Biomedical subjects

Luis Menéndez-Arias

Publications and source records attributed to Luis Menéndez-Arias.

18 recordsLinked to original sources

PubMed2006

Mutational patterns associated with the 69 insertion complex in multi-drug-resistant HIV-1 reverse transcriptase that confer increased excision activity and high-level resistance to zidovudine.

Human immunodeficiency virus type 1 (HIV-1) strains having dipeptide insertions in the fingers subdomain and other drug resistance-related mutations scattered throughout their reverse transcriptase (RT)-coding region show high-level resistance to zidovudine (AZT) and other nucleoside analogues. Those phenotypic effects have been correlated with their increased ATP-dependent phosphorolytic activity on chain-terminated primers. Mutations T69S and T215Y and a dipeptide insertion (i.e. Ser-Ser) between positions 69 and 70 are required to achieve low-level resistance to thymidine analogues. However, additional amino acid substitutions are necessary to achieve the high-level phenotypic resistance to AZT shown by clinical HIV isolates carrying a dipeptide insertion in their RT-coding region. In order to identify those mutations that contribute to resistance in the sequence context of an insertion-containing RT derived from an HIV clinical isolate (designated as SS RT), we expressed and purified a series of chimeric enzymes containing portions of the wild-type or SS RT sequences. ATP-mediated excision activity measurements using AZT- and stavudine (d4T)-terminated primers and phenotypic assays showed that molecular determinants of high-level resistance to AZT were located in the fingers subdomain of the polymerase. Further studies, using recombinant RTs obtained by site-directed mutagenesis, revealed that M41L, A62V and in a lesser extent K70R, were the key mutations that together with T69S, T215Y and the dipeptide insertion conferred high levels of ATP-dependent phosphorolytic activity on AZT and d4T-terminated primers. Excision activity correlated well with AZT susceptibility measurements, and was consistent with phenotypic resistance to d4T. Structural analysis of the location of the implicated amino acid substitutions revealed a coordinated effect of M41L and A62V on the positioning of the beta3-beta4 hairpin loop, which plays a key role in the resistance mechanism.

Adenosine Triphosphate↗

PubMed2006

HIV protease cleaves poly(A)-binding protein.

The PABP [poly(A)-binding protein] is able to interact with the 3' poly(A) tail of eukaryotic mRNA, promoting its translation. Cleavage of PABP by viral proteases encoded by several picornaviruses and caliciviruses plays a role in the abrogation of cellular protein synthesis. We report that infection of MT-2 cells with HIV-1 leads to efficient proteolysis of PABP. Analysis of PABP integrity was carried out in BHK-21 (baby-hamster kidney) and COS-7 cells upon individual expression of the protease from several members of the Retroviridae family, e.g. MoMLV (Moloney murine leukaemia virus), MMTV (mouse mammary tumour virus), HTLV-I (human T-cell leukaemia virus type I), SIV (simian immunodeficiency virus), HIV-1 and HIV-2. Moreover, protease activity against PABP was tested in a HeLa-cell-free system. Only MMTV, HIV-1 and HIV-2 proteases were able to cleave PABP in the absence of other viral proteins. Purified HIV-1 and HIV-2 proteases cleave PABP1 directly at positions 237 and 477, separating the two first RNA-recognition motifs from the C-terminal domain of PABP. An additional cleavage site located at position 410 was detected for HIV-2 protease. These findings indicate that some retroviruses may share with picornaviruses and caliciviruses the capacity to proteolyse PABP.

Amino Acid Sequence↗

PubMed2006

DR_SEQAN: a PC/Windows-based software to evaluate drug resistance using human immunodeficiency virus type 1 genotypes.

BACKGROUND: Genotypic assays based on DNA sequencing of part or the whole reverse transcriptase (RT)- and protease (PR)-coding regions of the human immunodeficiency virus type 1 (HIV-1) genome have become part of the routine clinical management of HIV-infected individuals. However, the results are difficult to interpret due to complex interactions between mutations found in viral genes. RESULTS: DR_SEQAN is a tool to analyze RT and PR sequences. The program output includes a list containing all of the amino acid changes found in the query sequence in comparison with the sequence of a wild-type HIV-1 strain. Translation of codons containing nucleotide mixtures can result in potential ambiguities or heterogeneities in the amino acid sequence. The program identifies all possible combinations of 2 or 3 amino acids that derive from translation of triplets containing nucleotide mixtures. In addition, when ambiguities affect codons relevant for drug resistance, DR_SEQAN allows the user to select the appropriate mutation to be considered by the program's drug resistance interpretation algorithm. Resistance is predicted using a rule-based algorithm, whose efficiency and accuracy has been tested with a large set of drug susceptibility data. Drug resistance predictions given by DR_SEQAN were consistent with phenotypic data and coherent with predictions provided by other publicly available algorithms. In addition, the program output provides two tables showing published drug susceptibility data and references for mutations and combinations of mutations found in the analyzed sequence. These data are retrieved from an integrated relational database, implemented in Microsoft Access, which includes two sets of non-redundant core tables (one for combinations of mutations in the PR and the other for combinations in the RT). CONCLUSION: DR_SEQAN is an easy to use off-line application that provides expert advice on HIV genotypic resistance interpretation. It is coded in Visual Basic for use in PC/Windows-based platforms. The program is freely available under the General Public License. The program (including the integrated database), documentation and a sample sequence can be downloaded from http://www2.cbm.uam.es:8080/lmenendez/DR_SEQAN.zip.

Algorithms↗

PubMed2006

Insertions and deletions in HIV-1 reverse transcriptase: consequences for drug resistance and viral fitness.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target of drugs fighting HIV infection. The introduction of potent antiretroviral therapies based on the use of RT inhibitors and/or protease inhibitors has been an important achievement towards the control of AIDS. However, the development of drug resistance constitutes a major hurdle towards long-term efficacy of those therapies. With the increasing complexity of the antiretroviral regimens, novel mutational patterns conferring high-level resistance to nucleoside and nonnucleoside RT inhibitors have been identified in viral isolates. Among them, insertions and deletions in the beta3-beta4 hairpin-loop-coding region of HIV-1 RT have been identified in heavily-treated patients. Insertions of one, two or several residues appear to have a significant impact on nucleoside analogue resistance. The frequently found combination of a dipeptide insertion and thymidine analogue resistance mutations (i.e. T215Y) in the viral RT confers an ATP-dependent phosphorolytic activity that facilitates the removal of the inhibitor from primers terminated with zidovudine or stavudine. Furthermore, this mechanism appears to be relevant for resistance mediated by one amino acid-deletions appearing in combination with thymidine analogue resistance mutations. However, in other sequence contexts (i.e. in the presence of Q151M), the effects of the deletion are not fully understood. Drugs targeting the excision repair mechanism could be an important aid in the fight against multinucleoside-resistant HIV isolates bearing complex mutational patterns in their RT-coding region.

Amino Acid Sequence↗

PubMed2005

Suppression of multidrug-resistant HIV-1 reverse transcriptase primer unblocking activity by alpha-phosphate-modified thymidine analogues.

A dipeptide insertion between codons 69 and 70 together with the amino acid substitution T215Y in the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1 (HIV-1) strains are known to confer phenotypic resistance to zidovudine (AZT) and stavudine (d4T). Phenotypic resistance correlates with an increased ATP-dependent phosphorolytic activity. Nucleoside alpha-boranophosphate diastereoisomers derived from AZT and d4T were tested as substrates of a multidrug-resistant HIV-1 RT (designated as SS RT) bearing a Ser-Ser insertion at codons 69-70 and other drug resistance-related mutations, in DNA polymerization assays and ATP-mediated excision reactions. Using pre-steady-state kinetics, we show that SS RT can incorporate both R(p) and S(p) diastereoisomers, although R(p) is the preferred isomer. Chirality at the internucleotidic linkage formed upon incorporation of nucleoside alpha-boranophosphate did not affect ATP-mediated excision. As reported for AZT and d4T-terminated primers, substituting Thr, Asn or Ser for Tyr215 abrogates the ATP-dependent phosphorolytic activity on primers terminated with alpha-boranophosphate derivatives of thymidine analogues. However, unlike in the case of AZT, eliminating the dipeptide insertion in SS RT had no effect on the ATP-mediated excision of primers terminated with alpha-boranophosphate derivatives of d4T. Studies with ATP analogues showed that exchanging a non-bridging oxygen atom at the gamma-phosphate group for sulfur causes a significant reduction of the ATP-dependent phosphorolytic activity of SS RT. Interestingly, SS RT's excision activity is completely eliminated upon phosphorothioate substitution at the 3' end of primers terminated with AZT. These results suggest that phosphorothioate derivatives of currently approved drugs could be useful against excision-proficient HIV-1 strains.

Adenosine Triphosphate↗

PubMed2005

Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114.

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3'-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent K(m) values for dNTP. Discrimination between AZTTP (3'-azido-3'-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5-6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G-->A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

Alanine↗

PubMed2004

Relative replication fitness of multi-nucleoside analogue-resistant HIV-1 strains bearing a dipeptide insertion in the fingers subdomain of the reverse transcriptase and mutations at codons 67 and 215.

A two-serine insertion at position 69 (i69SS) of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) appears to be critical to enhance multi-nucleoside RT inhibitor resistance (MNR) in the sequence context of multiple zidovudine (AZT) resistance mutations (i.e., M41L, L210W, T215Y). In this study, we measured the replication capacity relative to the wild-type (WT) HIV-1 of a series of recombinant viruses carrying the i69SS in the background of a clinical isolate with MNR in which we introduced mutations D67N, Y215T, Y215S, or Y215N. In vitro measurements included replication kinetics and growth competition assays at different multiplicities of infection (MOI). While the addition of D67N had a minor effect on replication capacity, the reversion of Tyr-215 to Thr, Ser, or Asn was sufficient to increase the virus ability to replicate in a drug-free environment. The same genotypic changes at position 215 rendered the MNR virus susceptible to AZT and stavudine. Interestingly, the presence of the insertion together with mutation T215Y in an otherwise WT sequence background was not sufficient to confer high-level resistance to AZT, although its replication capacity was clearly impaired. Therefore, the RT residue 215 plays a critical role in both replication capacity and drug resistance of multidrug-resistant viruses containing the i69SS.

Adult↗

PubMed2004

Tryptophan scanning mutagenesis of aromatic residues within the polymerase domain of HIV-1 reverse transcriptase: critical role of Phe-130 for p51 function and second-site revertant restoring viral replication capacity.

The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.

Animals↗

PubMed2004

Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers.

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Adenosine Triphosphate↗

PubMed2004

Increased G-->A transition frequencies displayed by primer grip mutants of human immunodeficiency virus type 1 reverse transcriptase.

A genetic screen based on the blue-white beta-galactosidase complementation assay designed to detect G-->A mutations arising during RNA-dependent DNA synthesis was used to compare the fidelity of mutant human immunodeficiency virus type 1 reverse transcriptases (RTs) with the mutations M230L and M230I with the wild-type enzyme, in the presence of biased deoxynucleoside triphosphate (dNTP) pools. The mutant RTs with the M230L and M230I changes were found to be 20 to 70 times less faithful than the wild-type RT in the presence of low [dCTP]/[dTTP] ratios but showed similar fidelity in assays carried out with equimolar concentrations of each nucleotide. Biased dNTP pools led to short tandem repeat deletions in the target sequence, which were also detectable with the assay. However, deletion frequencies were similar for all of the RTs tested. The reported data suggest that RT pausing due to the low dNTP levels available in the RT reaction mixture facilitates strand transfer, in a process that is not necessarily mediated by nucleotide misinsertion.

Base Sequence↗

PubMed2004

HIV-resistance to viral entry inhibitors.

An increasing number of human immunodeficiency virus (HIV)-infected patients have detectable viraemia despite treatment with multiple-drug combinations or have developed resistance to all available classes of antiretroviral therapy. HIV entry has become an important pharmacological target. Enfuvirtide is the first HIV entry inhibitor to be approved for the treatment of drug-experienced patients but several other agents are progressing through preclinical and clinical trials. However, because different entry inhibitors target different parts of the entry process their combined effects could be synergistic or generate different distinct profiles of drug-resistance. The HIV envelope glycoprotein that drives HIV entry is highly variable. Its plasticity allows HIV to escape the immune system and its variability is associated with HIV tropism, fitness and replicative capacity. Thus, mutations that confer resistance to entry inhibitors will modify these parameters. Therapeutic strategies that aim at blocking virus entry may also be used to alter the natural evolution of HIV in an unprecedented way. Here, we will describe the structure and function of the envelope glycoprotein complex that constitute the basis for the emergence of resistance to HIV entry inhibitors, review those HIV entry inhibitors for which drug-resistance has been evaluated and discuss the interplay between viral resistance to inhibitors of HIV entry and the pathogenicity of HIV and AIDS.

Amino Acid Sequence↗

PubMed2003

Curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis.

BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) can be cured of virus by treatment with the antiviral nucleoside analogue ribavirin. To study whether the process involved an increase in the number of mutations in the mutant spectrum of the viral population, viral genomes were cloned from persistently infected cells treated or untreated with ribavirin. An increase of up to 10-fold in mutation frequencies associated with ribavirin treatment was observed in the viral genomes from the treated cultures as compared with parallel, untreated cultures. To address the possible mechanisms of enhanced mutagenesis, we investigated the mutagenic effects of ribavirin together with guanosine, and mycophenolic acid in the presence or absence of guanosine. Changes in the intracellular nucleotide concentrations were determined for all treatments. The results suggest that the increased mutation frequencies were not dependent on nucleotide pool imbalances or due to selection of preexisting genomes but they were produced by a mutagenic action of ribavirin.

Animals↗

PubMed2003

The eukaryotic translation initiation factor 4GI is cleaved by different retroviral proteases.

The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.

Animals↗

PubMed2003

Fitness variations and their impact on the evolution of antiretroviral drug resistance.

The human immunodeficiency virus (HIV) exhibits extensive heterogeneity due to its rapid turnover, high mutation rate, and high frequency of recombination. Its remarkable genetic diversity plays a key role in virus adaptation, including development of drug resistance. The increasing complexity of antiretroviral regimens has favored selection of HIV variants harboring multiple drug resistance mutations. Evolution of drug resistance is characterized by severe fitness losses, which can be partially overcome by compensatory mutations or other adaptive changes that restore virus replication capacity. Recent reports have addressed the impact of drug-resistance mutations on viral fitness. Methods include in vitro estimates based on the determination of viral replication kinetics, viral infectivity in single-cycle assays and growth competition experiments; as well as estimates of the relative fitness of viral populations in vivo calculated from standard population genetics theory. This review focuses on the effects in viral fitness of mutations arising during treatment with reverse transcriptase and protease inhibitors, and the molecular mechanisms (including compensatory mutations) that improve the viral fitness of drug-resistant variants.

Anti-HIV Agents↗

PubMed2002

Multidrug-resistant HIV-1 reverse transcriptase: involvement of ribonucleotide-dependent phosphorolysis in cross-resistance to nucleoside analogue inhibitors.

Human immunodeficiency virus type 1 (HIV-1) strains having a dipeptide insertion between codons 69 and 70 of the viral reverse transcriptase (RT) have been observed in isolates from patients treated with 3'-azido-3'-deoxythymidine (AZT) and other nucleoside analogues. These viruses contain additional mutations related to drug resistance and display reduced susceptibility to most nucleoside analogue inhibitors, including AZT. The mechanism of AZT resistance implies an increased ability of the multidrug-resistant (SS) RT to remove AZT-monophosphate (AZTMP) from blocked primers through a nucleotide-dependent reaction. We show that its higher ATP-dependent phosphorolytic activity is also detectable with primers terminated with 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) or 2',3'-dideoxythymidine-5'-monophosphate (ddTMP), but is significantly reduced when the dipeptide insertion is deleted. Removal of AZTMP, d4TMP and ddTMP can be inhibited by the next complementary deoxynucleoside triphosphate (dNTP). AZTMP removal reactions catalysed by SS RT were highly resistant to dNTP inhibition (IC(50)>0.25mM), while unblocking of d4TMP- and ddTMP-terminated primers was around tenfold more sensitive to inhibition by the next complementary dNTP. Both SS and mutant 2S0S RTs were able to unblock and extend primers terminated with 2',3'-dideoxycytidine-5'-monophosphate (ddCMP) in the presence of ATP, albeit very poorly. Under these conditions, none of the RTs was able to remove 2',3'-dideoxy-3'-thiacytidine-5'-monophosphate (3TCMP) from a terminated DNA primer. Resistance mediated by ATP-dependent phosphorolysis depends on the intracellular levels of dNTP. High levels as found in transformed cell lines (i.e. H-9, CEM lymphoblasts, SupT1 cells, etc.) may prevent repair of primers terminated with d4TMP. However, ATP-dependent phosphorolysis could be relevant for d4T resistance in cells having low levels of dNTPs. This proposal could explain why insertion-containing HIV-1 variants have been detected in the absence of AZT, during d4T treatment.

Amino Acid Sequence↗

PubMed2002

Molecular basis of fidelity of DNA synthesis and nucleotide specificity of retroviral reverse transcriptases.

Reverse transcription involves the conversion of viral genomic RNAinto proviral double-stranded DNA that integrates into the host cell genome. Cellular DNA polymerases replicate the integrated viral DNA and RNA polymerase II transcribes the proviral DNA into RNA genomes that are packaged into virions. Although mutations can be introduced at any of these replication steps, reverse transcriptase (RT) errors play a major role in retroviral mutation. This review summarizes our current knowledge on fidelity of reverse transcriptases. Estimates of retroviral mutation rates or fidelity of retroviral RTs are discussed in the context of the different techniques used for this purpose (i.e., retroviral vectors replicated in culture, misinsertion and mispair extension fidelity assay, etc.). In vitro fidelity assays provide information on the RT's accuracy during the elongation reaction of DNA synthesis. In addition, other steps such as initiation of reverse transcription, or strand transfer, and factors including viral proteins such as Vpr [in the case of the human immunodeficiency virus type 1 (HIV-1)] have been shown to influence fidelity. A comprehensive description of the effect of amino acid substitutions on the fidelity of HIV-1 RT is presented. Published data point to certain dNTP-binding residues, as well as to various amino acids involved in interactions with the template or the primer strand, and to residues in the minor groove-binding track as major components of the fidelity center of retroviral RTs. Implications of these studies include the design of novel therapeutic strategies leading to virus extinction, by increasing the viral mutation rate beyond a tolerable threshold.

Amino Acid Sequence↗

PubMed2002

Targeting HIV: antiretroviral therapy and development of drug resistance.

Inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease (PR) are widely used in the clinical treatment of AIDS. However, the emergence of drug-resistant variants of HIV-1 severely limits the long-term effectiveness of these drugs. In this review, the molecular basis of resistance to RT and PR inhibitors will be addressed, focusing on the mutations that confer resistance to nucleoside and non-nucleoside drugs. The emergence of multidrug-resistant viruses results from the introduction of potent antiretroviral therapy and involves the combined effects of different drug-resistance mutations. The optimization of current antiretroviral drug regimens and the development of new drugs are challenging issues in HIV chemotherapy.

Animals↗

PubMed2002

Insertions in the reverse transcriptase increase both drug resistance and viral fitness in a human immunodeficiency virus type 1 isolate harboring the multi-nucleoside reverse transcriptase inhibitor resistance 69 insertion complex mutation.

Recent studies have shown that the accumulation of multiple mutations associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance may be grouped as multi-NRTI resistance (MNR) complexes. In this study, we have examined the viral fitness of recombinant viruses carrying the reverse transcriptase (RT) of a human immunodeficiency virus type 1 (HIV-1) primary isolate harboring mutations comprising the MNR 69 insertion complex. Different RT mutants were prepared in the sequence context of either the wild-type RT sequence of the HIV-1(BH10) isolate or the sequence found in a clinical HIV-1 isolate with the MNR 69 insertion mutation. As expected, in the presence of zidovudine, recombinant viruses harboring the MNR RT from the patient were more fit than wild-type viruses. However, in the absence of drug, the virus with the RT from the original clinical isolate (SS) was more fit than (i) the wild-type virus with an engineered serine insertion between residues 69 and 70 (T69SSS) and (ii) the recombinant virus with the MNR RT where the insertion was removed (2S0S). These results suggest that RT insertions, in the right sequence context (i.e., additional mutations contained in the MNR 69 insertion complex), enhance NRTI resistance and may improve viral fitness. Thus, comparing complex mutation patterns with viral fitness may help to elucidate the role of uncharacterized drug resistance mutations in antiretroviral treatment failure.

Adult↗