPC molecular basis, initially constructed on the principle of nucleic acids backbone and charge adjustable imitation. This mechanism assuming reduction of the cytotoxicity risks, explained the experimentally observed fact of low cytotoxicity of MPCs and possible achievement of high SI. The MPC ability to penetrate into the cells without disruption of cellular membrane permeability was confirmed in experiments with the fluorescent-labeled derivate AS-679, structurally and functionally related to AS-688. In the light of the previously described HIV inhibiting properties of AS-688, AS-679 and MPC analogous, the results could be considered prospective in development of new highly effective agents for combined antiviral protection.
Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus Infections/drug therapy , Cytomegalovirus/physiology , Polymers/pharmacology , Virus Attachment/drug effects , Cell Line , Cytomegalovirus Infections/metabolism , DNA Replication/drug effects , DNA Replication/physiology , DNA, Viral/biosynthesis , Drug Evaluation, Preclinical , Fibroblasts/virology , Humans , Polyelectrolytes , Virus Replication/drug effects , Virus Replication/physiologyABSTRACT
We examined the anticytomegalovirus properties of four compounds: pristimerin, the pristimerin analogue, lupeol and 2-acetylphenol-1-beta-D-glucopyranosyl (1 --> 6)-beta-D-xylpyranoside (acetophenol glycoside), isolated from Maytenus heterophylla, a Kenyan medicinal plant. The effects were studied on human cytomegalovirus (HCMV) replication in the human embryonic fibroblast cell line, MRC-5. In a viral plaque-reduction assay, pristimerin showed dose-dependent inhibitory properties with a 50% inhibitory concentration of 0.53 microg/ml (selective index = 27.9). The cells treated with pristimerin inhibited the cytopathic effects in HCMV-infected cells. Moreover, pristimerin suppressed viral replication without affecting the cell growth. Pristimerin inhibited the synthesis of viral DNA but had no virucidal effect on cell-free HCMV. Furthermore, Western blot analysis demonstrated that pristimerin decreased the amount of immediate early (IE) antigen (especially IE2) expression in the infected cells. These results suggest that pristimerin is a unique compound with potential anti-HCMV activity.
Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus/drug effects , Triterpenes/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Blotting, Western , Cell Line , Cytopathogenic Effect, Viral/drug effects , DNA, Viral/biosynthesis , Fibroblasts/drug effects , Humans , Immediate-Early Proteins/biosynthesis , Inhibitory Concentration 50 , Maytenus/chemistry , Pentacyclic Triterpenes , Phenols/isolation & purification , Phenols/pharmacology , Trans-Activators/biosynthesis , Triterpenes/chemistry , Triterpenes/isolation & purification , Triterpenes/toxicity , Viral Plaque Assay , Virus Replication/drug effectsABSTRACT
OBJECTIVE: To study the inhibitory effect of traditional Chinese medicine Compound Liuyuxeue(CLYX) on duck hepatitis B virus (DHBV) DNA, and provide experimental basis for developing a new drug for the clinical treatment. METHODS: One - day old Guangxi brown spotted ducks infected with DHBV were used as the hepatitis B virus infected animal model. Positive ducks were detected by PCR at 13 days after the infection of DHBV, and were randomly divided into five groups: the high dose group, middle dose group and low dose group of Compound Liuyexue( CLYX) , model group, positive control group. Every group had 10 ducks and CLYX was given for 14 days. The content of DHBV DNA in serum were measured by Fluoresceence Quantitative PCR( FQ-PCR). RESULTS: The serum DHBV DNA content was decreased significantly by the treatment with CLYX. The high dose group and middle dose group of CLYX could significantly inhibit DHBV DNA replication in vivo( P <0. 01). DHBV DNA content in serum in high dose group and middle dose group did not return significantly 3 days after stopping treatment, and its inhibitory effects were dose-and tim-dependents. CONCLUSION: CLYX can inhibit significantly DHBV DNA.