Spontaneous Mutations in HIV-1 Gag, Protease, RT p66 in the First Replication Cycle and How They Appear: Insights from an In Vitro Assay on Mutation Rates and Types

Abstract

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.

Mutation Rates, Mutation Frequencies, and Proofreading-Repair Activities in RNA Virus Genetics

“…Nuances of mutation rate calculations apply both to viruses and cells, including bacteria. Problems comprise (i) the use of different units (mutations per nucleotide versus mutations per genome), (ii) not considering the mode of viral genome replication in terms of template utilization (multiple copies produced from the same template versus each progeny molecule becoming a new template; probably an intermediate situation occurs in most viruses), and (iii) bias caused by selection intervening between the biochemical event that defines the mutation rate and the actual mutant quantification to give the mutation frequency [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ].…”

To Plate or to Simply Unfreeze, That Is the Question for Optimal Plasmid Extraction

“…In-house competent E. coli DH5a cells were chemically transformed 16 with two ampicillin-resistant plasmids, pTT5 17 and pET21d (Thermo Fisher Scientific, Singapore). These plasmids hold the following gene inserts: HIV-1 protease (Hprot, accession number: AY622223.1), HIV-1 Gag (Hgag 18 ), human CD89 (Fc fragment of IgA receptor FCAR, accession number: NM_002000.4, 19,20 ), and human CD32 (Fc fragment of IgG receptor IIa FCG2A, accession number: NM_001136219.3 21 ).…”

Peering Into Avian Influenza A(H5N8) for a Framework towards Pandemic Preparedness

“…Certainly, the avian H5N8 virus were shown to be capable of genetic reassortment with human influenza viruses (H3N2, H1N1 and pandemic H1N1) for viral titers and replication kinetics analysis in vitro using various cell lines [62] or in vivo using mice and ferret models [60,61]. Through simulating the genetic reassortment of avian H5N8 with other human influenza viruses and their effects on viral replication and transmission, placing emphasis on the PB2 subunit and its possible reassortants, it could be possible to generate a predictive mutation platform as was performed for HIV [63].…”

“…Early methods to do these have been complicated by different escape mutations from polyclonal human immunity [70] confounding the analysis. Thereby, the use of an innate selection-free system [63] may provide a clearer insight on the influence of natural genetic code biases [71] to get a more accurate mutation rate as for HIV [72] geared towards Influenza reassortment.…”