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. 2009 Oct:1178:268-75.
doi: 10.1111/j.1749-6632.2009.05006.x.

APOBEC3 proteins inhibit LINE-1 retrotransposition in the absence of ORF1p binding

Nika Lovsin  1 , B Matija Peterlin
Affiliations

APOBEC3 proteins inhibit LINE-1 retrotransposition in the absence of ORF1p binding

Nika Lovsin et al. Ann N Y Acad Sci. 2009 Oct.

Abstract

Members of the apolipoprotein B mRNA editing complex polypeptide 1-like (APOBEC) family of enzymes exhibit inhibitory activity against a variety of exogenous and endogenous retroviruses including retrotransposons, such as long interspersed element 1 (LINE-1). Indeed, human APOBEC3A, APOBEC3B, and APOBEC3F inhibit retrotransposition of human LINE-1, mouse IAP and MusD retrotransposons. In our study, we examined whether the inhibitory effect of APOBEC3 proteins correlates with APOBEC3 ability to bind the LINE-1 ORF1 protein. We examined the interactions between the LINE-1 ORF1 protein and the most potent LINE-1 retrotransposon inhibitors, human APOBEC3A and APOBEC3B, by immunofluorescence and immunoprecipitation. Although human APOBEC3A shows the highest inhibitory potency against LINE-1 retrotransposon, no direct interactions were identified either by immunofluorescence or by co-immunoprecipitation. APOBEC3B binds to LINE-1 ORF1 protein, yet no co-localization was detected. We concluded that APOBEC3 proteins interfere indirectly with the LINE-1 retrotransposition pathway, probably through interference with RNA targeting.

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Figures

Figure 1
Figure 1
Neo-based retrotransposition assay. HeLa cells were cotransfected with L1 construct (JM101) and respective A3 protein. Five days after transfection, cells were subjected to G418 selection for 12 days. For each experiment colonies were counted from two dishes and retrotransposition efficiency was determined relative to the control, which was set to 100%.
Figure 2
Figure 2
Localization of L1 ORF1, A3A, and A3B proteins. HeLa cells were transfected with plasmids encoding L1 ORF1 (pcDNA3.1.ORF1.V5), A3A (pKA3A.HA), or A3B (pKA3B.HA) proteins. At 48 h after transfection, cells were fixed and stained with rabbit anti-HA or mouse monoclonal anti-V5 antibody and a mouse-specific secondary antibody conjugated to Alexa-Fluor 488 or rabbit specific secondary antibody conjugated to Alexa-Fluor 568. Samples were viewed by fluorescence microscopy. (Left) Immunofluorescence with primary anti-HA (red), or primary anti-V5 antibodies (green). (Right) DNA visualized by DAPI staining, merge with immunofluorescence is shown.
Figure 3
Figure 3
Co-localization studies ORF1, A3A, and A3B proteins. HeLa cells were cotransfected with plasmids encoding L1 ORF1 (pcDNA3.1ORF1.V5) and A3A (pKA3A.HA) (A) and L1 ORF1 and A3B (pKA3B.HA) proteins (B). Samples were processed as in Figure 2. Merged views are displayed on the right.
Figure 4
Figure 4
Co-immunoprecipitation of L1 ORF1 and A3 proteins. 293T cells were cotransfected with plasmids encoding L1 ORF1 (pcDNA3.1ORF1.V5) and A3A (pKA3A.HA), A3B (pKA3B.HA), mA3 (pFLAG.mA3) proteins. At 48 h after transfection cells were lysed and immunoprecipitation with anti-HA or anti-FLAG antibodies. Immunobloting was performed with anti-V5 antibodies. Addition of RNase A during the immunoprecipitation is indicated.
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