Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition

摘要

BackgroundLINE-1s (L1, Long Interspersed Element-1) are the most abundant autonomousnon-LTR retrotransposons in the human genome and replicate by reversetranscription of an RNA intermediate. Full-length L1 encodes two open readingframes (ORF1, ORF2) and ORF2 has reverse transcriptase activity.ResultsHere we expressed human L1 RT in E.coli and the purified protein displayed thesame RT activity as that of ORF2p expressed in insect cells. We tested the effectof different reverse transcriptase inhibitors on L1 RT and found that all four testednucleoside inhibitors efficiently inhibited L1 RT activity competitively. The Ki valuesof NRTIs were calculated (AZTTP, 16.4±4.21 nM; d4TTP, 0.73±0.22 nM; ddCTP,0.72±0.16 nM; 3TCTP, 12.9±2.07 nM). L1 RT was less sensitive to non-nucleoside reverse transcriptase inhibitors, among these nevirapine had no effect,even at concentrations up to 500 μM. We also examined the effect of RT inhibitorson L1 retrotransposition efficiency in vivo using a cell-based retrotranspositionassay. Similarly, all analog inhibitors decreased L1 retrotransposition frequencywith different potencies whereas nevirapine had little or no effect on L1retrotransposition. For comparison, we also tested the same inhibitors to highlypurified RT of an LTR-retrotransposon (Ty1) and found it was less sensitive toNRTIs than L1 RT and has the same inhibition profile as L1 RT to NNRTIs.ConclusionsThese data indicate that bacterially expressed L1 RT is an active reversetranscriptase sensitive to nucleoside RT inhibitors but not to non-nucleosideinhibitors.