An Efficient Identification Of Resistant Mutants To Integrase Inhibitors Using A Saturated Random Mutant Library Of HIV-1 Intenrase

An efficient screening method to select mutants resistant to rategravir(an integrase strand transfer inhibitor,or INSTI)and BI-D and KF116(two allosteric integrase inhibitors or ALLINIs)using a random mutant library of HIV-1 integrase was described here.Methods:1.To construct a library of integrase variants with adequate coverage and diversity to permit efficient selection of resistant mutants against integrase inhibitors,we applied a mutagenesis method called transposon-directed base-exchange mutagenesis(TDEM).In TDEM,transposons were used to generate gaps in random locations of the target gene(integrase)and the usage of predesigned mutation insertion cassettes can replace three bases of the gene near the gap generated by the transposon with three preset bases within the insertion cassettes.2.To screen mutants conferring resistance to the integrase inhibitors,we introduced the mutated integrase library into the plasmid containing the HIV-1 genome,pNL4.3.A pool of HIV-1 strains encoding the mutant integrase library was generated by transfecting 293T cells with the mutant pNL4.3,and then the HIV-1 mutant strains were used to infect the CEM cells in the presence of one of the three drugs to obtain selected pools of viral strains resistant to the drug.Sanger sequencing and Illumina sequencing were both utilized to identify the mutants in the selected pools.When analyzing the Illumina sequencing results,we used a factor called FC,which stood for the fold change between the frequency of one mutation in the selected pool of resistant mutants and the frequency of this mutation in the input library.Results:1.A saturated random library of HIV-1 integrase(288 amino acids)was constructed with the goal of replacing every amino acid residue with all other possible substitutions.The diversity of the resultant library covers 65.8%of the total of 5,472(288 x 19)possible variants.2.IIllumina sequencing results identified mutations E35K,C56G,Q221K among others in the selected library in the presence of 40 nM raltegravir.FC of almost every reported major primary resistant mutant was larger than 1.The Illumina sequencing of 400 nM BI-D selection identified mutations C56G,G82E and G272E among others.The Sanger sequencing identified mutations in residues T124 and T125 in the selected pool of viral strains under the pressure of 50 nM KF116 which is consistant with other researches.Conclusions:A random mutant library with substitutions distributed evenly in integrase was constructed by applying TDEM.Using HIV-1 viral strains containing this library we identified residues that made contributions to drug resistance.Almost every reported major primary resistant mutants to raltegravir were identified in the Illumina sequencing data of the screening assay under the pressure of raltegravir.This fact supported the method described here.Combining the results of Sanger sequencing and Illumina sequencing we identified some intriguing residues that have never been reported previously.We chose five such mutations to verify directly their resistance to the drugs.Unfortunately,due to the limit of time I have not finished the verification on my own.I have constructed the five mutations by site-directed mutagenesis PCR,and my colleagues will complete the analyses after my graduation.