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

Extended Analysis of HIV Infection in Cisgender Men and Transgender Women Who Have Sex with Men Receiving Injectable Cabotegravir for HIV Prevention: HPTN 083

HPTN 083 demonstrated that injectable cabotegravir (CAB) was superior to oral tenofovir disoproxil fumarate-emtricitabine (TDF-FTC) for HIV prevention in cisgender men and transgender women who have sex with men. We previously analyzed 58 infections in the blinded phase of HPTN 083 (16 in the CAB arm and 42 in the TDF-FTC arm). This report describes 52 additional infections that occurred up to 1 year after study unblinding (18 in the CAB arm and 34 in the TDF-FTC arm). Retrospective testing included HIV testing, viral load testing, quantification of study drug concentrations, and drug resistance testing. The new CAB arm infections included 7 with CAB administration within 6 months of the first HIV-positive visit (2 with on-time injections, 3 with ≥1 delayed injection, and 2 who restarted CAB) and 11 with no recent CAB administration. Three cases had integrase strand transfer inhibitor (INSTI) resistance (2 with on-time injections and 1 who restarted CAB). Among 34 CAB infections analyzed to date, diagnosis delays and INSTI resistance were significantly more common in infections with CAB administration within 6 months of the first HIV-positive visit. This report further characterizes HIV infections in persons receiving CAB preexposure prophylaxis and helps define the impact of CAB on the detection of infection and the emergence of INSTI resistance

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