P05. Subtype-Specific Dolutegravir Inhibition in Treatment naïve HIV-1 Integrase
Kamalendra Singh1*, Ujjwal Neogi2*, Leonard C Rogers1*, Shambhu G. Aralaguppe2, Seongmi Kim1, Stefan Sarafianos1, Anders Sönnerborg2
Affiliates: 1University of Missouri, US 2Karolinska Institutet, Stockholm Sweden *Equal contributions
Dolutegravir (DTG) is a second generation HIV-1 integrase (IN) strand transfer inhibitor (INSTI). DTG (together with tenofovir and emtricitabine) is currently included in first-line antiviral treatment regimens in resource-rich settings. DTG has been shown to be a promising alternative to non-nucleoside reverse transcriptase inhibitors (NNRTIs) since its introduction in resource-limited sectors of the United States (downtown Washington, DC, the South Bronx, and downtown Los Angeles) in patients infected with HIV-1 subtype B (HIV-1B). However, its effect among patients from low- and middle-income countries, which mainly have non-B HIV-1 is poorly studied. In order to fill this gap, we cloned IN genes from patient isolates from HIV-1B, HIV-1C and CRF01_AE and determined inhibitory profiles of DTG in biochemical assays.
Patient-derived IN genes were cloned into the pRSFDuet plasmid to prepare recombinant IN proteins from different subtypes. All proteins were purified to near homogeneity using Ni-affinity chromatography. Gel-based assays were used for monitoring strand-transfer and 3’-end processing assays in the presence and in the absence of DTG. Synthetic template-primers (21-mer duplex and 19/21-mer partial duplex) were used for determining DTG binding affinity. Molecular models of IN-DNA complexes were generated for all subtypes. These models were then used for DTG docking to determine the binding energies of DTG in IN-DNA complex. Therapy naïve patient derived gag-pol (n=15), representing HIV-1C, B, and 01_AE, was cloned into pNL4.3Δgag_pol to prepare recombinant replication competent viruses. TZM_bl cells were infected with viruses at an M.O.I of 0.005 in the presence of serial DTG dilutions and then assayed for dose-response curve after 48 hours post infection. Fold-change in EC50 was calculated against NL43 virus.
Our biochemical characterization showed that all IN enzymes from different HIV-1 subtypes have comparable DNA binding, 3’-end processing, and strand-transfer activities. The DNA binding affinities for HIV-1B, HIV-1C and 01_AE were 8.2, 10.1 and 7.8 nM, respectively. The Kd.DTG as determined in strand-transfer assays was 8.3, 3.7 and 5.9 nM for HIV-1B, HIV-1C and 01_AE, respectively. The molecular docking showed that integrases from all subtypes favorably bind DTG near the active site. In addition, the docking score for the HIV-1C IN-DNA complex was slightly better than HIV-1B IN-DNA and 01_AE IN-DNA complexes. All the therapy naïve chimeric viruses gave an EC50 fold change ≤1 compared to NL4-3 with EC50 values < 3nM.
Our biochemical, molecular modeling and virological data suggest that DTG binds to HIV-1C IN with slightly better affinity that to HIV-1B and 01_AE integrases. These results suggest that DTG can be a strong choice-drug for non-B HIV-1 subtype.