Carlos Briones

Role of a dipeptide insertion between codons 69 and 70 of HIV‐1 reverse transcriptase in the mechanism of AZT resistance

The 3′‐azido‐3′‐deoxythymidine (AZT)‐resistant pheno type of a heavily mutated human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) carrying a dipeptide (Ser‐Ser) insertion between codons 69 and 70 as well as other mutations related to resistance to RT inhibitors has been studied. Recombinant virus carrying this variant RT (termed SS RT) showed reduced susceptibility to all nucleoside RT inhibitors in clinical use, particularly to AZT. In the presence of ATP, recombinant SS RT had an increased ability to remove the 3′‐terminal nucleotide from AZT‐ terminated primers and extend the unblocked primer, compared with wild‐type HIV‐1 RT (BH10 isolate). Insertion of two serines in the sequence context of BH10 RT did not affect the ATP‐dependent phosphorolytic activity of the enzyme, and had no influence in resistance to RT inhibitors. However, SS RT mutants lacking the dipeptide insertion or bearing a four‐serine insertion showed reduced ATP‐dependent phosphorolytic activity that correlated with increased AZT sensitivity, as determined using a recombinant virus assay. Therefore, the insertion appears to be critical to enhance AZT resistance in the sequence context of multidrug‐resistant HIV‐1 RT.

Primary genotypic and phenotypic HIV-1 drug resistance in recent seroconverters in Madrid.

Objective: Transmission of drug‐resistant HIV‐1 strains is increasing with widespread use of antiretroviral drugs in developed countries. This study examined the prevalence of resistant viruses in recent seroconverters in Madrid, Spain. Design: HIV isolates from 30 consecutive participants with positive or indeterminate HIV antibody test results and a negative test result at a mean of 6.6 months earlier were examined for HIV drug resistance. All study subjects admitted to having very recently engaged in high‐risk practices. All were therapeutically naive and were recruited between 1997 and 1999 in a referring health care facility for sexually transmitted diseases. Methods: Population‐based sequencing of the viral reverse transcriptase (RT) and protease (PR) regions derived from plasma viral RNA was performed. Phenotypic resistance was assessed by a recombinant virus assay. Results: Overall prevalence of genotypes associated with reduced susceptibility was 26.7% (8 of 30 participants). Resistance mutations were seen against nucleoside analogues in 7 (23.3%), nonnucleoside reverse transcriptase inhibitors in 1 (3.3%), and protease inhibitors in 2 (6.7%). Zidovudine‐resistance mutations M41L and/or T215Y were the commonest, found in 20% (6 of 30 participants). Resistance mutations to at least two antiretroviral families (multidrug‐resistance) were detected in 2 (6.7%) study subjects. A median infectious dose (IC50) increase of fourfold for any drug was found in 7 patients, and in 2 was > tenfold for zidovudine (genotype M41L + T215Y) and lamivudine (genotype M184V), respectively. Conclusions: Drug‐resistant HIV variants were present in over one quarter of individuals recently diagnosed as infected in Madrid, Spain. Therefore, resistance testing at baseline should be considered for the optimal design of first‐line antiretroviral combinations.

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain.

ObjectiveTo examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain. MethodsA cross-sectional study was conducted including 601 HIV-infected patients who attended 20 Spanish hospitals in June 1998. Drug resistant mutations were examined using hybridization line probe assays (LiPA). The 6 bp insert at position 69 and the codon 75 mutant were examined by sequencing analysis in specimens lacking reactivity to 69/70 and 74 bands on LiPA, respectively. ResultsPrimary resistance to NRTI was recognized in nine out of 52 (17%) naïve individuals, whereas primary resistance to PI was found in seven out of 126 (6%) PI-naïve patients. The codons most frequently involved in NRTI resistance were at positions 70 (66%), 184 (44%), 215 (33%), and 41 (11%), whereas the most common PI resistance mutation was at codon 82 (6/7 subjects). In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI. The most frequent NRTI mutations occurred at codons 184 (38.5%), 215 (30.1%), and 41 (22.5%), whereas the most frequent PI mutations in pre-treated subjects were found at positions 82 (15.8%) and 84 (11.4%). Overall, patients who began triple combinations as initial therapy showed a lower number of key resistance mutations than those who began highly active antiretroviral therapy (HAART) after being exposed to NRTI for a period of time (mean number of mutations, 0.1 versus 1.8, P  < 0.05). Codon 75 mutant was found in three out of 387 patients (0.7%), whereas no insertions at codon 69 were recognized. ConclusionThe prevalence of primary genotypic resistance to NRTI and PI in Spain was 17% and 6%, respectively. Zidovudine, lamivudine, indinavir and ritonavir were the drugs most frequently affected. These data support the use of resistance testing prior to the introduction of first-line antiretroviral therapies in Spain. Among pre-treated subjects, drug resistance genotypes were less prevalent in those who began HAART as initial therapy.

Dynamics of dominance of a dipeptide insertion in reverse transcriptase of HIV-1 from patients subjected to prolonged therapy.

A small proportion (0.8%) of individuals of a cohort of HIV-1 infected patients subjected to prolonged therapy with nucleoside analogues included a recently recognised dipeptide insertion in their RT (Ser-Ser or Ser-Gly between RT codons 69 and 70). To study the dynamics of dominance of genomes with this genetic change, sequential HIV-1 isolates from two patients were analyzed with regard to consensus sequences and complexity of mutant spectra. The two patients displayed completely different, complex evolutionary patterns leading to temporary dominance of dipeptide insertions. In one patient, a virus very closely related to an ancestor virus from the same patient overtook the population at late times, displacing genomes encoding a Ser-Ser insertion. In another patient the sequential dominance of genomes with Ser-Ser insertion-->no insertion-->Ser-Gly insertion was observed. These three types of genomes coexisted in the mutant spectrum of one HIV-1 isolate. Complexity was also reflected in the shape of phylogenetic trees derived with genomes from the mutant spectrum at each time point. The results suggest that HIV-1 genomes encoding a dipeptide insertion between RT codons 69 and 70 do not show a clear selective advantage over other genomes lacking the insertion. Such an absence of a clear selective advantage will favor that such genomes encoding this RT insertion become dominant only in a transient fashion, and following disparate kinetics in different patients.

Prevalence and genetic heterogeneity of the reverse transcriptase T69S-S-X insertion in pretreated HIV-infected patients.

The emergence of resistance to antiretroviral drugs represents one of the main reasons for treatment failure in HIV-infected persons. Resistance to multiple nucleoside analogues may result from rearrangements in the HIV pol gene, in particular one insertion of two amino acids at position 69. Herein, we examined the prevalence of this resistant genotype and its genetic heterogeneity in a group of 475 healthy pretreated HIV-positive subjects in Spain. Only 4 (0.8%) carried the codon 69 insertion. It was always found coupled to the T69S mutation. The extra amino acids were S-S in 2 subjects and S-G in the other 2. The presence of the insert was seen only in subjects previously exposed to AZT monotherapy for at least 6 months.

Usefulness of genotypic analysis of resistance to nucleoside analogues in the clinical setting.

Abstract The introduction of drug resistance testing during clinical care of patients infected with the human immunodeficiency virus (HIV) is still a matter for discussion. Thirty-seven HIV-infected subjects who had positive plasma viraemia despite undergoing antiretroviral therapy were tested for the presence of genotypic mutations linked to resistance to nucleoside analogues. Thirty (81.1%) individuals showed one or more mutations conferring drug resistance. Eight harboured mutants at codons 215 and 41, which confer resistance to zidovudine and are associated with the worst prognosis. One individual carried the codon 69-SSS insertion, which confers multidrug resistance. Seven (18.9%) subjects did not show any mutation, but all of them failed to adhere to the treatment regimen and/or experienced diarrhea that likely caused malabsorption of the drugs. Clinicians referred to the resistance profile when they adjusted or prescribed subsequent combination regimens for their patients.