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

Abstract

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 Ki values 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 μM. 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.

Figure 1

Expression of human L1 reverse transcriptase in E. coli. (A) Domain structure of human L1 ORF2 protein. The conserved domains are shown as shaded rectangles. L1 RT domain (Aa 238-1061) was cloned into pMal-c2X downstream of MBP gene in frame. (B) Human L1 RT domain was expressed in E. coli as a fusion protein (MBP-RT) and purified as described in materials and methods. The sample was resolved by 4-20% SDS/PAGE with Coomassie blue staining. Protein molecular mass markers are to the right (kDa). The arrow indicates the mobility of MBP-RT fusion protein.

Figure 2

Effect of RT inhibitors on the activities of HIV-1, L1 and Ty1 RTs. The reverse transcriptase activities of HIV-1 RT (blue diamond), L1 RT (red square) and Ty1 RT (green triangle) were measured as described in the materials and methods. The RT activity of control assay without inhibitors was considered as 1.0. The activity in the presence of inhibitors was indicated as relative activity with respect to the control. All experiments were done at least three times and standard deviations are shown. (A) AZTTP. (B) d4TTP. (C) ddCTP. (D) 3TCTP. (E) NVP. (F) DLV. (G) EFV. (H) Alignment of partial sequences of HIV-1 p66, HIV-2, L1 and Ty1 RTs. Residues (101, 106, 181, 188, 190) important to NNRTI pocket motif are highlighted. Sequence from 122-174 is not shown.

Figure 3

Kinetic analysis of inhibition of L1 RT by NRTIs. The L1 RT activity was measured as described in materials and methods. (A) Double reciprocal plot of the velocity of the L1 RT activity as a function of [³²P]-dTTP substrate concentration. Increasing concentrations of substrate in the absence (diamond) or presence of 2 nM (square), 5 nM (triangle), 10 nM (circle) or 15 nM (+) AZTTP. (B) Double reciprocal plot of the velocity of the L1 RT activity as a function of [³²P] dTTP substrate concentration. Increasing concentrations of substrate in the absence (diamond) or presence of 0.1 nM (square), 0.2 nM (triangle), 0.5 nM (circle) or 1 nM (+) d4TTP. (C) Double reciprocal plot of the velocity of the L1 RT activity as a function of [³²P] dCTP substrate concentration. Increasing concentrations of substrate in the absence (diamond) or presence of 0.5 nM (square), 1 nM (triangle), 2 nM (circle) or 5 nM (+) ddCTP. (D) Double reciprocal plot of the velocity of the L1 RT activity as a function of [³²P] dTTP substrate concentration. Increasing concentrations of substrate in the absence (diamond) or presence of 5 nM (square), 10 nM (triangle), 20 nM (circle) or 50 nM (+) 3TCTP.

Figure 4

Effect of RT inhibitors on ORFeus-Hs retrotransposition efficiency. A tissue culture cell-based L1 retrotransposition assay was done as described in materials and methods. Example plates from retrotransposition assay indicating different effects of RT inhibitors on L1 retrotransposition. See Table 2 for details.