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Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

ABSTRACT: HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), ¹⁹ F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66_L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66_L289K and p51 were predicted to form a heterodimer while p66 and p51_L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

SUBMITTER: Xi Z

PROVIDER: S-EPMC8996465 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

Xi Zhaoyong Z Ilina Tatiana V TV Guerrero Michel M Fan Lixin L Sluis-Cremer Nicolas N Wang Yun-Xing YX Ishima Rieko R

Protein science : a publication of the Protein Society 20220501 5

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), <sup>19</sup> F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66<sub>L289K</sub> . NMR and SAXS experiment ...[more]

PMID: 35481647

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Project description:BackgroundThe catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.Methods and findingsThe prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 x 10(-12)). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43-4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance.ConclusionsThis study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patient's response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure.

Project description:Once HIV-1 enters a cell, the viral core is uncoated by a poorly understood mechanism and the HIV-1 genomic RNA is reverse transcribed into DNA. Host cell factors are essential for these processes, although very few reverse transcription complex binding host cell factors have been convincingly shown to affect uncoating or reverse transcription. We previously reported that cellular eukaryotic translation elongation factor 1A (eEF1A) interacts tightly and directly with HIV-1 reverse transcriptase (RT) for more efficient reverse transcription. Here we report that the surface-exposed acidic residues in the HIV-1 RT thumb domain alpha-J helix and flanking regions are important for interaction with eEF1A. Mutation of surface-exposed acidic thumb domain residues D250, E297, E298, and E300 to arginine resulted in various levels of impairment of the interaction between RT and eEF1A. This indicates that this negatively charged region in the RT thumb domain is important for interaction with the positively charged eEF1A protein. The impairment of RT and eEF1A interaction by the RT mutations correlated with the efficiency of reverse transcription, uncoating, and infectivity. The best example of this is the strictly conserved E300 residue, where mutation significantly impaired the interaction of RT with eEF1A and virus replication in CD4+ T cells without affecting in vitro RT catalytic activity, RT heterodimerization, or RNase H activity. This study demonstrated that the interaction between surface-exposed acidic residues of the RT thumb domain and eEF1A is important for HIV-1 uncoating, reverse transcription, and replication.IMPORTANCE HIV-1, like all viruses, requires host cell proteins for its replication. Understanding the mechanisms behind virus-host interactions can lay the foundation for future novel therapeutic developments. Our lab has identified eEF1A as a key HIV-1 RT binding host protein that is important for the reverse transcription of HIV-1 genomic RNA into DNA. Here we identify the first surface-exposed RT residues that underpin interactions with eEF1A. Mutation of one strictly conserved RT residue (E300R) delayed reverse transcription and viral core uncoating and strongly inhibited HIV-1 replication in CD4+ T cells. This study advances the structural and mechanistic detail of the key RT-eEF1A interaction in HIV-1 infection and indicates its importance in uncoating for the first time. This provides a further basis for the development of an RT-eEF1A interaction-inhibiting anti-HIV-1 drug and suggests that the surface-exposed acidic patch of the RT thumb domain may be an attractive drug target.

Dataset Information

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

Publications

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

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