Figure 4. SAMHD1 Inhibits LINE-1 ORF2p-Mediated Endogenous Reverse Transcription in LINE-1 RNP.

(A) SAMHD1 did not affect the expression of the LINE-1 ORF1 protein. HEK293T cells were transfected with the pc-L1-1FH vector plus the empty vector VR1012 or the expression vector for SAMHD1, HnRNPL, or MOV10. ImageJ software (NIH) was used to quantitate ORF1 band intensities, and their absolute readings are indicated above the immunoblot.

(B) A diagram of the LEAP assay. The LINE-1 RNP (from pc-L1-1FH) was produced from transfected HEK293T cells and purified by ultracentrifugation through a sucrose cushion as previously described. The LEAP primer, containing a linker region (dashed line), was used to precisely target onto LINE-1 mRNA, and the reverse transcription occurred with the assistance of the ORF2 protein. Synthesized cDNA was then amplified through standard PCR with two primers (dash arrows) targeting to LINE-1 and the linker.

(C) SAMHD1 reduced the reverse transcription efficiency mediated by ORF2p in LINE-1 RNPs. The amount of ORF1 proteins in isolated LINE-1 RNPs in the absence or presence of SAMHD1 was determined by immunoblotting using an anti-HA antibody. LINE-1 RNA was examined by using the LEAP primer and MuLV reverse transcriptase for cDNA synthesis, followed by PCR amplification.

(D) Quantitative real-time PCR analysis of LEAP products and LINE-1 RNA RT-PCR products.

(E) ORF2p level was lowered by 62% in average, with the presence of exogenous SAMDH1 protein. The bar chart was based on five independent experiments. The error bars indicated the SD.

See also Figure S4.