**1. Introduction**

Dolutegravir (DTG) is a widely used second-generation integrase strand transfer inhibitor (INSTI) with a high genetic barrier to resistance [1–5]. It prevents the HIV integrase enzyme from incorporating viral DNA into the host cell genome [1]. However, resistance mutations within the integrase enzyme can cause reduced susceptibility to

**Citation:** Seatla, K.K.; Maruapula, D.; Choga, W.T.; Morerinyane, O.; Lockman, S.; Novitsky, V.; Kasvosve, I.; Moyo, S.; Gaseitsiwe, S. Limited HIV-1 Subtype C *nef* 3-PPT Variation in Combination Antiretroviral Therapy Naïve and Experienced People Living with HIV in Botswana. *Pathogens* **2021**, *10*, 1027. https:// doi.org/10.3390/pathogens10081027

Academic Editor: Hezhao Ji

Received: 28 May 2021 Accepted: 11 August 2021 Published: 13 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

DTG [1,6]. Most major DTG resistance mutations amongs<sup>t</sup> therapy-experienced individuals are usually located within the HIV-1 catalytic core domain of the integrase region [1,7]. However, a handful of studies have suggested associations between DTG failure and mutations outside the integrase region in the 3--polypurine tract (3-PPT) of the HIV-1 *nef* gene [8,9].

The *nef* gene of HIV-1 is a small accessory protein of about 206 amino acids that contributes to HIV disease progression mainly by downregulating the expression of CD4 and major histocompatibility complex class I molecules, amongs<sup>t</sup> other functions [10–13]. It has a highly conserved purine-dominated 15-nucleotide sequence (3-PPT) that is involved in the reverse transcription process, resulting in the production of double-stranded viral DNA, enabling the integration into the host cell genome [14–16].

Malet et al. unexpectedly cultured a virus that had mutations in the 3-PPT motif conferring resistance to DTG [8]. Similar mutations were identified in the guanine-tract (G-tract) motif at the 3- end of 3-PPT of one patient with virologic failure (VF) while on a DTG monotherapy trial [9].

A subsequent study by Malet et al. with a larger number of individuals failing INSTI-based regimens revealed a highly conserved 3-PPT with no associations with DTG failure discernible [17]. Further in vitro work went on to confirm this [18]. A recent study from Cameroon amongs<sup>t</sup> INSTI-naïve individuals also showed a highly conserved 3-PPT region [19]. Furthermore, analysis of publicly available HIV-1 *nef* gene sequences from the Los Alamos HIV-1 database reveals a highly conserved 3-PPT region across various subtypes [17].

Given these inconsistent study results and the fact that there are limited 'real-world' data on the contribution of 3-PPT to failure of DTG-based regimens, we conducted this study to address these knowledge gaps. In our study, we sought to determine the diversity of 3-PPT of the HIV-1 subtype C (HIV-1C) *nef* gene amongs<sup>t</sup> cART-naïve and cART-treated individuals with and without VF. We also assessed whether HIV-1C mutations in 3-PPT contribute to VF amongs<sup>t</sup> individuals failing INSTI-based cART regimens regardless of the presence of mutations in the integrase region.

#### **2. Materials and Methods**

#### *2.1. Selection of Study Population and HIV-1 Genotyping*

Participant samples were obtained from two studies conducted in Botswana. The first study consisted of sequences generated from residual plasma specimens obtained from therapy-experienced individuals experiencing VF while on DTG- or raltegravir (RAL)- based cART described elsewhere (BOSELE study; Figure 1) [7]. VF was defined as two or more consecutive plasma HIV-1 RNA levels (viral loads (VL)) > 400 copies/mL as per standard of care guidelines in Botswana. The HIV-1 integrase region was amplified using nested reverse transcription-polymerase chain reactions (RT-PCRs) where necessary and sequenced using a BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) on a 3130xl Genetic Analyser (Life Technologies Corporation, Applied Biosystems, Carlsbad, CA, USA) as previously described [7,20]. Sequencing of the *nef* gene was attempted from the same HIV-1 extracts that integrase sequences were successfully generated from. Briefly, products of about 620 base pairs were amplified using nested RT-PCRs where necessary using the following primers numbered relative to HIV-1 reference strain (HxB2) nucleotide positions (shown in brackets): NEF8683F\_pan TAGCAGTAGCT-GRGKGRACAGATAG (8683–8707), NEF9536R\_pan TACAGGCAAAAAGCAGCTGCT-TATATGYAG (9507–9536), NEF8746\_SgrI\_AscI\_F AGAGCACCGGCGCGCCTCCACAT-ACCTASAAGAATMAGACARG (8736–8772), and NEF9474\_SacII\_ClaI\_R GCCTCCGCG-GATCGATCAGGCCACRCCTCCCTGGAAASKCCC (9449–9491) [21]. Amplicons were bidirectionally Sanger-sequenced as described above. The Sequencher software, version 5.0 (Gene Codes Corporation, Ann Arbor, MI, USA), was used to manually edit our electropherograms and form contigs with further downstream analysis carried using the BioEdit software.

**Figure 1.** Study schema depicting the selection of study sequences. (**A**), selection and analysis of 3-PPT *nef* gene sequences according to participant cART status and HIV-1 RNA levels; (**B**), selection and analysis of 3-PPT of *nef* gene amongs<sup>t</sup> individuals with VF while on DTG/RAL-based cART. Seqs, sequences; PID, participant identification number; VL, viral load; VF, virologic failure; RAL, raltegravir; DTG, dolutegravir; cART, combination antiretroviral therapy; 3-PPT, 3--polypurine tract; RT, reverse transcriptase HIV-1 gene; PR, protease HIV-1 gene; DRMs, drug resistance mutations; HIV-1C, HIV-1 subtype C.

> The second group consisted of full-genome HIV-1C sequences obtained from participants enrolled in a large community-randomised HIV-1 prevention trial described elsewhere (BCPP study) [22,23]. The sequences were aligned to the HIV reference strain (HxB2) at the nucleotide level using virulign [24] and block-trimmed to the *nef* gene of HxB2 in the BioEdit, version 7.2.0, software [25]. We included two nucleotides before and one nucleotide after the 3-PPT *nef* gene sequence to have a complete amino acid coding for the 3-PPT tails. The sequences were assessed for hypermutations using the Hypermut tool at the Los Alamos National Laboratory HIV Database website (http://www.hiv.lanl.gov/ accessed on 8 March 2021). All sequences were exported to Microsoft® Excel® for Microsoft 365 MSO (16.0.13901.20148) 32-bit for further downstream analysis, and graphs were created. Additional statistical computations were performed using Stata version 14 (Stata Corporation, College Station, TX, USA) and R version 4.0.3; Pearson's chi-square test was used to compare the proportion of 3-PPT of *nef* gene mutations per position by ART status and VL suppression. *p*-Values < 0.05 were considered statistically significant.

> The Abbott RealTime HIV-1 assay, Cobas TaqMan/Cobas AmpliPrep HIV test (Roche Molecular Systems, Branchburg, NJ, USA), or Aptima HIV-1 Quant assay on Panther Systems (Hologic Inc., San Diego, CA, USA) was used to quantify HIV-1 RNA levels.

## *2.2. Ethical 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.
