DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO METHOD TO FORCE AND SELECT FOR FUNCTIONAL HIV-1 INTERSUBTYPE pol RECOMBINANTS

Abstract

Introduction Inter subtype recombination in the pol gene of the HIV-1 affects the enzymatic structure and integrity which greatly impacts their function. These may be silent, deleterious or beneficial for the survival and adaptation of the virus. Aim This study was set out to induce intersubtype recombination within the pol and characterize phenotypes and recombination breakpoints. Materials and methods To create pRECnfl5`LTRΔenv/URA3, we amplified URA3 inserts using our designed primers, the inserts and 5`LTR shuttle vector (pREC5`LTRsbfI) were chemically transformed into the yeast, S. cerevisae for homologous recombination to occur. Cloning of URA3 into the env was confirmed by PCR and sequencing. Results URA3 integration into the 5`LTR shuttle vector was done and confirmed by a URA3 PCR and sequencing of the env gene of HIV-1 in the plasmid. Conclusion Data showed successful integration of the URA3 in the env part of the HIV-1 genome in the vector plasmid and hence pRECnfl5`LTRΔenv/URA3 created.