**4. Discussion**

We analysed 12 HIV-1C *nef* 3-PPT sequences (eight had paired integrase sequences without INSTI drug resistance mutations and four had paired IN sequences with INSTI drug resistance mutations) from patients with VF while on DTG/RAL-based cART to search for changes in 3-PPT sequence that could be linked to DTG VF as previously reported, and we did not find any. Amongst the eight patients who were failing an INSTI-based regimen but who did not have INSTI resistance mutations in the integrase region, all had a 100% conservation in their 3-PPT sequences (they had no mutations at the nucleotide or amino acid level). In addition, we analysed 1263 3-PPT sequences from patients who were cART naïve to investigate HIV-1C 3-PPT variability. We further analysed 4696 3-PPT sequences from patients on cART (but not on a DTG-based regimen) stratified according to virological suppression and found no 3-PPT region variability.

Malet et al. found some significant variability at position 9071 (25% and 10% in HIV-1 subtype B and CRF01, respectively) [17] and position 9075 (10% variability in HIV-1 subtype D). In our analysis, position 9071 revealed a variability of 0.29% (*n* = 6009) and 0% (*n* = 12) of sequences from patients not exposed to DTG cART and those failing DTG/RAL cART, respectively. Position 9075 was also conserved with a variability of 0.4% (*n* = 6009) and 0% (*n* = 12) amongs<sup>t</sup> the two groups. Perhaps this difference in variability could be explained by the different HIV-1 subtypes—all our sequences were HIV-1C.

We observed a high conservation amongs<sup>t</sup> the six nucleotides of the G-tract residues of 3-PPT (mean of 99.47%) similar to what others have found (99.95%) [8,17–19].

We did not explore the entire HIV-1C genome (5- long terminal repeat (LTR), gag, protease, reverse transcriptase, vif, vpr, vpu, envelope, and 3- LTR) for other mutations that could be linked to INSTI resistance. We did not measure plasma DTG or RAL levels to check whether issues of nonadherence could be contributing to VF.

In conclusion, we conducted one of the largest analyses of the HIV-1C 3-PPT region, showing grea<sup>t</sup> conservation of the region at the nucleotide and amino acid level. Although we did not detect any association of 3-PPT mutations with VF on INSTI-based cART, our data were limited on the number of 3-PPT sequences generated from patients failing an INSTI-containing regimen without INSTI mutations. However, our data add to a growing list of studies that have found no association of 3-PPT mutations with INSTI resistance [17,18]. Future studies should also investigate the role of other HIV-1 genes outside of Pol as this might enhance our understanding of mechanisms associated with INSTI resistance.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pathogens10081027/s1, Table S1: HIV-1C nef 3-PPT variability amongs<sup>t</sup> 12 therapy experienced individuals experiencing VF while on DTG/RAL ART. We included the two proximal and last distal nct positions to the 3-PPT to complete aminoacid picture of the region; 3polypurine tract, 3-PPT; nct, nucleotide; VL, viral load; seqs., sequences; VF, virological failure; DTG, dolutegravir; RAL, raltegravir; cART, combination antiretroviral therapy, Adapted from Figure 2a of Malet I et al. [7]; Figure S1: HIV-1C nef 3-PPT variability amongs<sup>t</sup> sequences from individuals on cART and not on ART, *n* = 5959. cART, combination antiretroviral therapy; 3-PPT, 3polypurine tract; Figure S2: HIV-1C nef 3-PPT variability amongs<sup>t</sup> cART naive individuals, *n* = 1263; Figure S3: HIV-1C nef 3-PPT variability amongs<sup>t</sup> individuals on ART with VL ≤ 400 copies/mL, *n* = 4483; Figure S4: HIV-1C nef 3-PPT variability amongs<sup>t</sup> individuals on ART with VL > 400 copies/mL, *n* = 213; Figure S5: HIV-1C nef 3-PPT variability amongs<sup>t</sup> sequences from individuals on ART and not on ART, *n* = 2992 (hypermutations removed); Figure S6: HIV-1C nef 3-PPT variability amongs<sup>t</sup> ART naive individuals, *n* = 762 (hypermutations removed); Figure S7: HIV-1C nef 3-PPT variability amongs<sup>t</sup> individuals on ART with VL ≤ 400 copies/mL, *n* = 2074 (hypermutations removed); Figure S8: HIV-1C nef 3-PPT variability amongs<sup>t</sup> individuals on ART with VL > 400 copies/mL, *n* = 119 (hypermutations removed). **Author Contributions:** Writing–original draft preparation, K.K.S.; conceptualization, S.G., K.K.S.; investigation, K.K.S.; writing—reviewing and editing, K.K.S., S.G., I.K., S.L., S.M., O.M., V.N., D.M., W.T.C., data curation, K.K.S., S.G.; formal analysis, K.K.S., O.M., S.M., S.G.; project administration, S.G., S.L.; supervision, S.G., I.K; funding acquisition, S.G., K.K.S., S.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This publication was made possible with help from the Harvard University Center for AIDS Research (CFAR), an NIH-funded program (P30 AI060354), which is supported by the following NIH cofunding and participating institutes and centres: National Institute of Allergy and Infectious Diseases; National Cancer Institute; Eunice Kennedy Shriver National Institute of Child Health and Human Development; National Heart, Lung, and Blood Institute; National Institute on Drug Abuse; National Institute of Mental Health; National Institute on Aging; National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of General Medical Sciences; National Institute on Minority Health and Health Disparities; National Institute of Dental and Craniofacial Research; Office of AIDS Research; and Fogarty International Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was supported through the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS Africa Initiative (grant # DEL-15-006). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences' (AAS) Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa's Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (grant # 107752/Z/15/Z) and the UK government. The views expressed in this publication are those of the author(s) and not necessarily those of AAS, NEPAD Agency, Wellcome Trust, or the UK government. 'Research reported in this publication was supported by the Fogarty International Center and National Institute of Mental Health of the National Institutes of Health under Award Number D43 TW010543. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.' S.L. was supported by the National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases K24 mentoring grant—NIH K24 AI131928. All funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Botswana Combination Prevention Project Impact Evaluation was supported by the President's Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC) under the terms of cooperative agreements U01 GH000447 and U2G GH001911. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official positions of the funding agencies.

**Institutional Review Board Statement:** Both study protocols were approved by the health research and development division of the Botswana Ministry of Health and Wellness (Botswana's IRB of authority). For the BOSELE study participants, a waiver of informed consent was obtained, and for the BCPP study, all the participants provided informed consent. The BCPP study was approved by the IRB at the U.S. Centers for Disease Control and Prevention and is registered at ClinicalTrials.gov (NCT01965470). All studies were conducted according to the principles stated in the Declaration of Helsinki.

**Informed Consent Statement:** For the BOSELE study participants, a waiver of informed consent was obtained, and for the BCPP study, all the participants provided informed consent.

**Data Availability Statement:** Sequences available at national centre for biotechnology information (NCBI) GenBank, accession numbers MW690052-MW690089, MG989443.1, MG989444.

**Acknowledgments:** We thank our patients and the staff of all infectious disease care clinics (PMH IDCC) that we visited throughout Botswana. We thank the Botswana Harvard HIV Reference Laboratory staff, Botswana Harvard Partnership, and Botswana Ministry of Health and Wellness for their collaboration. We thank the study participants from both study cohorts without whom this study would not have been possible.

**Conflicts of Interest:** All authors have no conflict of interest to declare.
