RT Gene Research Articles

HIV-1 drug resistance genotyping quality assessment: Results of the ENVA7 Genotyping Proficiency Programme

Background Drug-resistance testing plays a critical role in selection of optimal treatment regimens for HIV infected individuals. Laboratories performing testing must implement quality control measures including external quality assessment. Objectives The ENVA7 Programme (2007) was organised by QCMD to assess the performance of laboratories testing for drug-resistance mutations in the HIV-1 Protease and Reverse Transcriptase genes. Study design The ENVA7 panel consisted of 5 lyophilised plasma samples (HIV-1 subtypes B, C and F). The viruses harboured wild type or resistant genotypes at various positions of the PR and RT genes. All IAS-defined resistance-associated codons were scored in comparison to the consensus sequence for each sample using a scoring system developed to allow simple and standardised comparisons between laboratories and/or technologies. Results 111 laboratories from 44 countries participated of which 95 submitted 98 datasets. 36 datasets were generated using ViroSeq (Abbott), 27 using TruGene (Siemens) and 35 using in-house assays. Conclusions All technologies successfully genotyped each of the panel samples, irrespective of the virus subtype. While the assays for genotypic HIV drug-resistance determination have evolved into reliable and technically capable procedures of generating high quality results, variation in the quality of results is still observed between laboratories.

Genotypic and phenotypic characterization of human immunodeficiency virus type 1 isolates circulating in pregnant women from Mozambique

This work evaluated HIV-1 subtypes from different geographic regions and phenotypic data from drug-naïve HIV-positive pregnant women from Mozambique. We analyzed 75 pol sequences from patients and the distribution of the subtypes in three regions of Mozambique and found that the majority of samples analyzed clustered with subtype C. In the northern region, multiple variants were found 5 (approximately 18%) subtype A, 3 (approximately 11%) subtype D and 2 (approximately 7.1%) mosaics (A/C/D and C/D), whereas 18 (64.3%) isolates were subtype C, from a total of 28 samples. Already in the southern region, only one (5%) isolate of 20 samples was subtype D, and the other 19 (95%) isolates were subtype C. All 27 (100%) isolates from the central region grouped within clade C. No primary resistance mutations to IP, NNRTI or NRTI were found. There was no evidence of phenotypic resistance in any of the isolates tested, suggesting that neither the polymorphism in the protease, nor the one found at codon 215 of the RT gene caused an increase in phenotypic resistance. This finding suggests that HAART regimens indicated by WHO will probably be successful in Mozambique.

Prevalence of resistance-associated mutations in Human Immunodeficiency Virus type 1-positive individuals failing HAART in Rio de Janeiro, Brazil

We investigated the occurrence of HIV-1 antiretroviral resistance in individuals failing to respond to highly active antiretroviral therapy (HAART) attended by RENAGENO from 2001-2004. One hundred and seventeen patients were selected for this study; their plasma viral RNA was extracted and the PR and RT genes sequenced to examine subtype, genetic polymorphisms and mutations associated with resistance to antiretroviral drugs. HIV-1 sequence analysis showed that 86/100 (86%) were infected with subtype B, 7/100 (7%) with subtype F and 7/100 (7%) with RT/PR hybrid forms (2 D/B, 2 F/B, 2 B/F and 1 D/F). In 14 (12%) of the samples, the subtype was not determined. The prevalence of resistance mutations was high (93.1%), mainly in the RT gene. The most prevalent resistance mutations were: M184V (60.7%), T215Y (49.6%) and M41L (46.7%) in the RT gene and L90M (19.6%), M46I (16.2%) and D30N (12.8%) in the PR gene. The frequency of resistance mutations tended to increase from the first to the second therapeutic scheme failure (p=0.079); but it stabilized after subsequent failures (p=0.875). Our finding of a high frequency of drug resistant HIV-1 samples supports the need for continuous genotypic monitoring of patients failing HAART.

HIV Drug Resistance Tendencies in Latvia

The treatment of HIV infection in Latvia by using highly active antiretroviral therapy (HAART) was started in 1996. The prevalence and tendencies of HIV drug resistance among treated and treatment-naive patients in Latvia in the years 2006-2007 were evaluated in this study. Data of HIV genotyping, performed in 132 HIV-1 infected during years 2006-2007 by TRUGENE HIV-1 genotyping assay (BayerHealthCare-diagnostics) are included in the study. Analysis of data showed that in the group of treatment-naive individuals majority carried wild type virus. Prevalence of resistance-associated mutations (RAMs) in the treatment-naive group according to IAS list was 28%. In most cases it was NRTI mutation A62V that is associated with multinucleoside resistance caused by Q151M, its effect in the absence of Q151M is not known. By many authors A62V is supposed to be a result of polymorphism in RT gene and is excluded from the list of resistance mutations. High prevalence of A62V is typical for HIV-1 subtype A. As majority of treatment-naive cases (89%) in this study were with HIV-1 subtypes A or AE, we excluded A62V mutation and estimated RAMs prevalence in group of treatment-naive HIV-infected individuals as 7%. Minor PI mutations were not included in analyses. In Europe published rates generally very between 5% and 15%. In the group of treatment-experienced HIV infected people 25/75 were with HIV-1 subtype B, the rest part--with non-B subtypes: A/AE (35/75), CRF-01AE (7/75), B/AE (4/75) and others. In treatment-experienced patients RAMs prevalence was estimated as 58.6%. Most frequently RAMs were found for nucleoside reverse transcriptase inhibitors (NRTI) (49.3%) followed by non-nucleoside reverse transcriptase inhibitors (NNRTI) (22.6%) and protease inhibitors (PI) (16%). In the group of NRTI mutations M184V (26/75; 34.6%), A62V (12/75; 16.0%) and T215Y (8/75; 10.6%), in NNRTI mutations K103N (10/75; 13.3%), G190S (6/75; 8.0%), in PI group mutations L90M (6/75; 8.0%) and M461/L (6/75; 8.0%) occurred most frequently. The following drug susceptibility was predicted according to the Trugen expert interpretations: in 33/75 (44%) patients no evidence of resistance, in 21/75 (28%) patients resistance to 1 drug class (NRTI--16/75, NNRTI--4/75, PI--1/75), in 17 patients (22.6%) resistance to 2 drug classes (NRTI+NNRTI--9/75, NRTI+PI--7/75, NNRTI+PI--1/75) and in 3/75 (4%) patients resistance to all 3 classes of drugs (NRTI+NNRTI+PI). We conclude, that prevalence of RAMs in treatment-naive HIV infected persons in Latvia is comparable with prevalence in Europe. The origin of predominated mutation A62V associated with NRTI at present is not clear. In more than half of treated HIV infected patients HIV resistance to at least one HAART class was predicted.

Profile of Primary Resistance in HIV-1-Infected Treatment-Naive Individuals from Western India

The majority of the HIV drug resistance (HIVDR) testing studies have focused on subtype B virus. The predominance of subtype C in the Indian subcontinent along with greater access to antiretroviral therapy (ART) necessitates studies on HIVDR genotyping. We determined the prevalence of mutations associated with protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI), and nonnucleoside reverse transcriptase inhibitors (NNRTI) from plasma of 40 antiretroviral drug-naive study participants in Indian HIV-1 pol gene sequences. Of these, 36 sequences belonged to subtype C, two to subtype A1, and two were subtype A1C recombinants. The heterosexual route was the most common route of transmission. Drug resistance-associated mutations were observed in 10% (4/40) of the study participants. The resistance mutation observed in the protease gene was V82A, whereas in the RT gene, M41L, D67N, M184V, and A98G were documented. This is the first study reporting major protease mutations by genotyping in ART-naive individuals from western India.

Viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate HIV infection

Although highly active antiretroviral therapy (HAART) can reduce HIV-1 viremia to levels that are below the limit of detection of clinical assays, the virus persists in reservoirs, and trace levels of free virions can be found in the plasma. Whether this residual viremia represents ongoing cycles of replication continuing despite HAART or simply the release of virus from stable reservoirs has been controversial. Here we summarize the evidence that HAART can stop ongoing cycles of replication. The evidence comes from a detailed analysis of the residual viremia, which shows it to be archival and nonevolving in character. In addition, new pharmacodynamic measures incorporating a previously ignored slope parameter have provided the first real indication of how well HAART actually suppresses viral replication in vivo. Together, these results argue that the ultimate theoretical potential of HAART to control viral replication has already been reached. Progress toward eradication of the infection will require novel approaches to target the stable reservoirs that persist even when viral replication is completely halted.

Profile of drug resistance mutations among HIV-1-infected Tunisian subjects failing antiretroviral therapy

Three years after the introduction of antiretroviral therapy (ART) in Tunisia (North Africa), we aimed to determine the prevalence of drug resistance mutations in Tunisian HIV-1-infected patients failing ART. Plasma samples of 80 patients were tested for genotypic resistance using two distinct line probe assays, LiPA HIV-1 reverse transcriptase RT and LiPA HIV-1 protease assay. Of the 80 patients, 82.5% showed resistance to at least one antiretroviral molecule. In the RT gene, resistance to nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) were recognized in 66.25 and 37.5%, respectively, with M184V, T215Y and K103N being the codons most frequently involved. Resistance to protease inhibitors (PIs) was found in 46.25% of cases. Despite the presence of different mutations, the viral variants were still susceptible to other RTIs and PIs that are currently not available in Tunisia. Thus, alternative therapeutic options exist but are not yet accessible.

Drug Resistance-Associated Genotypic Alterations in the pol Gene of HIV Type 1 Isolates in ART-Naive Individuals in North India

ABSTRACT We genotyped the RT and PI regions of the pol gene of HIV-1 from treatment-naive infected individuals in North India and evaluated their possible physiological relevance and association with drug resistance. Plasma samples from 52 newly diagnosed HIV-1-infected drug-naive individuals were subjected to CD4(+) cell count and plasma viral load. For genotyping, the protease and RT regions of the pol gene were amplified from cDNA reverse transcribed from plasma viral RNA by single or nested polymerase chain reaction (PCR). Sequences of amplified products were analyzed for mutations using the Stanford DR and REGA database. Two out of 49 amplicons showed mutations at known "major" subtype B drug resistance positions (one each in protease and RT). In the protease region it showed a major drug resistance mutation at M46I as well as "minor" positions F53L and T74P. In the RT gene, one patient showed a mutation at major NNRTI position G190V. Forty-nine percent had mutations in the hinge (M36I, R41K, H69K) and alpha-helix (L89M) regions of the C-virus protease, which has been linked to increased catalytic activity. Our study indicates that a number of major mutations associated with resistance to PIs, NNRTIs, and NRTIs do exist, though at a low frequency, among HIV-1 isolates from treatment-naive individuals in North India. Many minor or accessory mutations related to resistance to PIs and NRTIs are also present as the variants. These results point to the greater biochemical fitness of subtype C protease and faster decrease in drug sensitivity.

Characterization, genomic organization and chromosomal distribution of Ty1- copia retrotransposons in species of Hypochaeris (Asteraceae)

This study aims to analyze the diversity of Ty1- copia retrotransposons in 18 taxa of Hypochaeris, including two Old World species H. maculata (2 n = 2 x = 10) and H. angustifolia (2 n = 2 x = 8), and representatives of the South American species (16 accessions of 15 species; all 2 n = 2 x = 8). Analysis of 380 PCR-amplified sequences, corresponding to a conserved domain of the subset of Ty1 -copia reverse transcriptase ( rt) gene amplifiable with degenerate standard primers, showed high levels of intra- and interspecific heterogeneity. Nucleotide diversity (Pi) of the copia fragments was high in all species and varied from 0.229 ( H. angustifolia) to 0.412 ( H. chillensis). Higher sequence heterogeneity correlates positively with larger genome size among analyzed species. Phylogenetic analyses of amplified fragments revealed different patterns of intraspecific heterogeneity within species, with most sequences forming one well-supported main clade while a few sequences fall into small clades or are left ungrouped. The combined analysis of all sequences revealed the presence of three main clades and showed that highly diverged species contain closely related Tyl- copia group retrotransposons. One of the main clades includes rt sequences of all South American species and three sequences of their putative ancestor, H. angustifolia, but no sequence of the Old World H. maculata. FISH with copia retrotransposons in four Hypochaeris species, including H. maculata and H. angustifolia and New World H. apargioides and H. spathulata, revealed differences in the chromosomal distribution between the two groups. In Old World species copia retroelements are distributed over the whole length of the chromosomes, excluding rDNA sites and some centromeres. In the South American species the two largest chromosome pairs are enriched in copia, while most of the long arms of the two small pairs of chromosomes are devoid of these elements. The patterns of heterogeneity and chromosomal distribution of Ty1- copia retrotransposons in Hypochaeris are discussed in the context of the origin, genome evolution and organization of the South American species.

HIV-1 genotypic resistance profiles in children failing highly active antiretroviral therapy

To study the genotypic resistance profiles of HIV-1 children failing highly active antiretroviral therapy (HAART) so as to provide helpful information for the treatment regime of Chinese children infected with HIV-1. Peripheral venous blood samples were collected from 20 HIV-1 infected children of Henan province, aged 9 (3 - 14). Nested RT-PCR was used to amplify part of the RT (40 -250 aa) gene. The PCR products of RT gene underwent nucleotide sequencing, the resulting nucleotide sequences were analyzed by the HIVdb data offered by the Stanford University web site to find the drug resistance mutations. (1) Phylogenetic analysis revealed that 20 of the RT sequences were classified as subtype B. (2) According to the genotypic analysis, 20 , 15, and 13 children showed high level resistance to the nevirapine. (NVP), delavirdine (DLV), and efavirenz (EFV) respectively; 7 and 5 children showed high and intermediate level resistance to azidothymidine (AZT) respectively. Five children showed potential low-level and intermediate level resistance to lamivudine (3TC), and 11 showed high level resistance to 3TC; 11 showed intermediate and high level resistance to stavudine (d4T) and didanoside (ddI) respectively; and 19 and 12 children showed resistance to abacavir (ABC) and tenofovir (TDF) which had never been taken by these children. The emergence of HIV resistant strains during antiretroviral therapy is one of the main reasons for treatment failure in HIV-infected children.

Genetic Polymorphisms and Resistance Mutations of HIV Type 2 in Antiretroviral-Naive Patients in Burkina Faso

Natural polymorphisms in the pol gene of HIV-2 may influence the susceptibility to antiretroviral drugs and the choice of treatment. We collected samples in centers for anonymous HIV testing in Ouagadougou, Burkina Faso, in patients supposedly naive for any antiretroviral treatment. Eighty-four samples were first tested as HIV-2 positive in Burkina Faso and then shipped to Brussels, Belgium, for confirmation of the serological status and plasma viral load. Fifty-two samples were confirmed as HIV-2 positive in Belgium. Twelve others were HIV-1 positive and 20 were dually reactive. Twenty-one of HIV-2 confirmed samples had an HIV-2 plasma viral load higher than 1000 copies/ml. These viruses were sequenced in the protease and reverse trancriptase genes and 17 sequences of the pol gene were obtained. Highly polymorphic positions were identified in protease and RT genes. Two samples harbored known resistance mutations: M184V RT mutation in one and Q151M with M184V in the other. Phylogenetic analysis showed that viruses in Burkina Faso did not cluster separately from published sequences from neighboring countries. The two resistant strains were unrelated. Our findings imply either that resistant viruses are circulating in Burkina Faso or that some individuals take unsupervised treatment. Both hypotheses present problems.

Selection of human immunodeficiency virus type 1 resistance against the pyranodipyrimidine V-165 points to a multimodal mechanism of action

We have previously identified the pyranodipyrimidines (PDPs) as a new class of integrase (IN) inhibitors. The most potent congener V-165 inhibits HIV-1 integration at low micromolar concentrations by inhibiting the binding of IN to the DNA. As part of pre-clinical studies with PDP, we wanted to investigate HIV resistance development against V-165 and to further characterize the physicochemical properties of the compound. We selected PDP-resistant HIV-1 strains by growing the virus in the presence of increasing concentrations of V-165. The selected strains were analysed genotypically and phenotypically. Mutant IN enzymes were generated and evaluated in an enzymatic oligonucleotide-based assay for their activity and sensitivity to the different IN inhibitors. In addition, the antiviral effect of the compound on viral entry and integration was measured using quantitative PCR. Numerous mutations were detected in the RT, IN and env genes of the virus selected in the presence of V-165. Although V-165 inhibited integration in vivo as indicated by a decrease in the number of integrated proviruses, the compound also inhibited viral entry at a concentration of 19 microM. V-165 was poorly recovered from human hepatic microsomal matrix and 1% BSA. These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.

Sequence analysis of Arthrospira maxima based on fosmid library

Arthrospira (Spirulina) (Setchell & Gardner) is an important cyanobacterium not only in its nutritional potential but in its special biological characteristics. An unbiased fosmid library of Arthrospira maxima FACHB438 that contains 4300 clones was constructed. The size distribution of insert fragments is from 15.5 to 48.9 kb and the average size is 37.6 kb. The recombination frequency is 100%. Therefore the library is 29.9 equivalents to the Arthrospira genome size of 5.4 Mb. A total of 719 sample clones were randomly chosen from the library and 602 available sequences, which consisted of 307,547 bases, covering 5.70% of the whole genome. The codon usage of A. maxima was not strongly biased. GC content at the first position of codons (46.9%) was higher than the second (39.8%) and the third (45.5%) positions. GC content of the genome was 43.6%. Of these sequences, 287 (47.7%) showed high similarities to known genes, 63 (10.5%) to hypothetical genes and the remaining 252 (41.8%) had no significant similarities. The assigned genes were classified into 22 categories with respect to different biological roles. Remarkably, the high presence of 25 sequences (4.2%) encoding reverse transcriptase indicates the RT gene may have multiple copies in the A. maxima genome and might play an important role in the evolutionary history and metabolic regulation. In addition, the sequences encoding the ATP-binding cassette transport system and the two-component signal transduction system were the second and third most frequent genes, respectively. These genomic features provide some clues as to the mechanisms by which this organism adapts to the high concentration of bicarbonate and to the high pH environment.

Chromosomal locations of the maize (Zea mays L.) HtP and rt genes that confer resistance to Exserohilum turcicum

We used 125 microsatellite markers to genotype the maize (Zea mays L.) near isogenic lines (NIL) L30HtPHtPRtRt and L30htphtpRtRt and the L40htphtprtrt line which contrast regarding the presence of the recently described dominant HtP and the recessive rt genes that confer resistance to Exserohilum turcicum. Five microsatellite markers revealed polymorphisms between the NIL and were considered candidate linked markers for the HtP resistance gene. Linkage was confirmed by bulked segregant sample (BSS) analysis of 32 susceptible and 34 resistant plants from a BC1F1 population derived from the cross (L30HtPHtPRtRt x L40htphtprtrt) x L40htphtprtrt. The bnlg198 and dupssr25 markers, both located on maize chromosome 2L (bin 2.08), were polymorphic between bulks. Linkage distances were estimated based on co-segregation data of the 32 susceptible plants and indicated distances of 28.7 centimorgans (cM) between HtP and bnlg198 and 23.5 cM between HtP and dupssr25. The same set of susceptible plants was also genotyped with markers polymorphic between L30HtPHtPRtRt and L40htphtprtrt in order to find markers linked to the rt gene. Marker bnlg197, from chromosome 3L (bin 3.06), was found linked to rt at a distance of 9.7 cM. This is the first report on the chromosomal locations of these newly described genes.

Detection of a Group II Intron without an Open Reading Frame in the Alpha-Toxin Gene ofClostridium perfringensIsolated from a Broiler Chicken

A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.

Diversity and evolution of Ty1- copia and Ty3- gypsy retroelements in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae)

We present the first study on the diversity and evolution of Ty1- copia and Ty3- gypsy retroelements in a group of non-photosynthetic flowering plants. To this end partial sequences of the reverse transcriptase ( rt) gene were obtained from 20 clones for each retroelement type from seven and six accessions of Orobanche and Phelipanche (Orobanchaceae), respectively. Overall sequence similarity is higher in Ty3- gypsy elements than in Ty1- copia elements in agreement with the results from other angiosperm groups. Higher sequence diversity and stronger phylogenetic structure, especially of Ty1- copia sequences, in Orobanche species compared to Phelipanche species support the previously suggested hypothesis (based on karyological and cytological data) that genomes of Orobanche species are more dynamic than those of Phelipanche species. No evidence was found for intraspecific differences of retroelement diversity nor for differences between pest taxa and their putative wild relatives, e.g., O. crenata and O. owerini. The occurrence of a few sequences from Phelipanche species in clades otherwise comprising sequences from Orobanche species might be due to horizontal gene transfer, but the alternative of vertical transmission cannot be rejected unambiguously.

Selection of a Multiple Drug-Resistant Hepatitis B Virus Strain in a Liver-Transplanted Patient

Sequential anti-hepatitis B virus (HBV) therapy may lead to the selection of complex mutants. We analyzed the genetic and phenotypic evolution of the viral quasispecies of a patient who received successively lamivudine, add-on adefovir+lamivudine, followed by lamivudine+adefovir+hepatitis B immunoglobulins (HBIg) after orthotopic liver transplantation. For genotypic analysis, a 1310-bp region of the polymerase gene was amplified, cloned, and sequenced. Huh-7 cells were transfected to compare the replication fitness of HBV mutants and their susceptibility to drugs. At baseline, all HBV genomes carried a wild-type (wt) RT gene but 22% harbored the sP120S and 55% the sC107stop mutations within the surface (S) gene associated with vaccine escape. Following viral breakthrough to lamivudine monotherapy, a complex mixture of lamivudine-resistant HBV strains prevailed. Interestingly, among these mutants emerged a population harboring only the rtL180M+A181V mutations, conferring lamivudine-resistance in vitro. After addition of adefovir to the ongoing treatment, viral load dropped, and the patient underwent an orthotopic liver transplantation and received HBIg. As viral load rose again, a single viral population was progressively selected, harboring the rtV173L+L180M+A181V+N236T and sP120S mutations. In vitro, this last mutant showed a level of replication reduced by only 30% compared to wt HBV and a strong resistance to both lamivudine (>1000-fold) and adefovir (>10-fold). It remained sensitive to tenofovir both in vitro and in vivo. We report the selection of a complex HBV mutant that escaped the antiviral pressure of lamivudine, adefovir, and HBIg, and provide insight on the process of selection via genotypic and phenotypic analysis.

Novel Broad-Spectrum Thiourea Non-Nucleoside Inhibitors for the Prevention of Mucosal HIV Transmission

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTI) are an integral part of combination therapy comprising three classes of antiretroviral drugs for the management of HIV/AIDS. NNRTIs are chemically diverse compounds that bind to a common allosteric site of HIV-1 RT and noncompetitively inhibit DNA polymerization. Resistance to NNRTIs arises rapidly upon drug treatment and results from mutation of the amino acids lining the HIV-1 RT binding pocket. Nevertheless, rationally designed NNRTIs deduced from changes in binding pocket size, shape, and residue character that result from clinically observed NNRTI resistance mutations exhibit broad-spectrum anti-HIV-1 activity. Notably, membrane permeable tight binding NNRTIs have utility as topical microbicides since they are capable of blocking cell-free and cell-associated mucosal HIV-1 infection without metabolic activation. This review summarizes the discovery of highly potent tight binding phenethyl-thiazolyl-thiourea (PETT) derivatives targeting the NNI binding pocket of HIV-1 RT. These NNRTIs were rationally designed by molecular docking using a composite binding pocket constructed by superimposing the crystal structure coordinate data of several NNI/RT ligand-binding site complexes. Molecular modeling and score functions such as molecular surface area, the buried surface, and binding affinity values were used to analyze how drug-resistant mutations would change the RT binding pocket shape, volume, and chemical make-up of these NNRTIs, and how these changes could affect drug binding. Several ligand derivatization sites were identified for docked compounds that fit the binding pocket. The best fit was determined by calculating an inhibition constant (Ludi Ki) of the docked compound for the composite binding pocket. Compounds with a Ludi Ki of <1 microM were identified as the most promising tight binding NNRTIs. This review highlights novel lipophilic thiourea NNRTIs that display high binding affinity and selective indices with robust anti-HIV-1 activity against the wild type as well as drug-resistant isolates carrying multiple RT gene mutations. The increasing prevalence of drug-escape mutants among recent HIV seroconverters makes the discovery of these broad-spectrum thiourea NNRTIs useful as a component of topical microbicide for the prevention of mucosal HIV transmission.

Natural polymorphism in protease and reverse transcriptase genes and in vitro antiretroviral drug susceptibilities of non-B HIV-1 strains from treatment-naive patients

Background Most studies on antiretroviral (ARV) resistance of human immunodeficiency virus (HIV) have been done on subtype B which only represent a limited proportion of infections worldwide. Objective Understand baseline susceptibilities to ARVs in non-B strains. Methods To explore in greater detail possible intrinsic resistance to antiretroviral drugs in non-B subtypes, phenotypic resistance was tested in 35 non-B (A, D, F, G, J; CRF02, 06, 09, 11, 13) HIV-1 isolates obtained from ARV treatment naive patients. The panel includes strains with an increasing number of minor mutations in the protease gene and/or with atypical mutations at positions associated with resistance in protease and RT. Results We detected phenotypic resistance (fold-change values equal or superior to biological test cut-offs (BCO) in 14 of the 35 strains, 4 strains had decreased in vitro susceptibility to more than one drug. However, it is important to note that in most cases the increased fold-changes were close to the BCOs. Phenotypic resistance was observed against each of the three ARV drug classes: ritonavir ( n = 3), nelfinavir ( n = 2), saquinavir ( n = 2), zidovudine ( n = 2), stavudine ( n = 1), didanosine ( n = 1); delavirdine ( n = 6), efavirenz ( n = 1) and nevirapine ( n = 1). Some mutations could be associated with decreased in vitro susceptibility: 1 of 3 strains only with mutations M46I/L in protease, 1/2 A98S, K101N, V108I, V179I, and P236L in reverse transcriptase. Interestingly, the presence of an increasing number of minor mutations in the protease gene was not associated with decreased in vitro susceptibility to protease inhibitors. Conclusion It is necessary to continue phenotypic studies on non-subtype B strains to identify the role of all polymorphisms present in protease and RT genes and to optimize interpretation algorithms. Data obtained from large, diverse populations of HIV-1 infected individuals is critical for defining and standardizing the quantification of resistance (phenotypic and genotypic testing).

HIV Type 1polGene Diversity and Antiretroviral Drug Resistance Mutations in the Democratic Republic of Congo (DRC)

To study recombination and the natural polymorphism in pol of HIV-1 strains in the Democratic Republic of Congo (DRC) we sequenced the protease and RT genes for 70 HIV-1 strains previously characterized in the env V3-V5 region from a sentinel surveillance study in 2002. For 41 of the 70 (58.6%) strains, the same subtype/ CRF designations were observed in pol and env. Twenty-three (32.9%) of 70 pol sequences were complex recombinants involving two to five subtypes as well as fragments that could not be classified into any of the known subtypes. All subtypes were involved in recombination events. Unclassified (U) and env subtype H strains were very likely to be recombinant strains. Overall, many minor mutations were identified in the protease sequences. Although at the time of our study ARV use was not yet widespread in DRC, three strains were identified with one major mutation associated with drug resistance: L90M and M46L in protease and K103N in RT.