The MOV10 helicase inhibits LINE-1 mobility

Abstract

LINE-1 (long interspersed element 1) is an autonomous non-long terminal repeat retrotransposon. Its replication often causes mutation and rearrangement of host genomic DNA. Accordingly, host cells have evolved mechanisms to control LINE-1 mobility. Here, we report that a helicase named MOV10 effectively suppresses LINE-1 transposition. Mutating the helicase motifs impairs this function of MOV10, suggesting that MOV10 requires its helicase activity to suppress LINE-1 replication. Further studies show that MOV10 post-transcriptionally diminishes the level of LINE-1 RNA. The association of MOV10 with both LINE-1 RNA and ORF1 suggests that MOV10 interacts with LINE-1 RNP and consequently causes its RNA degradation. These data demonstrate collectively that MOV10 contributes to the cellular control of LINE-1 replication.

Keywords: Host-Pathogen Interactions; LINE-1; MOV10; Post-transcriptional; RNA Helicase; Restriction; Retrotransposon; Reverse Transcription; Viral Replication; Virology.

FIGURE 1.

Overexpression of MOV10 inhibits LINE-1 retrotransposition in HeLa cells. A, illustration of the CMV-L1-neo^RT, IAP-neo^TNF, and MusD-neo^TNF reporter (left panel) and the generation of a functional neomycin resistance mRNA (right panel). Following transcription from the 5′-UTR promoter of LINE-1, the intron in the neomycin resistance gene is excised. This intronless mRNA is then reverse transcribed into cDNA that is able to produce a functional neomycin resistance mRNA. B, domain structure of the MOV10 helicase. The positions of the mutated amino acids Lys and Glu in the Walk A (GKT) and Walker B (DEAG) motifs are indicated. C, Western blots to show the expression of MOV10 and its mutants in transiently transfected HeLa cells. Tubulin was probed as an internal control. D, G418 resistance colony assays. HeLa cells were transfected with 750 ng of the CMV-L1-neo^RT, IAP-neo^TNF, MusD-neo^TNF, or pcDNA3.1 DNA with or without MOV10 DNA or its mutants. Control represents untransfected HeLa cells that also underwent G418 selection. pcDNA3.1 is a plasmid DNA that carries the neomycin resistance gene and thus confers resistance to G418. Colonies were visualized with crystal violet staining. The results of a representative colony assay are shown. The data from three independent experiments are summarized in the bar graph. The number of cell colonies in the absence of MOV10 is arbitrarily set as 100. E, cellular location of MOV10 and its mutants. FLAG-tagged MOV10 and its mutants (MOV10EQ and MOV10KR) were transiently expressed in HEK293 cells and detected by immunostaining with anti-FLAG antibodies (pseudocolored in green).

FIGURE 2.

Overexpression of MOV10 inhibits LINE-1 activity in HEK293 cells. A, effects of MOV10 on the activity of IAP and MusD in transiently transfected HEK293 cells. B and C, effects of MOV10 and its mutants on LINE-1 (B) and IAP (C) retrotransposition. Expression levels of MOV10 and its mutants were assessed by Western blotting (top panels). One representative colony assay is shown (middle panels). The results from three independent experiments are summarized in the bar graph (bottom panels).

FIGURE 3.

Knockdown of MOV10 increases LINE-1 retrotransposition in HEK293 and HeLa cells. A, Western blots to measure levels of endogenous MOV10 in HEK293 cells that were transfected with control siRNA or siRNA oligonucleotides targeting MOV10 mRNA. B, HEK293 cells were transfected with siRNA oligonucleotides prior to transfection with 750 ng of the CMV-L1-neo^RT, IAP-neo^TNF, MusD-neo^TNF, or pcDNA3.1 DNA. The G418-resistant cell colonies were scored for each transfection. Control represents untransfected HEK293 cells that underwent G418 selection. C, results of three independent transfection experiments shown in B are summarized in the bar graph. D, nest RT-PCR to measure endogenous LINE-1 RNA in HeLa cells. HeLa cells were transfected with siRNA oligonucleotides targeting endogenous MOV10. Equal amounts of total cellular RNA normalized by the GAPDH internal control was used to generate cDNA with (+) or without (−) reverse transcriptase. The cDNA was then amplified using a nest PCR method with primers specific for LINE-1, as described under “Experimental Procedures.” The first lane is molecule weight markers.

FIGURE 4.

Overexpression of MOV10 diminishes the production of LINE-1 cDNA. A, location of primers that were used to amplify cDNA of L1-neo^RT, IAP-neo^TNF, and MusD-neo^TNF (49). Primer 1 binds only to cDNA that is generated from spliced neomycin resistance mRNA, not to the plasmid DNA that was used in transfection. B, equal amounts of total cellular DNA normalized by the GAPDH internal control (Con) were used to amplify the target cDNA, Results of PCR to show the levels of cDNA products of LINE-1, IAP, and MusD. The same amounts of total cellular DNA were used as the template in PCR that was run for various cycles to show the linear range of the amplification reactions. The results shown represent those from three independent transfection experiments.

FIGURE 5.

MOV10 diminishes LINE-1 RNA level. A, effects of ectopic MOV10 on the RNA levels of LINE-1, IAP, and MusD. HEK293 cells were co-transfected with CMV-L1-neo^RT, IAP-neo^TNF, or MusD-neo^TNF DNA together with wild type or mutant MOV10 DNA. Viral RNA levels were measured by Northern blotting using ³²P-labeled DNA probes specific for the neomycin resistance gene. The bands indicated are the major splicing forms of L1-neoRT, IAP-neoTNF, and MusD-neoTNF. The cellular GAPDH mRNA served as internal controls. B, knockdown of MOV10 increases LINE-1 RNA level in HEK293 cells. HEK293 cells were transfected with control siRNA or MOV10-targeting siRNA prior to transfection with CMV-L1-neo^RT, IAP-neo^TNF, and MusD-neo^TNF DNA. Northern blotting was performed to measure the RNA levels of L1-neo^RT, IAP-neo^TNF, and MusD-neo^TNF. A representative result of three independent experiments is shown. C, MOV10 impairs L1.3 RNA expression. JM101/L1.3 DNA was either transfected into HEK293 cells together with wild type MOV10 DNA or transfected into HEK293 cells that had been treated with control or MOV10 siRNA oligonucleotides. Levels of L1.3 RNA were measured by Northern blotting. GAPDH mRNA was detected to serve as an internal control. The overexpression and knockdown of MOV10 in all transfection experiments were confirmed by Western blotting (data not shown). D, HEK293 cells were co-transfected with CMV-L1-neoRT and MOV10 expressing plasmid. Equal amount of total cellular RNA was treated with DNase to remove the potential contamination of plasmid DNA. The cDNA products were normalized by the GAPDH. The unspliced form of LINE-1 RNA was detected by PCR using the primers Fneo and Rintron, whereas the spliced form of LINE-1 RNA was detected by PCR using the primers Fneo and Rneo.

FIGURE 6.

Effects of MOV10 overexpression on LINE-1 5′-UTR promoter activity and RNA stability. A, HEK293 cells were co-transfected with the wild type or mutant MOV10 DNA together with L1-FL. The firefly luciferase activity was measured to represent the activity of LINE-1 5′-UTR promoter. The pGL3-basic contains the firefly luciferase gene that lacks a promoter at its 5′ end. Firefly luciferase activity from this vector was measured to reflect the basal expression of firefly luciferase gene. The TK-RL plasmid DNA was included in each transfection to control the efficiency of different transfections. The results shown are the ratios of firefly luciferase activity versus Renilla luciferase activity. B, Western blots to show the expression of MOV10 and its mutants in the transiently transfected HEK293 cells. C, the same amounts of HEK293 cells were co-transfected with CMV-L1-neo^RT and either MOV10 DNA or pcDNA3.1 and then treated with actinomycin D for various time periods as indicated. The RNA level of CMV-L1-neo^RT was measured by Northern blotting as described previously. The cellular GAPDH mRNA served as internal controls. D, HEK293 cells were co-transfected with MOV10 DNA and plasmid encoding Myc-tagged ORF1. The cells were collected and lysed 48 h post-transfection. Equal amounts of supernatant were incubated with MOV10-specific antibody or anti-Myc antibody. The immunoprecipitated materials were analyzed by Western blots using anti-Myc or MOV10-specific antibody, respectively. E, HEK293 cells were transfected with FLAG/HA-tagged MOV10 and CMV-L1-neo^RT DNA. The presence of LINE-1 RNA in the transfected cells as well as in the immunoprecipitated MOV10 complex was detected by RT-PCR using primers that amplify the neo coding sequence. W.B., Western blot.

FIGURE 7.

MOV10 inhibits LINE-1 retrotransposition when Ago2 is knocked down. Western blots were performed to determine the efficiency of Ago2 knockdown in each transfection experiment. Colony assays were conducted to assess the retrotransposition efficiency of LINE-1 (A), IAP (B), or MusD (C) in HeLa cells that had been transfected with either control siRNA or Ago2-targeting siRNA oligonucleotides. The results of three independent transfection experiments are summarized in each bar graph.