Background: LINE-1s (L1, Long Interspersed Element-1) are the most abundant autonomous non-LTR retrotransposons in the human genome and replicate by reverse transcription of an RNA intermediate. Full-length L1 encodes two open reading frames (ORF1, ORF2) and ORF2 has reverse transcriptase activity. Results: Here we expressed human L1 RT in E. coli and the purified protein displayed the same RT activity as that of ORF2p expressed in insect cells. We tested the effect of different reverse transcriptase inhibitors on L1 RT and found that all four tested nucleoside inhibitors efficiently inhibited L1 RT activity competitively. The Kivalues of 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 M. We also examined the effect of RT inhibitors on L1 retrotransposition efficiency in vivo using a cell-based retrotransposition assay. Similarly, all analog inhibitors decreased L1 retrotransposition frequency with different potencies whereas nevirapine had little or no effect on L1 retrotransposition. For comparison, we also tested the same inhibitors to highly purified RT of an LTR-retrotransposon (Ty1) and found it was less sensitive to NRTIs than L1 RT and has the same inhibition profile as L1 RT to NNRTIs. Conclusions: These data indicate that bacterially expressed L1 RT is an active reverse transcriptase sensitive to nucleoside RT inhibitors but not to non-nucleoside inhibitors.
ASJC Scopus subject areas
- Molecular Biology