The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) plays a major role in treatment resistance, from the development of vaccines to therapeutic drugs. In addressing the crux of the issue, various attempts to estimate the mutation rate of HIV-1 resulted in a large range of 10−5–10−3 errors/bp/cycle due to the use of different types of investigation methods. In this review, we discuss the different assay methods, their findings on the mutation rates of HIV-1 and how the locations of mutations can be further analyzed for their allosteric effects to allow for new inhibitor designs. Given that HIV is one of the fastest mutating viruses, it serves as a good model for the comprehensive study of viral mutations that can give rise to a more horizontal understanding towards overall viral drug resistance as well as emerging viral diseases.

While drug resistant mutations in HIV-1 are largely credited to its error prone HIV-1 RT, the time point in the infection cycle that these mutations can arise and if they appear spontaneously without selection pressures both remained enigmatic. Many HIV-1 RT mutational in vitro studies utilized reporter genes (LacZ) as a template to investigate these questions, thereby not accounting for the possible contribution of viral codon usage. To address this gap, we investigated HIV-1 RT mutation rates and biases on its own Gag, protease, and RT p66 genes in an in vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease, and RT p66 at 4.71 × 10−5, 6.03 × 10−5, and 7.09 × 10−5 mutations/bp, respectively. Gag and p66 genes showed a large number of ‘A to G’ mutations. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 × 10−4 mutations/bp) and that ‘A to G’ mutations occurred in regions reminiscent of ADAR neighbor sequence preferences. Mutational free energies of the ‘A to G’ mutations revealed an avoidance of destabilizing effects, with the natural p66 gene codon usage providing barriers to disruptive amino acid changes. Our study demonstrates the importance of studying mutation emergence in HIV genes in a RT-PCR in vitro selection pressure free system to understand how fast drug resistance can emerge, providing transferable applications to how new viral diseases and drug resistances can emerge.

Drug resistant mutants of HIV-1 is largely credited to its error prone HIV-1 RT, making it an important aspect for investigation. Previous HIV-1 RT studies rely on reporter genes (LacZ) as template, leaving the effects on HIV genes still uncharacterized. To address this, we studied HIV-1 RT mutation rates and bias on the Gag, protease, and RT p66 in an in-vitro selection pressure free assay and found clinical mutations with a general avoidance of key sites and detrimental mutations in the backdrop of mutations rates: 4.71 x 10^-5, 6.03 x 10^-5, and 7.09 x 10^-5 mutations/bp for Gag, protease and RT p66 respectively. Gag and p66 had significant A to G hypermutations we attributed to cellular adenosine deaminases and from comparisons with silently mutated p66 sequences, we observed an increase in mutation rates (1.88 x 10^-4 mutations/bp) and A to G mutations in regions reminiscent of ADAR recognition motifs. Analysis of change in mutational free energies of the A to G mutations revealed a general tendency to avoid destabilizing effects with the natural p66 gene codon usage providing barriers to effects of ADAR. Our study demonstrates the importance of studying mutation emergence in HIV genes to understand how fast drug resistance can emerge, and in this, provide potential transferable understanding to other viruses to how new viral disease and drug resistance can emerge.