Genome-wide Screening of Cellular Suppressors against HIV-1 Protease in Fission Yeast (Schizosaccharomyces pombe)

Abstract

The HIV-1 Protease (PR) is a necessary enzyme for the replication of HIV. PR cleaves the long chain HIV proteins into smaller individual mature proteins. A PR inhibitor (PI) suppresses this cleavage activity. All current PIs were developed based on the structural design against the PR active site. Therefore, any PR mutation that results in conformational changes of the PR enzymatic site will lead to drug resistance to all current PIs. Due to the increasing emergence of drug resistant viruses, additional PIs that inhibit PR with different mechanisms such as allosteric inhibitors are needed. With this specific goal in mind, genome-wide searches for cellular proteins capable of suppressing the PR activity were conducted in the fission yeast Schizosaccharomyces pombe. The search for multicopy suppressors of PR-induced cell death in fission yeast was performed by transformation of a S. pombe expression mouse cDNA library into the RE294 strain, which carries a single integrated copy of HIV PR in the yeast chromosome. The use of RE294 to measure PR-induced cell death is based on the fact that, in fission yeast, PR-induced cell death can normally be detected by its inability to form colonies on an agar plate. Thus the PR-resistant yeast cells can be directly detected by colony formation on PR-inducing agar plate. A total of 5.18X106 transformants were screened which statistically covers the entire mouse genome. Sixty-two PR-resistant transformants were initially identified and 6 of them (4 unique clones) were confirmed as suppressors. The cDNA inserts carried on the plasmids were sequenced and one of them (W19) was further characterized. Over the 1.8kb gene insert of the plasmid W19, a 1.5kb fragment was found to be the minimum region to confer full suppression of PR-induced death of fission yeast.