Structural determinants of murine leukemia virus (MLV) reverse transcriptase (RT) important for fidelity and drug resistance in vivo

Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations generating mutants that exhibit altered host tropisms, resistance to antiviral drugs, and the ability to escape the host\u27s immune system. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity and drug-resistance in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P cell line also contains an MLV-based retroviral vector (GA-1), which encodes a wild-type bacterial beta-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with X-Gal and the frequencies of inactivating mutations in lacZ were quantified. Several structural determinants of MLV RT were identified that were important for fidelity which included position V223 of the YVDD box, several residues of the dNTP-binding site, as well as residues residing in the RNase H domain of MLV RT. A number of these MLV RT mutants resulted in statistically significant decreases in fidelity (1.2 to 2.8-fold) whereas two mutants showed a statistically significant increase in fidelity (0.8-fold) relative to wild-type MLV RT. Furthermore, these amino acid residues were observed to play critical roles in catalysis and viral replication, which was exhibited by the reductions in both viral titers as well as RT activities of these mutants. In addition to identifying structural determinants important for fidelity, we also showed that the V223 position of MLV RT may not be the only structural determinant important for resistance to the antiviral nucleoside analog, 2\u27, 3\u27-dideoxy-3 \u27-thiacytidine (3TC). This is in contrast to results observed with human immunodeficiency virus type 1 (HIV-1) RT. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis as well as dug-resistance and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT

Intact HIV proviruses persist in children seven to nine years after initiation of antiretroviral therapy in the first year of life

In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact

Nevirapine- Versus Lopinavir/Ritonavir-Based Initial Therapy for HIV-1 Infection among Women in Africa: A Randomized Trial

In a randomized control trial, Shahin Lockman and colleagues compare nevirapine-based therapy with lopinavir/ritonavir-based therapy for HIV-infected women without previous exposure to antiretroviral treatment

Sensitive Phenotypic Detection of Minor Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Variants

Detection of drug-resistant variants is important for the clinical management of human immunodeficiency virus type 1 (HIV-1) infection and for studies on the evolution of drug resistance. Here we show that hybrid elements composed of the Saccharomyces cerevisiae retrotransposon Ty1 and the reverse transcriptase (RT) of HIV-1 are useful tools for detecting, monitoring, and isolating drug-resistant reverse transcriptases. This sensitive phenotypic assay is able to detect nonnucleoside reverse transcriptase inhibitor-resistant RT domains derived from mixtures of infectious molecular clones of HIV-1 in plasma and from clinical samples when the variants comprise as little as 0.3 to 1% of the virus population. Our assay can characterize the activities and drug susceptibilities of both known and novel reverse transcriptase variants and should prove useful in studies of the evolution and clinical significance of minor drug-resistant viral variants

Multiple, Linked Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Treatment-Experienced Patients Are Missed by Standard Genotype Analysis

To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis