**3. Results**

A total of 6021 HIV-1C *nef* gene sequences were available for analysis from both studies, and their basic demographics are shown in Table 1.


**Table 1.** Basic demographics of 6009 HIV-1C diagnosed, cART-naïve, and cART-experienced individuals.

\* Participants from BCPP study, \*\* We generated 40 sequences representing 34 'unique' individuals from an ongoing study characterizing therapy-experienced participants experiencing VF while on DTG/RAL cART. † Analysable gender data available for 5998 individuals; rest of dataset contained 'ND' shown as 'Unknown'. ND, not documented; VF, virologic failure; DTG, dolutegravir; cART, combination antiretroviral therapy; BCPP, Botswana Combination Prevention Project. Column 3 of this table is the same dataset represented in Column 3 of Table 1 of Seatla et al. [7]. This table has been modified with permission from Seatla et al. [7].

> We included two nucleotides before (HxB2 nct position 9067 and 9068) and one nucleotide after 3-PPT (HxB2 nct position 9084) in our analysis of the 15-nucleotide 3-PPT region (5- AAAAGAAAAGGGGGG 3--HXB2 numbering 9069 to 9083) to complete the amino acid translation of the flanking regions of 3-PPT (Table 2, Figure 2).

> All nucleotide positions of our HIV-1C *nef* gene 3-PPT sequences were highly conserved regardless of whether cART-naïve (*n* = 1263), on ART with VL < 400 (*n* = 4483) copies/mL, or on cART with VL ≥ 400 copies/mL (*n* = 213) groups (Figure 2). In addition, there was no statistically significant difference between 'cART naïve' and 'on cART' groups (*p* = 0.81), 'on cART < 400- and 'on cART ≥ 400- groups (*p* = 0.88), 'ART naïve' and 'on cART < 400- groups (*p* = 0.72), 'cART naïve' and 'on cART ≥ 400- groups (*p* = 0.99), 'on cART and cART < 400- groups (*p* = 0.86), 'on cART and cART ≥ 400- groups (*p* = 0.92), and 'on ART' and individuals with VF while on DTG/RAL cART group (*p* = 0.81). Analysis of sequences derived from buffy coat (*n* = 6009) adjusted for hypermutations (*n* = 2992) also revealed highly conserved *nef* 3-PPT residues with no statistically significant difference determined. The terminal six guanine stretch of 3-PPT also showed a high degree of conservation with all nucleotide residues having a mean rate of 99.47% (Figure 2, Supplementary Materials).

**Figure 2.** HIV-1C *nef* gene 3-PPT variability amongs<sup>t</sup> 6009 sequences from individuals on cART and not on cART. 3-PPT, 3polypurine tract; cART, combination antiretroviral therapy; HIV-1C, HIV-1 subtype C.



each patient. ± Historical DRMs denoted with '±' retrieved from electronic databases and/or patients' medical charts. Mutations listed within brackets '()' are accessory mutations. ¥ denotes the same participants as listed in Table 2 of Seatla et al. [7]. ND, no mutations detected; cART, combination antiretroviral therapy; GRT, genotypic resistance testing; RT, reverse transcriptase; NRTI, nucleoside/nucleotide reverse transcriptase inhibitors; NNRTI, non-nucleoside reverse transcriptase inhibitors; PR, protease; PI, protease inhibitor; HxB2, HIV reference sequence\_K03455; INSTI, integrase strand transfer inhibitors; DRMs, drug resistance mutations. Light blue colour depicts the 3- PPT of the HIV-1 *nef* gene, yellow and orange colours depict the amino acid translation of the 3-nucleotide sequence. Adapted from Figure 2a of Malet et al. [8], Figure 1 of Malet I et al. [17], and with permission from Table 2 of Seatla et al. [7].
