Reply to Das and Berkhout, “How Polypurine Tract Changes in the HIV-1 RNA Genome Can Cause Resistance against the Integrase Inhibitor Dolutegravir”

Strand transfer inhibitors are potent molecules targeting HIV-1 integration, a critical step involved in retroviral replication. To date, three inhibitors, raltegravir, elvitegravir, and dolutegravir (DTG), are available to treat infected patients. Unfortunately, many resistance pathways have been described for raltegravir and elvitegravir, and all mutations leading to strand transfer resistance have been located in the integrase gene. In the case of dolutegravir, the last integrase inhibitor, only a few mutations described from patients or from in vitro selection are reported to confer resistance (1–3). Following the publication of the article describing a virus having selected mutations outside the integrase gene and conferring resistance to HIV-1 integrase inhibitors (4), Das and Berkhout propose a model for the replication of this 3=-polypurine-tract (3=-PPT)-mutated virus (18). Integration of the viral DNA would be possible due to the modification of the 5= long terminal repeat (LTR) end which makes the virus insensitive to dolutegravir action, allowing it to integrate into the host genome. This hypothesis is plausible since an optimal binding of dolutegravir on the integrase/DNA complex is required for inhibition of the compound and the binding requires the canonical LTR end (5). Since integration is a concerted mechanism occurring in the intasome composed of integrase and both LTR ends, the deficient binding of dolutegravir on the 5= LTR could allow the unmodified 3= LTR end to become insensitive to dolutegravir. Indeed, as reported in the literature, mutations at one end could have consequences for the other end (6). Consequently, integration of both 5= LTR and 3= LTR could occur despite lower efficiency, explaining the resistance of the 3=-PPT mutant to dolutegravir. Since 2-LTR circles are formed by ligation of the two LTR ends by the nonhomologous end joining (NHEJ) pathway, the LTR-LTR junction should reflect the integrity of the LTR ends (7). To test this hypothesis, sequencing of the U3-U5 junction of 2-LTR circles was carried out from MT-4 cells infected with the wild-type (WT) and 3=-PPTmutated viruses with or without DTG (without DTG at day 6 for WT and day 8 for mutated virus and with DTG at day 15 for 3=-PPT-mutated virus). Briefly, after extraction of DNA, a fragment of 307 nucleotides encompassing more than 100 nucleotides from either side of the junction was amplified using primers specific for U3 and U5 sequences, and pyrosequencing on a GS Junior sequencer (Roche 454 Life Sciences) was performed. A total of 1,675 and 1,280 reads per nucleotide position was amplified for WT and mutant viruses, respectively. Interestingly, we found that the LTR-LTR junction Published 29 May 2018 Citation Malet I, Subra F, Richetta C, Charpentier C, Collin G, Descamps D, Calvez V, Marcelin A-G, Delelis O. 2018. Reply to Das and Berkhout, “How polypurine tract changes in the HIV-1 RNA genome can cause resistance against the integrase inhibitor dolutegravir.” mBio 9:e00623-18. https://doi.org/10.1128/ mBio.00623-18. Editor Dimitrios Paraskevis, Medical School, University of Athens Copyright © 2018 Malet et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Olivier Delelis, delelis@lbpa.ens-cachan.fr. This is a response to a letter by Das and Berkhout (https://doi.org/10.1128/mBio.00006 -18). AUTHOR REPLY