Early dynamic changes of quasispecies in the reverse transcriptase region of hepatitis B virus in telbivudine treatment.

BACKGROUND: Telbivudine (LdT) - a synthetic thymidine ß-L-nucleoside analogue (NA) - is an effective inhibitor for hepatitis B virus (HBV) replication. The quasispecies spectra in the reverse transcriptase (RT) region of the HBV genome and their dynamic changes associated with LdT treatment remains largely unknown. METHODS: We prospectively recruited a total of 21 treatment-naive patients with chronic HBV infection and collected sequential serum samples at five time points (baseline, weeks 1, 3, 12, and 24 after LdT treatment). The HBV RT region was amplified and shotgun-sequenced by the Ion Torrent Personal Genome Machine (PGM)® system. We reconstructed full-length haplotypes of the RT region using an integrated bioinformatics framework, including de novo contig assembly and full-length haplotype reconstruction. In addition, we investigated the quasispecies' dynamic changes and evolution history and characterized potential NAs resistant mutations over the treatment course. RESULTS: Viral quasispecies differed obviously between patients with complete (n = 8) and incomplete/no response (n = 13) at 12 weeks after LdT treatment. A reduced dN/dS ratio in quasispecies demonstrated a selective constraint resulting from antiviral therapy. The temporal clustering of sequential quasispecies showed different patterns along with a 24-week observation, although its statistic did not differ significantly. Several patients harboring pre-existing resistant mutations showed different clinical responses, while NAs resistant mutations were rare within a short-term treatment. CONCLUSION: A complete profile of quasispecies reconstructed from in-depth shotgun sequencing may has important implications for enhancing clinical decision in adjusting antiviral therapy timely.

Complex genetic encoding of the hepatitis B virus on-drug persistence.

Tenofovir disoproxil fumarate (TDF) is one of the nucleotide analogs capable of inhibiting the reverse transcriptase (RT) activity of HIV and hepatitis B virus (HBV). There is no known HBV resistance to TDF. However, detectable variation in duration of HBV persistence in patients on TDF therapy suggests the existence of genetic mechanisms of on-drug persistence that reduce TDF efficacy for some HBV strains without affording actual resistance. Here, the whole genome of intra-host HBV variants (N = 1,288) was sequenced from patients with rapid (RR, N = 5) and slow response (SR, N = 5) to TDF. Association of HBV genomic and protein polymorphic sites to RR and SR was assessed using phylogenetic analysis and Bayesian network methods. We show that, in difference to resistance to nucleotide analogs, which is mainly associated with few specific mutations in RT, the HBV on-TDF persistence is defined by genetic variations across the entire HBV genome. Analysis of the inferred 3D-structures indicates no difference in affinity of TDF binding by RT encoded by intra-host HBV variants that rapidly decline or persist in presence of TDF. This finding suggests that effectiveness of TDF recognition and binding does not contribute significantly to on-drug persistence. Differences in patterns of genetic associations to TDF response between HBV genotypes B and C and lack of a single pattern of mutations among intra-host variants sensitive to TDF indicate a complex genetic encoding of the trait. We hypothesize that there are many genetic mechanisms of on-drug persistence, which are differentially available to HBV strains. These pervasive mechanisms are insufficient to prevent viral inhibition completely but may contribute significantly to robustness of actual resistance. On-drug persistence may reduce the overall effectiveness of therapy and should be considered for development of more potent drugs.

Evolutionary trends of resistance mutational patterns of HBV reverse transcriptase over years (2002-2012) of different treatment regimens: The legacy of lamivudine/adefovir combination treatment.

Antiviral therapy has revolutionized treatment of chronic HBV infections. First generation compounds, lamivudine and adefovir, displayed a high rate of treatment failures, and have been replaced by more potent compounds with high genetic barrier to resistance. However, the evolution of the virus towards resistance due the use of first generation compounds may still provide useful information for a better management of current antivirals. A single center sequence database including 705 HBV reverse transcriptase sequences from patients failing antiviral treatments (2002-2012) has been statistically analyzed to highlight viral evolution in relationship to the use of antiviral compounds and to their associations/sequencing in those years. The influence of viral genotypes and polymorphisms on resistance-related mutational patterns was also investigated. This study documents how, after the first years of antiviral therapy, the use of adefovir as an add-on strategy allowed a consistent reduction treatment failures. It also documents the effects of the initial misuse of entecavir in lamivudine experienced patients. In the latest years, the correct use of entecavir and the introduction of tenofovir allowed further curbing of resistance-related treatment failures, which virtually disappeared. Furthermore, the study allows a better understanding of how viral genotype (A vs D) conditions specific mutational pathways to resistance against lamivudine and entecavir, and demonstrates that the use of adefovir in lamivudine experienced patients is associated to peculiar mutational patterns, in particular A181V + F/Y221L. Despite some concern may arise for patients previously treated with lamivudine/adefovir, in sequence or combination, where the virus may have developed a lower genetic barrier against resistance to tenofovir, the outlook of antiviral treatment of HBV infection should be quite optimistic.

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.

Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells.

Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and the development of breast cancer.

A single dose of a MIV-150/Zinc acetate gel provides 24 h of protection against vaginal simian human immunodeficiency virus reverse transcriptase infection, with more limited protection rectally 8-24 h after gel use.

Previously we showed that repeated vaginal application of a MIV-150/zinc acetate carrageenan (MIV-150/ZA/CG) gel and a zinc acetate carrageenan (ZA/CG) gel significantly protected macaques from vaginal simian human immunodeficiency virus reverse transcriptase (SHIV-RT) infection. Gels were applied either daily for 2 weeks or every other day for 4 weeks, and the animals were challenged 4-24 h later. Herein, we examined the effects of a single vaginal dose administered either before or after virus challenge. Encouraged by the vaginal protection seen with MIV-150/ZA/CG, we also tested it rectally. Vaginal applications of MIV-150/ZA/CG, ZA/CG, and CG gel were performed once 8-24 h before, 1 h after, or 24 h before and 1 h after vaginal challenge. Rectal applications of MIV-150/ZA/CG and CG gel were performed once 8 or 24 h before rectal challenge. While vaginal pre-challenge and pre/post-challenge application of MIV-150/ZA/CG gel offered significant protection (88%, p<0.002), post-challenge application alone did not significantly protect. ZA/CG gel reduced infection prechallenge, but not significantly, and the effect was completely lost post-challenge. Rectal application of MIV-150/ZA/CG gel afforded limited protection against rectal challenge when applied 8-24 h before challenge. Thus, MIV-150/ZA/CG gel is a highly effective vaginal microbicide that demonstrates 24 h of protection from vaginal infection and may demonstrate efficacy against rectal infection when given close to the time of HIV exposure.

Hepatitis B virus variant with the a194t substitution within reverse transcriptase before and under adefovir and tenofovir therapy.

Retrospective analysis of our local HBV reverse transcriptase (rt) sequence database including 973 sequences recovered from 616 patients identified one unique HBV DNA sequence harbouring amino acid (aa) substitution rtA194T, which has been suspected to confer reduced susceptibility to tenofovir but whose implication in antiviral resistance has not been confirmed. This sequence has been recovered from the blood sample of a 35-year-old man presenting with chronic hepatitis B and cirrhosis, at time of initiation of HBV therapy with adefovir (ADV). Retrospective analysis showed that viruses were rt194A eleven months earlier. Nonetheless, rt sequences recovered from the two sequential serum samples showed 98% nucleotide identity and were clustered in phylogenetic reconstruction. Clonal sequencing was performed retrospectively, which showed that rt194A HBV sequences were the only detected in the earliest sample and rt194T HBV sequences were the only detected in the later sample. HBV rtA194T mutants were still the majoritary quasi-species 17 months after being identified for the first time. HBV genotype determined by means of population sequencing then phylogeny reconstruction was E. HBV harboring a rt194T can be naturally observed, although very rarely, in absence of any prior therapy. Indeed, they represent six (0.2%) of three 110 sequences recovered from drug naive patients in the Stanford HBV sequence database (http://hivdb.stanford.edu/HBV/DB/cgi-bin/MutPrevByGenotypeRxHBV.cgi). In the present observation, we cannot interpret the virological response under anti-HBV therapy due to short follow-up and nonoptimal drug compliance, as indicated by patient's interview and TDF plasma Ctrough determination.

Effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus.

The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). At 42 °C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 °C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 °C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.

[Dissection of mechanism for the adefovir dipivoxil resistance in chronic hepatitis B patients].

OBJECTIVE: To explore the mechanism for adefovir dipivoxil (ADV) resistance occurred in chronic hepatitis B patients of a series of phase III clinical trails. METHODS: 30 resistant HBV strains were selected out from 177 cases of ADV treated chronic hepatitis B patients. HBV polymerase RT region were amplified by nested PCR and analyzed with the standard nucleotide sequence of HBV strains deposited in GeneBank. RESULTS: 21 out of 30 HBV strains were primary resistant strains, among them 5 HBV strains (23.8%, 5/21) had the polymorphism site of rtN118H. While the other 9 HBV strains showed secondary resistance, variations in conservative region C (rtM207V) and other non-conservative regions were found. The classic mutation sites such as rtN236T and rtA181V/T were not found. CONCLUSIONS: Polymorphism site of rtN118H might be responsible for HBV primary resistance to ADV therapy. rtM207V variation in HBV RT C domain and other variation sites might play a role in HBV secondary resistance to ADV treatment, and natural resistant quasispecies may be the basis for the ADV quick resistance. These conclusions await further confirmation by phenotype test.

Design strategies of novel NNRTIs to overcome drug resistance.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are very potent and most promising anti-AIDS drugs that specifically inhibit HIV-1 reverse transcriptase (RT). However, to a great extent, the efficacy of NNRTI drugs is impaired by rapid emergence of drug-resistance mutations. Fortunately, detailed analysis of a wide range of crystal structures of HIV-1 RT/NNRTI complexes together with data on drug resistance mutations has identified factors important for design of inhibitors and resilience to mutations, such as, exhibiting conformational flexibility and positional adaptability of NNRTIs, forming extensive main chain hydrogen bonding, targeting highly conserved residues in HIV-1 RT and possessing unconventional mechanisms for NNRTI-mediated inhibition of RT. Besides, the plasticity of NNRTIs binding pocket (NNIBP) also provides a broad space for the discovery of new generations of NNRTIs. For instance, the composite binding pocket, integrated all available crystal structure information about the NNRTI binding site of HIV RT, was demonstrated to be an effective tool to better understand the flexible nature of the binding pocket and to identify specific inhibitors. The RT/solvent interface proved to be an attractive site for incorporating a moiety to improve water solubility and pharmacokinetics or introducing a second pharmacophore to construct multifunctional ligand. Totally, the characterization of NNRTIs and NNIBP may help in the design of more effective drugs that are potent toward wild type and drug-resistant strains of RT. In this paper we attempt to translate the general knowledge gained from a large number of related literature into a set of medicinal chemistry strategies to improve the drug resistance profile of NNRTIs.

Antiretroviral drug resistance in HIV-2: three amino acid changes are sufficient for classwide nucleoside analogue resistance.

Genotypic surveys suggest that human immunodeficiency virus type 1 (HIV-1) and HIV-2 evolve different sets of mutations in response to nucleoside reverse-transcriptase inhibitors (NRTIs). We used site-directed mutagenesis, culture-based phenotyping, and cell-free assays to determine the resistance profiles conferred by specific amino acid replacements in HIV-2 reverse transcriptase. Although thymidine analogue mutations had no effect on zidovudine sensitivity, the addition of Q151M together with K65R or M184V was sufficient for high-level resistance to both lamivudine and zidovudine in HIV-2, and the combination of K65R, Q151M, and M184V conferred classwide NRTI resistance. These data suggest that current NRTI-based regimens are suboptimal for treating HIV-2 infection.

Evolution of hepatitis B virus polymerase mutations in a patient with HBeAg-positive chronic hepatitis B virus treated with sequential monotherapy and add-on nucleoside/nucleotide analogues.

BACKGROUND: Nucleoside/nucleotide analogues are a fundamental tool for the treatment of chronic hepatitis B virus (HBV). Sequential anti-HBV treatment might lead to the selection of mutations. OBJECTIVE: This report aimed to analyze the genetic evolution of the reverse-transcriptase (RT) gene of viral quasispecies in a patient with hepatitis B e antigen (HBeAg)-positive chronic HBV who received, sequentially, lamivudine (LAM), adefovir dipivoxil (ADV), and ADV + telbivudine (LDT) combination treatment over a total of 108 weeks. METHODS: A 20-year-old Chinese man presented to Huashan Hospital, Fudan University, Shanghai, People's Republic of China, with hepatitis B surface antigen-positive and HBeAg-positive chronic HBV and was sequentially treated with LAM 100 mg/d for 18weeks,ADV 10mg/d for 68weeks, and ADV 10mg/d + LDT 600 mg/d combination treatment for 22 weeks. Compliance was monitored every 4 weeks using a pill count. For genotypic analysis, the RT region of the polymerase gene from the serum of this patient was amplified, cloned, and sequenced. Fifty clones with HBV insert were selected for sequencing at weeks 0 (baseline), 18, 22, 60, 70, 86, and 108. RESULTS: The rtM204V/L LAM-resistance mutation was detected in 4.4% (2/45) of clones prior to LAM treatment. At week 18 during LAM treatment, the rtM204I mutation became predominant, being present in 79.5% (35/44) of clones. The rtM204I mutation was associated with compensatory mutations (rtL180M and rtT184L). A total of 9.1% (4/44) of the clones harbored the rtL180M + rtT184L + rtM204I mutations. Two new mutations, rtL229V and rtV191I, were detected in 75.0% (33/44) and 11.4% (5/44) of clones, respectively. At week 22 during ADV treatment, LAM-resistance mutations (rtL180M, rtT184L, rtM204I, rtV191I, and rtL229V) were not detected. At week 86 during ADV therapy, the rtN236T ADV-resistance mutation was detected in 58.8% (20/34) of clones. A total of 20.6% (7/34) of the clones harbored the rtK212T + rtM250L mutation, and rtA181V was found in 2.9% (1/34) of the clones. At week 108, after the patient had been receiving ADV + LDT combination therapy for 22 weeks, rtS202G and rtI269T had emerged, representing 28.9% (13/45) and 8.9% (4/45), respectively, of the viral population during ADV + LDT combination treatment. We also detected several polymorphic sites,including rtF221Y, rtS223A, rtI224V, rtN238H, rtL267Q, and rtQ271M, during the sequential treatment. After 22 weeks of combination treatment, HBV DNA count was decreased to less than the lower limit of quantitation (<200 copies/mL). CONCLUSIONS: This report identified HBV mutations that escaped the antiviral pressure of LAM, ADV, and ADV + LDT in this patient and provided insight into the process of mutation selection through genotypic analysis during antiviral treatment. Mutations selected under sequential treatments of LAM, ADV, and ADV + LDT can lead to a series of compensatory mutations, which partially restore the level of viral replication. ADV administered in combination with LDT appeared to be effective in this selected case with clinical or virologic resistance to sequential treatment with LAM and ADV.

Characterization of cyclodextrin inclusion complexes of the anti-HIV non-nucleoside reverse transcriptase inhibitor UC781.

The highly potent anti-HIV agent UC781 is being evaluated for use in topical microbicides to prevent HIV transmission. However, UC781 is extremely hydrophobic with poor water solubility, a property that may complicate appropriate formulation of the drug. In this study, we examined the ability of several cyclodextrins, beta-cyclodextrin (beta CD), methyl-beta-cyclodextrin (M beta CD), and 2-hydroxylpropyl-beta-cyclodextrin (HP beta CD), to enhance the aqueous solubility of UC781. Each of the cyclodextrins provided dramatic increases in UC781 aqueous solubility, the order being M beta CD>HP beta CD>beta CD. The complexation constants (K (1:1)) of the inclusion complexes were determined via a phase solubility technique using high-performance liquid chromatography and showed that UC781 solubility increased linearly as a function of cyclodextrin concentration. Ultraviolet spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and 2D (1)H ROESY NMR spectroscopy were used to further characterize these UC781/cyclodextrin complexes. The inhibitory potency of UC781 and its HP beta CD inclusion complex were evaluated using an in vitro HIV-1 reverse transcriptase inhibition assay The inhibitory potency of the UC781/HP beta CD complex was 30-fold greater than that of UC781 alone, showing that the complexed drug is able to provide substantial inhibition of its target. The enhancement of UC781 aqueous solubility is essential for the development of a useful vaginal microbicide dosage form, and our data suggest that UC781/cyclodextrin inclusion complexes may be useful in this context.

[YMDD motif variants detected by Inno-Lipa HBV DR assay in chronic hepatitis B patients during lamivudine therapy]. / Lamivudin tedavisi alan kronik hepatit B olgularinda Inno-Lipa HBV DR yöntemi ile saptanan YMDD motif degisiklikleri.

Lamivudine resistance is a result of mutations in YMDD motif in which rt203-206th codons (Y: tyrosine; M: methionin; D: aspartic acid; D: aspartic acid) of reverse trancriptase; the catalytic part of hepatitis B polymerase enzyme, takes place. In this study we aimed to show the YMDD motif variants in chronic hepatitis B patients who have presumably developed lamivudine resistance due to viral polymerase gene mutation during lamivudine therapy. Twenty lamivudine treated patients were included in the study, and HBV-DNA was extracted from the sera of the patients by using extraction kit (Invisorb, Instant Spin DNA/RNA Virus Mini Kit, Germany). A line probe assay (INNO-LIPA HBV DR; INNOGENETICS N.V, Ghent, Belgium) was used to determine YMDD motif variants at viral polymerase gene fragment in HBV-DNA samples of these patients and evaluated colorimetrically. In 12 (60%) patients' samples YMDD motif variants were detected leading to lamivudin resistance. Eleven (91.6%) of the 12 samples having motif variants were YMDD variant and wild-type combination. While YMDD + YIDD combination was determined in only one specimen, YMDD + YVDD combination was determined in six. Mixed combination of YMDD + YVDD + YIDD were detected in four samples. In 8 (66.6%) HBV-DNA samples YMDD variants were accompanied with L180M variants. It can be concluded that determination of genotypic resistance before the expression of phenotypic resistance during lamivudin therapy is important since addition of adefovir to the therapy at an early stage will prevent the occurence of hepatitis with mutant species. Thus lamivudin resistance should be followed up regularly in all of the chronic hepatitis B patients under lamivudin therapy.

Synthetic chemistry-led creation of a difluorinated biaryl ether non-nucleoside reverse transcriptase inhibitor.

In the process of developing a series of novel, fluorinated biaryl ether NNRTIs, we fortuitously discovered derivative 20, which possesses excellent potency against both wild-type and clinically relevant mutations of the reverse transcriptase enzyme.

A 21-base pair insertion/duplication at codon 69 of the HIV type 1 reverse transcriptase in a patient undergoing multiple nucleoside therapy.

We describe the selection of a previously unreported 21-base pair insertion following codon 69 of the HIV-1 reverse transcriptase (RT) from a patient undergoing multiple nucleoside analogue therapy. This insertion was a direct duplication of the preceding 21 bases of HIV-RT, and was selected in a background of NRTI-resistance mutations including substitutions at RT codons 41, 67, 184, 210, and 215. Longitudinal genotypic and phenotypic resistance tests performed before and after selection of the insertion suggested that the insertion conferred an additional decrease in susceptibility to some nucleoside analogues, most notably didanosine, stavudine, abacavir, and tenofovir. However, phenotypic analysis of an insertion-containing site-directed mutant constructed in an HIV-1 HXB2 background revealed no direct association between the 21-base pair insertion and decreased susceptibility to NRTIs, suggesting that the insert requires the context of the patients' virus in order to confer resistance. These observations may offer new insight into the relative contribution of HIV-RT codon 69 insertion mutations to antiretroviral resistance.

All-trans retinoic acid attacks reverse transcriptase resulting in inhibition of HIV-1 replication.

We previously reported that all-trans retinoic acid (ATRA) inhibited growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. Interestingly, ATRA significantly inhibited reverse transcriptase (RT) activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. To clarify whether ATRA has an inhibitory effect on the replication of HIV, we examined HIV proviral DNA in a HIV-1-positive cell line (8E5) using real time PCR. ATRA as well as AZT reduced the proviral DNA load of 8E5 in a dose-dependent manner. These results suggest that there is a common element of ATRA signaling in both HTLV-1 and HIV. Furthermore, we examined the effects of ATRA on viral replication in primary lymphocytes of three individuals infected with HIV. ATRA reduced viral replication significantly similar to AZT. These findings suggested that ATRA acts as a RT inhibitor, reducing the HIV-1 proviral DNA load. Finally, we conclude that ATRA may be a potential therapeutic agent for HIV infection.

Efavirenz in plasma from HIV-infected patients does not directly block reverse transcriptase activity in cell-free assays but inhibits HIV replication in cellular assays.

The aim of this study was to characterize the potent nonimmunoglobulin (Ig) inhibitory activity defected in plasma from some HIV-infected, efavirenz (EFV)-treated patients. Concentration of EFV in plasma was measured by HPLC and correlation with reverse transcriptase (RT) inhibition or decrease in virus replication in cellular assays was searched. After plasma protein elimination by ethanol extraction, an inhibitory activity is measurable on RT in vitro that correlates with EFV concentration determined by HPLC. However, total plasma-containing EFV does not inhibit RT activity in cell-free assay, but it does efficiently inhibit virus replication in cell culture assays. Thus, despite being bound to plasma proteins (retention of EFV after extensive dialysis), EFV in plasma conserves its antiviral activity on infected cells. This observation precludes the use of crude sera and plasmas from EFV-treated patients for the study of antibody-mediated neutralizing activity.

Synthesis and biological evaluation of phosphonated dihydroisoxazole nucleosides.

Phosphonated isoxazolinyl nucleosides have been prepared via 1,3-dipolar cycloaddition reaction of nitrile oxides with corresponding vinyl or allyl nucleobases for antiviral studies. The cytotoxicity, the anti-HSV activity and the RT-inhibitory activity of the obtained compounds were evaluated and compared with those of AZT and diethyl{(1'SR,4'RS)-1'-[[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)]-3'-methyl-2'-oxa-3'-azacyclopent-4'-yl]}methylphosphonate, a saturated phosphonated dihydroisoxazole nucleoside analogue.