Abstract
The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.
Highlights
As APOBEC3G, a member of the recently discovered human cytidine deaminase family named apolipoprotein B-editing catalytic polypeptide 3 (APOBEC3), deaminates cytosine residues to uracil in the growing minus strand viral DNA during retroviral reverse transcription, we wanted to evaluate whether members of the APOBEC3 family interfere with L1 retrotransposition, which is dependent on reverse transcription of template RNA
To measure the effects of the APOBEC3 proteins on L1 retrotransposition, we cotransfected the L1 reporter plasmid pJM101/ L1RP with plasmids expressing A3A, A3B, A3C, A3F, A3G, or A3H into HeLa cells
By determining the number of G418r colonies, we found differential activity of the single APOBEC3s against L1 retrotransposition
Summary
Cell Culture—HeLa cells were maintained in Dulbecco’s high glucose modified Eagle’s medium supplemented with 10% fetal bovine serum, 0.29 mg/ml L-glutamine, and 100 units/ml penicillin/streptomycin (Invitrogen; Dulbecco’s modified Eagle’s medium complete). Western Blot Analysis—For analysis of APOBEC3 protein expression, 2 ϫ 105 HeLa cells were seeded and transfected with 2 g of plasmid DNA the day using FuGENE 6 transfection reagent (Roche Applied Science). The following day, each well was cotransfected with 0.5 g of reporter plasmid pJM101/L1RP [62] and 0.5 g of the respective APOBEC3 expression plasmid using 3 l of FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer’s protocol. The following day, cells were cotransfected with 0.5 g of the respective APOBEC3 expression plasmid or empty vector pcDNA3.1/Zeo(ϩ) with 0.5 g of pcDNA3.1(ϩ) expressing the neomycin resistance gene using FuGENE 6 transfection reagent (Roche Applied Science). Annealing temperatures were as follows: 58 °C for APOBEC3A, APOBEC3G, and 2-microglobulin; 50 °C for APOBEC3B and APOBEC3H; and 60 °C for APOBEC3C and APOBEC3F
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