HIV-1 Protease Evolvability is Affected by Synonymous Nucleotide Recoding

Maria Nevot; Ana Jordan-Paiz; Glòria Martrus; Cristina Andrés; Damir García-Cehic; Josep Gregori; Sandra Franco; Josep Quer; Miguel Angel Martinez

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/315366 doi: bioRxiv preprint 5 mechanisms within the innate immune response (11, 29, 30) , as well as to resolve the 87 importance of codon usage in the temporal regulation of viral gene expression (31). In (Table 1) . WTp32 and MAXp32, respectively, showed 5-129 fold and 13-fold increases in IC 50 for ATV, and 6-fold and 10-fold increases in IC 50 for 130 DRV (Table 1) . Although MAXp32 displayed a higher resistance to ATV and DRV 131 than WTp32 (Table1), these differences were not significant (P = 0.4816 and P = The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/315366 doi: bioRxiv preprint compared the frequencies of resistant mutations. For each of the two studied viruses and 137 the two tested drugs, we sequenced between 1.9 × 10 7 and 4.1 × 10 7 individual protease 138 nucleotides (Table 2) . Sequence clonal analysis revealed no resistance-associated 139 substitutions in viruses propagated without drugs (Table 3) . On the other hand, both WT 140 and MAX viruses propagated in the presence of PIs developed previously described 141 resistance mutations to ATV and DRV (Table 3) . Moreover, the resistance variant 142 repertoire differed between the MAX and WT viruses (Table 3) . Specifically, the G16E (Table 2) , was also included in this analysis. We found that the five generated mutants 165 displayed comparable IC 50 values to ATV in both backgrounds, WT and MAX (Table 166 3). We also determined the replication capacity of these two ten mutant viruses in MT-4 167 cells with and without ATV (Fig. 2) . Similar to the IC50 results, the five tested variants The amino acid mutant repertoire also differed between the WT and MAX 176 viruses when they were propagated without drugs (Table 2) . Only one variant, D30N, 177 was detected in both virus populations. We do not know whether the observed variants 178 are adaptive or neutral mutations. Regardless, completely different mutant spectra were 179 detected in these two viruses. 180 We performed a maximum likelihood phylogenetic reconstruction of all WT and 181 MAX unique amino acid variants that were recovered after 32 MT-4 cell passages in the 182 presence of ATV or DRV. Remarkably, the results showed that the two viruses, which 183 shared an identical starting amino acid sequence, followed different evolutionary 184 trajectories (Fig. 3) . Upon visual inspection of these phylogenetic trees, it was also 185 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/315366 doi: bioRxiv preprint 9 apparent that the MAX protease generated higher amino acid variant diversity (see 186 below). 187 We next compared the overall population nucleotide diversity of the WT and 188 MAX proteases after 32 passages in the absence or presence of ATV or DRV (Table 4) . 189 Overall, nucleotide sequence diversity was significantly higher in MAX populations such that viral proteins are translated from mRNAs using host cellular machinery. 221 We found that the WT and MAX protease viruses displayed different patterns of The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/315366 doi: bioRxiv preprint In some instances, the different mutant repertoire within the MAX protease 235 backgroun