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. 2016 Jul 18;11(7):e0157220.
doi: 10.1371/journal.pone.0157220. eCollection 2016.

APOBEC3DE Inhibits LINE-1 Retrotransposition by Interacting with ORF1p and Influencing LINE Reverse Transcriptase Activity

Weizi Liang  1 , Jiwei Xu  1 , Wensu Yuan  1 , Xuan Song  1 , Jianyong Zhang  1 , Wei Wei  2   3 , Xiao-Fang Yu  1   3 , Ying Yang  1
Affiliations

APOBEC3DE Inhibits LINE-1 Retrotransposition by Interacting with ORF1p and Influencing LINE Reverse Transcriptase Activity

Weizi Liang et al. PLoS One. .

Abstract

Human long interspersed elements 1 (LINE-1 or L1) is the only autonomous non-LTR retroelement in humans and has been associated with genome instability, inherited genetic diseases, and the development of cancer. Certain human APOBEC3 family proteins are known to have LINE-1 restriction activity. The mechanisms by which APOBEC3 affects LINE-1 retrotransposition are not all well characterized; here, we confirm that both A3B and A3DE have a strong ability to inhibit LINE-1 retrotransposition. A3DE interacts with LINE-1 ORF1p to target LINE-1 ribonucleoprotein particles in an RNA-dependent manner. Moreover, A3DE binds to LINE-1 RNA and ORF1 protein in cell culture system. Fluorescence microscopy demonstrated that A3DE co-localizes with ORF1p in cytoplasm. Furthermore, A3DE inhibits LINE-1 reverse transcriptase activity in LINE-1 ribonucleoprotein particles in a cytidine deaminase-independent manner. In contrast, A3B has less inhibitory effects on LINE-1 reverse transcriptase activity despite its strong inhibition of LINE-1 retrotransposition. This study demonstrates that different A3 proteins have been evolved to inhibit LINE-1 activity through distinct mechanisms.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. APOBEC3B and APOBEC3DE inhibit human LINE-1 retrotransposition.
(A) Schematic diagram of the full-length LINE-1 element and pc-L1RP EGFP plasmids. Canonical L1 contains a 5’ untranslated region (5’UTR), two open reading frames (ORF1 and ORF2), and a 3’UTR, including a polyadenylation signal (AATAAA) and polyA tail (A)n. pc-L1RP EGFP contains an antisense cassette of EGFP that is interrupted by an intron. The LINE-1 5’UTR promoter initiates ORF1 and ORF2 expression, and EGFP is under the control of the CMV promoter in trans. (B) Retrotransposition assay of LINE-1. EGFP can only be expressed when LINE-1 is transposed into the genome with the intron removed during RNA splicing. (C) Inhibitory effect of A3B and A3DE on LINE-1 mobility. The A3B- or A3DE-expressing vector was co-transfected with L1RP EGFP, and flow cytometry was used to detect the EGFP-positive cells. The pcDNA3.1 vector was used as the control. The bar charts represent the results from three independent experiments; error bars indicate the S.D. of three replicates within one experiment. (D) Representative flow cytometry dot diagrams for A3B and A3DE in LINE-1 restriction. The A3B-HA- or A3DE-HA-expressing vector was co-transfected with L1RP EGFP into HEK293T cells. JM111 transfected cells, as a negative control, were used to gate the EGFP-positive cells. The percentage of EGFP-positive events is shown in the upper right corner of each panel.
Fig 2
Fig 2. Neither APOBEC3B nor APOBEC3DE affects EGFP expression, and both do not cause cytotoxicity in HEK293T cells.
(A) No cytotoxicity was observed in HEK293T cells transfected with L1RP-EGFP together with A3B or ADE at 48 hr post-transfection, when compared to the control. Cells were stained with trypan blue, and the percentage of living cells was calculated as the % survival. (B) The empty vector, A3B and A3DE was co-transfected with, pcDNA3.1-EGFP plasmid in HEK293T cells and EGFP-positive cells were detected by flow cytometry at 48 hr post-transfection.
Fig 3
Fig 3. APOBEC3DE interacts with ORF1p in a RNA-dependent manner.
(A) Diagram of the co-IP assay. Cleared whole-cell lysate was used for these assays. Protein antigens were pulled down by specific antibodies. The antibodies were coupled to solid substrates. (B) A3B or A3DE interacts with ORF1p, and RNase disrupts the binding of A3F to ORF1p. The pc-L1-1FH plasmid was co-transfected with A3B-V5 or A3DE-V5 or the control vector into HEK293T cells. The cleared cell lysate was split into two halves, and RNase A was added to one half (final concentration, 50 μg/ml); both samples were then incubated with Flag-tagged beads. Western blotting was performed to identify the input, RNase A-treated, and untreated IP products. (C, D) IP of A3DE for endogenous L1 RNP. A3DE-HA was transfected into HEK293T cells and HA beads were used to pull down A3DE. IP assay was conducted at 48 hours post-transfection. IP product was aliquoted, one for Western Blotting and the other one for RNA extraction and L1 mRNA detection. (E) Immunofluorescence staining of A3DE and ORF1p in HEK293T cells.
Fig 4
Fig 4. APOBEC3DE targets LINE RNP particles and inhibits ORF2p function.
(A) Flow chart for the LEAP assay. pc-L1-1FH was co-transfected with A3B or A3DE or empty vector into HEK293T cells. The purified LINE-1 RNPs from pc-L1-1FH were used for three assays: LEAP, MLV RT-PCR, and western blotting. The LEAP primer was used to target LINE-1 mRNA through the designed linker region. Real-time PCR was used to amplify the L1 cDNA. The ratio of LEAP to MLV is expressed as the relative L1 cDNA amount. (B) Quantitative real-time PCR analysis of LEAP and MLV RT products. A3B or A3DE was co-transfected with the pc-L1-1FH plasmid into HEK293T cells, and cell lysates were prepared at 48 hr post-transfection. The relative amounts of synthesized cDNA from LEAP and MLV-RT were detected with the Linker primer and the L1-LEAP-R primer (as indicated in the Methods) by real-time PCR. A3DE reduced the reverse transcription activity of the L1 ORF2p when compared to the vector control.
Fig 5
Fig 5. APOBEC3DE CDA mutations maintained the inhibitory ability on LINE-1 retrotransposition.
(A) Diagram of A3DE cytidine deaminase domains. (B) The effects of A3DE mutations on LINE-1 restriction by flow cytometry. The effects of the WT and mutant A3DE on LINE-1 restriction were compared under the similar A3DE protein expression levels.
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Grants and funding

This work was supported by Tianjin Municipal Science and Technology Commission, Grant number: 13JCYBJC40800 (http://www.tstc.gov.cn/) to YY. The Tianjin Municipal Science and Technology Commission provided the funding for materials.

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