Viral DNA Strand Research Articles

Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS invitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the invitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.

A Review of Recent Research on Bio-inspired Structures and Materials

As result of advancements in DNA technology, nearly any two- or three-dimensional architecture may now be precisely built. The selection of DNA sequences creates new opportunities to predictably generate the structure and size of anodized monomolecular assemblies, which is crucial for successful self-assembly. Designer nucleoside analogues can be added to DNA nonmaterial’s to further functionalize them with accessible groups that affect their activity. This article highlights the latest developments young discipline a rundown of potential future directions and uses. The ability to create nearly any three dimensional geometry using a DNA-based scaffolding approach is a significant benefit; the only real restriction is the researcher's creativity. Examples of building blocks include engineered Holliday junctions, DNA scissoring, ion-triggered switches, DNA tube regulation DNA linkers, and Small-molecule-mediated DNA junction induction. A self-assembled crystalline DNA structure developed by Seaman, others is undoubtedly a recent key structure in three-dimensional design. [3] Tensor triangles, which are composed of three connected non-coplanar double helices of DNA, are essentially hard 3D triangular units. They come together with the addition of sticky vertices, eventually forming a symmetric 3D lattice. Surprisingly, at temperatures when genomic DNA is entirely reduced, the DNA will be unusually branched, with small sticky ends combining into a highly ordered substance if branch geometry and connector stiffness promote crystallization. Can be long (usually viral) DNA strands joined main strands to produce well-defined anodized as illustrated by Rothmans' pioneering work on DNA origami tiles. The successful fabrication of smiling faces on well-known maps and surfaces is proof that DNA origami only needs a few hundred basic strands to form. [12] Because rectangular origami tiles may be connected together by hanging DNA around the edges, they are now beginning to play a significant assembly line. Grasp the crucial elements DNA is involved in the construction of origami superstructures requires an understanding of origami tile design.

A mid-throughput HBV replication inhibition assay capable of detecting ribonuclease H inhibitors

The hepatitis B virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Inhibiting the RNaseH blocks viral reverse transcription by truncating the minus-polarity DNA strand, causing accumulation of RNA:DNA heteroduplexes, and abrogating plus-polarity DNA synthesis. Screening for RNaseH inhibitors is complicated by the presence of the minus-polarity DNA strand even when replication is fully inhibited because this residual DNA can be detected by standard screening assays that measure reduction in total HBV DNA accumulation. We previously developed a strand-preferential qPCR assay that detects RNaseH replication inhibitors by measuring preferential suppression of the viral plus-polarity DNA strand. However, this assay employed cells grown in 6- or 12-well plates and hence was of very low throughput. Here, we adapted the assay to a 96-well format and conducted a proof-of-principle screen of 727 compounds. The newly developed assay is a valuable tool for anti-HBV drug discovery, particularly when screening for RNaseH inhibitors.

Persistence of Human Bocavirus 1 in Tonsillar Germinal Centers and Antibody-Dependent Enhancement of Infection.

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies.IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding.

The Peptide AmPep1 Derived from Amaranth Recognizes the Replication Hairpin of TYLCV Disturbing Its Replication Process in Host Plants.

Antiviral compounds targeting viral replicative processes have been studied as an alternative for the control of begomoviruses. Previously, we have reported that the peptide AmPep1 has strong affinity binding to the replication origin sequence of tomato yellow leaf curl virus (TYLCV). In this study, we describe the mechanism of action of this peptide as a novel alternative for control of plant-infecting DNA viruses. When AmPep1 was applied exogenously to tomato and Nicotiana benthamiana plants infected with TYLCV, a decrease in the synthesis of the two viral DNA strands (CS and VS) was observed, with a consequent delay in the development of disease progress in treated plants. The chemical mechanism of action of AmPep1 was deduced using Raman spectroscopy and molecular modeling showing the formation of chemical interactions such as H bonds and electrostatic interactions and the formation of π-π interactions between both biomolecules contributing to tampering with the viral replication.

Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet.

HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg(2+) ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg(2+) (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.

Hepatitis B virus replication is blocked by a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) inhibitor of the viral ribonuclease H activity

Nucleos(t)ide analog drugs profoundly suppress Hepatitis B virus (HBV) replication but rarely cure the infection, so therapy is usually life-long. The nucleos(t)ide analogs inhibit the viral DNA polymerase and often push HBV to the brink of extinction, so it may be possible to eradicate HBV by suppressing HBV replication further. The HBV ribonuclease H (RNaseH) is a logical new drug target because it is the second of only two viral enzymes essential for viral replication. We recently developed a low throughput screening pipeline for inhibitors of the HBV RNaseH and viral replication. Here, we screened a series of twenty-three nitrogen-based polyoxygenated heterocycles including sixteen 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives for anti-HBV RNaseH activity. Nine compounds inhibited the HBV RNaseH, but activity was marginal for eight of them. Compound #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione, HID] was the best hit with an IC50 of 28.1μM and an EC50 of 4.2μM. It preferentially suppressed accumulation of the viral plus-polarity DNA strand in replication inhibition assays, indicating that replication was blocked due to suppression of HBV RNaseH activity. It had a CC50 of 75μM, yielding a therapeutic index of ∼18. The EC50 value was 7-fold lower than the IC50, possibly due to cellular retention or metabolism of the compound, or higher affinity for the full-length enzyme than the recombinant form used for screening. These data indicate that the 2-hydroxyisoquinoline-1,3(2H,4H)-diones will have different structure–activity relationships for the HBV and HIV RNaseHs. Therefore, HID compounds may provide a foundation for development of more effective RNaseH inhibitors of HBV replication.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.

Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

BackgroundWe applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins.Methodology/ResultsPhotoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.ConclusionAnalysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.

Retroviral Integrases Promote Fraying of Viral DNA Ends

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.

Nucleic Acid Interaction Kinetics of APOBEC3G Investigated Using Ensemble and Single Molecule Methods

Human APOBEC3G (A3G) is a host cell cytidine deaminase capable of restricting replication of retroviruses by deaminating ss viral DNA and also by directly inhibiting reverse transcriptase (RT)-catalyzed polymerization reactions. Only about 7 A3G molecules are packaged per HIV virion. Deamination by A3G may only happen during the short period that viral (-) strand DNA is available, necessitating rapid on/off nucleic acid binding kinetics. In contrast, in order for just a few A3G molecules to inhibit DNA polymerization by RT, they have to form a “roadblock”, requiring very slow protein dissociation from DNA. Here, we use SPR and single molecule DNA stretching to investigate the DNA/A3G interaction kinetics. Our results suggest that: (i) A3G binds ssDNA with moderate cooperativity (Hill constant ∼1.5), a binding site size of ∼15 nt, and a Kd of ∼60 nM; (ii) The on/off kinetics of A3G/ssDNA is unusually slow and multi-rate; the dominant “on” component has a bimolecular rate constant of ∼105 M−1.s−1; (iii) Dissociation of A3G from ssDNA has a fast and a slow component. The fraction of the slow component and the off times increase with longer incubation over ∼100 s. Taken together, our data are consistent with the existence of both “fast” and “slow” A3G/DNA binding modes. We hypothesize that the fast mode is a feature of protein dimers, whereas the slow mode is characteristic of multimeric A3G, with protein multimerization on ssDNA occurring over an ∼100 s time period.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS.

BackgroundTreatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs.MethodsPhylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR.ResultsThe calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810.ConclusionWe report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agents ever tested in vitro. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans.

APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

A Three-dimensional Model of the Human Immunodeficiency Virus Type 1 Integration Complex

While the general features of HIV-1 integrase function are understood, there is still uncertainty about the composition of the integration complex and how integrase interacts with viral and host DNA. We propose an improved model of the integration complex based on current experimental evidence including a comparison with the homologous Tn5 transposase containing bound DNA and an analysis of DNA binding sites using Goodford's GRID. Our model comprises a pair of integrase dimers, two strands of DNA to represent the viral DNA ends and a strand of bent DNA representing the host chromosome. In our model, the terminal four base pairs of each of the viral DNA strands interact with the integrase dimer providing the active site, while bases one turn away interact with a flexible loop (residues 186-194) on the second integrase dimer. We propose that residues E152, Q148 and K156 are involved in the specific recognition of the conserved CA dinucleotide and that the active site mobile loop (residues 140-149) stabilises the integration complex by acting as a barrier to separate the two viral DNA ends. In addition, the residues responsible for DNA binding in our model show a high level of amino acid conservation.

Effects of tumor-associated mutations on Rad54 functions.

Yeast RAD54 gene, a member of the RAD52 epistasis group, plays an important role in homologous recombination and DNA double strand break repair. Rad54 belongs to the Snf2/Swi2 protein family, and it possesses a robust DNA-dependent ATPase activity, uses free energy from ATP hydrolysis to supercoil DNA, and cooperates with the Rad51 recombinase in DNA joint formation. There are two RAD54-homologous genes in human cells, hRAD54 and RAD54B. Mutations in these human genes have been found in tumors. These tumor-associated mutations map to conserved regions of the hRad54 and hRad54B proteins. Here we introduced the equivalent mutations into the Saccharomyces cerevisiae RAD54 gene in an effort to examine the functional consequences of these gene changes. One mutant, rad54 G484R, showed sensitivity to DNA-damaging agents and reduced homologous recombination rates, indicating a loss of function. Even though the purified rad54 G484R mutant protein retained the ability to bind DNA and interact with Rad51, it was nearly devoid of ATPase activity and was similarly defective in DNA supercoiling and D-loop formation. Two other mutants, rad54 N616S and rad54 D442Y, were not sensitive to genotoxic agents and behaved like the wild type allele in homologous recombination assays. Consistent with the mild phenotype associated with the rad54 N616S allele, its encoded protein was similar to wild type Rad54 protein in biochemical attributes. Because dysfunctional homologous recombination gives rise to genome instability, our results are consistent with the premise that tumor-associated mutations in hRad54 and Rad54B could contribute to the tumor phenotype or enhance the genome instability seen in tumor cells.

Phosphorothioate Di- and Trinucleotides as a Novel Class of Anti-Hepatitis B Virus Agents

Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC(90)], 1.4 microM) and ORI-9020 (EC(90), 1.2 microM) and trinucleotide ORI-7170 (EC(90), 7.2 microM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5'-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

Brivudin (bromovinyl deoxyuridine).

Brivudin is an oral thymidine analogue indicated for the early treatment of acute herpes zoster in immunocompetent adults. It has high, selective activity against varicella zoster virus (VZV), inhibiting VZV replication, possibly through competitive inhibition of viral DNA polymerase, or by acting as an alternative substrate to deoxythymidine triphosphate, causing viral DNA strand breakage. In a large, 7-day, phase III trial in immunocompetent patients with herpes zoster, once-daily brivudin 125mg was significantly more effective than oral acyclovir 800mg five times daily in reducing the mean time from start of treatment to last vesicular eruption, and was as effective as acyclovir at healing lesions and alleviating acute zoster-related pain. The likelihood of developing post-herpetic neuralgia (PHN) in immunocompetent patients aged > or =50 years was significantly lower with brivudin than with acyclovir. Brivudin was as effective as oral famciclovir 250mg three times daily in terms of the prevalence of PHN, the time to last vesicular eruption and lesion healing in another large, 7-day, phase III study in immunocompetent patients with herpes zoster. Oral brivudin is generally well tolerated, with a similar tolerability profile to those of oral acyclovir or famciclovir. Nausea was the most commonly reported adverse event.

Inhibitory activity of dioxolane purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses

To design combination strategies for chronic hepatitis B therapy, we evaluated in vitro the inhibitory activity of 4 nucleoside analogs, (−)FTC, L-FMAU, DXG, and DAPD, in comparison with lamivudine (3TC) and PMEA. In a cell-free assay for the expression of wild-type duck hepatitis B virus (DHBV) reverse transcriptase, DAPD-TP was found to be the most active on viral minus strand DNA synthesis, including the priming reaction, followed by 3TC-TP, (−)FTC-TP, and DXG-TP, whereas L-FMAU-TP was a weak inhibitor. In cell culture experiments, important differences in drug concentration allowing a 50% inhibition of viral replication or polymerase activity (IC50s) were observed depending on the cell type used, showing that antiviral effect of nucleoside analogs may depend on their intracellular metabolism. IC50s obtained for wild-type DHBV replication in primary duck hepatocytes were much lower than with DHBV transfected LMH cells. IC50s were also significantly lower in the 2.2.1.5 and HepG2 cells compared with HBV transfected HuH7 cells. Moreover, L-FMAU inhibited preferentially HBV plus strand DNA synthesis in these cell lines. The antiviral effect of these inhibitors was also evaluated against 3TC-resistant mutants of the DHBV and HBV polymerases. These mutants were found to be cross resistant to (−)FTC. By contrast, the double DHBV polymerase mutant was sensitive to DXG-TP and DAPD-TP. Moreover, both purine analogs remained active against DHBV and HBV 3TC-resistant mutants in transfected LMH and HepG2 cells, respectively. In conclusion, the unique mechanism of action of these new inhibitors warrants further evaluation in experimental models to determine their capacity to delay or prevent the selection of drug resistant mutants. (H EPATOLOGY 2002;36:710-722.)

Trends in the design of nucleoside analogues as anti-HIV drugs.

HIV infection remains incurable although several anti-HIV drugs have been identified and developed. Among these the nucleoside analogues were and remain in the forefront of anti-HIV chemotherapeutic regimens. Most of these nucleoside analogues are modified mainly in the sugar moiety. In general, they lack a free hydroxy group at the 3'-position. Consequently, they cannot participate in the formation of a 3',5'-phosphodiester linkage, which renders the nucleoside 5'-triphosphates of such nucleoside analogues effective anti-HIV agents. Preventing the formation of 3',5'-phosphodiester linkages leads to inhibition of the viral DNA strand elongation and ultimately chain termination. The phosphorylation of nucleoside analogues is a key factor in their efficacy as anti-HIV agents. Efficient phosphorylation depends largely on the structure of the nucleoside. Therefore, modification of the structure of nucleoside analogues are sought to enhance their phosphorylation and ultimately, effective inhibition of the HIV reverse transcriptase. This review is concerned with the trends in the design of nucleoside analogues as anti-HIV agents.