A fission yeast cell-based system for multidrug resistant HIV-1 proteases

Abstract

BackgroundHIV-1 protease (PR) is an essential enzyme for viral production. Thus, PR inhibitors (PIs) are the most effective class of anti-HIV drugs. However, the main challenge to the successful use of PI drugs in patient treatment is the emergence of multidrug resistant PRs (mdrPRs). This study aimed to develop a fission yeast cell-based system for rapid testing of new PIs that combat mdrPRs.ResultsThree mdrPRs were isolated from HIV-infected patients that carried seven (M7PR), ten (M10PR) and eleven (M11PR) PR gene mutations, respectively. They were cloned and expressed in fission yeast under an inducible promoter to allow the measurement of PR-specific proteolysis and drug resistance. The results showed that all three mdrPRs maintained their abilities to proteolyze HIV viral substrates (MA↓CA and p6) and to confer drug resistance. Production of these proteins in the fission yeast caused cell growth inhibition, oxidative stress and altered mitochondrial morphologies that led to cell death. Five investigational PIs were used to test the utility of the established yeast system with an FDA-approved PI drug Darunavir (DRV) as control. All six compounds suppressed the wildtype PR (wtPR) and the M7PR-mediated activities. However, none of them were able to suppress the M10PR or the M11PR.ConclusionsThe three clinically isolated mdrPRs maintained their viral proteolytic activities and drug resistance in the fission yeast. Furthermore, those viral mdrPR activities were coupled with the induction of growth inhibition and cell death, which could be used to test the PI activities. Indeed, the five investigational PIs and DRV suppressed the wtPR in fission yeast as they did in mammalian cells. Significantly, two of the high level mdrPRs (M10PR and M11PR) were resistant to all of the existing PI drugs including DRV. This observation underscores the importance of continued searching for new PIs against mdrPRs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0131-5) contains supplementary material, which is available to authorized users.

Yeast for virus research

“…Expression of exogenous viral proteins also induce cell death and apoptosis in yeasts 64 174 . Viral proteins that induced yeast cell death and apoptosis include, but are not limited to, pro-apoptotic proteins such as adenovirus E4orf4 protein 175 , HIV-1 Vpr 67 176 , HIV-1 protease (PR) 177 178 179 180 , and ZIKV proteins 36 . Anti-apoptotic viral proteins include baculovirus p35 174 and DPV022 protein of the Deerpox virus 181 .…”

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors

“…Finally, we would like to point out that the methodology described in this study could be exploited as an assay system for inhibitors against any human proteins of interest. Provided that overproduction of those human proteins display some defective phenotypes including lethality, which is often the case (Benko et al, 2017;Matsuyama et al, 2006;Nkeze et al, 2015), small molecule libraries, natural products or extracts from any organism could be used to identify potential inhibitors (Figure 4). In fact, we have recently obtained small molecules derived from plants that specifically rescue lethality caused by HSET overproduction with very little cytotoxicity on their own (M.Y., T.T., and K.K., unpublished results).…”

“…those promoting lifespan extension (Sarnoski et al, 2017). Fission yeast has been used for several screenings, such as purification of specific compounds produced by Actinomycetes (Lewis et al, 2017) and a functional assay for Indinavir, an inhibitor against Human Immunodeficiency Virus Type-1 (Benko et al, 2016;Benko et al, 2017;Yang et al, 2012). Having seen toxicity derived from overproduced HSET in fission yeast cells (see Figure 1A), we exploited this lethal phenotype for the biological evaluation of known HSET inhibitors described earlier (AZ82, CW069 and SR31527) (Cosenza and Kramer, 2016;Xiao and Yang, 2016;Zhang et al, 2016).…”

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors

“…Provided that overproduction of those human proteins display some defective phenotypes including lethality, which is often the case (Benko et al, 2017;Matsuyama et al, 2006;Nkeze et al, 2015), small molecule libraries, natural products or extracts from any organism could be used to identify potential inhibitors (Fig. 5A).…”

“…those promoting lifespan extension (Sarnoski et al, 2017). Fission yeast has been used for several screenings, such as purification of specific compounds produced by Actinomycetes (Lewis et al, 2017) and a functional assay for Indinavir, an inhibitor against Human Immunodeficiency Virus Type-1 (Benko et al, 2016;Benko et al, 2017;Yang et al, 2012). Having seen toxicity derived from overproduced HSET in fission yeast cells (see Fig.…”