APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m601716200
ISSN1083-351X
AutoresHeide Muckenfuß, Matthias Hamdorf, Ulrike Held, Mario Perković, Johannes Löwer, Klaus Cichutek, Egbert Flory, Gerald G. Schumann, Carsten Münk,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe 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 ∼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 ∼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. 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 ∼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 ∼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. The long interspersed nuclear element 1 (LINE-1, L1) 4The abbreviations used are: LINE-1, L1, long interspersed nucleotide element-1; APOBEC3A, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A; A3A, APOBEC3A; Vif, virion infectivity factor; HIV-1, human immunodeficiency virus 1; HA, hemagglutinin; UTR, untranslated region; ORF, open reading frame; RNP, ribonucleoprotein; CCAA, mutation of Cys-101 and Cys-106 to Ala; E72A, mutation of Glu-72 to Ala; RT, reverse transcription; LTR, long terminal repeat; PBMC, peripheral blood mononuclear cells; SIV, simian immunodeficiency virus; siRNA, small interfering RNAs; IAP, intracisternal A particle. is the only autonomous non-LTR retrotransposon in the human genome. Approximately 520,000 L1 copies compose ∼17% of the chromosomal DNA (1Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. Kann L. Lehoczky J. LeVine R. McEwan P. 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Nucleic Acids Res. 2003; 31: 4929-4940Crossref PubMed Scopus (136) Google Scholar, 30Yu F. Zingler N. Schumann G. Stratling W.H. Nucleic Acids Res. 2001; 29: 4493-4501Crossref PubMed Scopus (150) Google Scholar). In this study, we demonstrate that the presence of A3A reduced the L1 retrotransposition frequency in HeLa cells by up to 85%. Expression of A3B, A3C, and A3F inhibited retrotransposition activity by ∼75%, whereas A3G and A3H had no influence. Furthermore, we were able to enhance L1 retrotransposition frequency in the absence of cotransfected APOBEC3s by knocking down endogenously expressed A3C via RNA interference. Although mutation of the putative catalytic deamination domain of APOBEC3A completely abolished its activity against L1 retrotransposition, we did not find editing of L1 de novo insertions in the presence of APOBEC3 proteins. In addition, sequence analyses of preexisting L1 insertions did not reveal any enhanced frequency of G-to-A transitions. Our data suggest a novel mode of action of APOBEC3 activity against the non-LTR retrotransposon LINE-1. 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). Human A3.01 T cells (National Institute for Biological Standards and Control, UK) were grown in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.29 mg/ml l-glutamine, and 100 units/ml penicillin/streptomycin. Human keratinocytes were kindly provided by Dr. August Bernd (Zentrum der Dermatologie und Venerologie, Johann-Wolfgang Goethe-Universität Frankfurt/Main, Germany) and cultivated in Hanks' medium supplemented with 5% fetal bovine serum and 100 units/ml penicillin/streptomycin. Cells were incubated at 37 °C with 100% humidity in 5-7% CO2 and passaged using standard cell culture techniques. PBMCs were isolated from EDTA-treated whole blood of healthy donors by Histopaque-1077 (Sigma) gradient centrifugation. Plasmids—L1 retrotransposition reporter construct pJM101/L1RP (62Kimberland M.L. Divoky V. Prchal J. Schwahn U. Berger W. Kazazian Jr., H.H. Hum. Mol. Genet. 1999; 8: 1557-1560Crossref PubMed Scopus (135) Google Scholar) was a gift from Haig H. Kazazian, Jr. pLLrpII.6 is a plasmid carrying a de novo L1 integrant with a spliced neor cassette that is localized on a plasmid backbone derived from the L1 rescue plasmid pCEP4/L1.3mneoI400/ColEI (20Gilbert N. Lutz-Prigge S. Moran J.V. Cell. 2002; 110: 315-325Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). The hemagglutinin (HA)-tagged human APOBEC3 proteins were expressed from the pcDNA3.1/Zeo(+) vector (Invitrogen). The original APOBEC3G-HA and APOBEC3A-HA expression constructs were generously provided by Nathaniel R. Landau (51Mariani R. Chen D. Schröfelbauer B. Navarro F. König R. Bollman B. Münk C. Nymark-McMahon H. Landau N.R. Cell. 2003; 114: 21-31Abstract Full Text Full Text PDF PubMed Scopus (772) Google Scholar) and Bryan R. Cullen (35Wiegand H.L. Doehle B.P. Bogerd H.P. Cullen B.R. EMBO J. 2004; 23: 2451-2458Crossref PubMed Scopus (410) Google Scholar), respectively. Plasmids expressing A3B, A3C, and A3F were generated by performing RT-PCR on RNA of PHA/IL2-activated human PBMCs. To isolate A3B (pcA3B-HA), forward primer CEM15-CM13 (5′-TAAGCGGAATTCTATCTAAGAGGCTGAACATG-3′) and reverse primer CEM15-HA-C (5′-TAGAAGCTCGAGTCAAGCGTAATCTGGAACATCGTATGGATAGTTTTCCTGATTCTGGAG-3′) were used. The amplicon was cloned into pCR4Blunt-TOPO (Invitrogen), and the NotI- and XbaI-restricted fragment was transferred into the NotI and SpeI sites of pcDNA3.1(+) (Invitrogen). To clone A3C (pcA3C-HA), forward primer CEM15-CM13 (see above) and reverse primer CEM15-CM28 (5′-AGCTCGAGTCAAGCGTAATCTGGAACATCGTATGGATACTGGAGACTCTCCCGTAGCCTT-3′) were applied. The amplicon was cloned into pCR4Blunt-TOPO and inserted into the EcoRI and XhoI restriction sites of pcDNA3.1(+). To clone A3F (pcA3F-HA), forward primer CEM15-CM12B (5′-TAAGCGAAGCTTCTTAGTCGGGACTAGCCGGC-3′) and reverse primer CEM15-CM29 (5′-AGTCTAGATCAAGCGTAATCTGGAACATCGTATGGATACTCGAGAATCTCCTGCAGCTTGCTGTC-3′) were used, and the amplicon was cloned into pCR4Blunt-TOPO. After HindIII and SpeI digestion, the resulting fragment was transferred into the HindIII and XbaI sites of pcDNA3.1(+). To clone A3H (pcA3H-HA), the coding region was amplified by PCR using the forward primer CEM15-CM42 (5′-CAGGCGAATTCCTGCTAAGGAAGCTGTGGCC-3′) and the reverse primer CEM15-CM43 (5′-TTCAGCTCGAGTCAAGCGTAATCTGGAACATCGTATGGATAGGACTTTATCCTCTCAAGCCG-3′) with the plasmid IRALp962F0354Q (obtained from the German Resource Center for Genome Research, Berlin) as template. The PCR product was transferred into the EcoRI and XhoI sites of pcDNA3.1(+). APOBEC3 expression constructs used in the retrotransposition reporter assays were generated by subcloning the respective APOBEC3-HA sequence from the above-described pcDNA3.1(+) vectors into pcDNA3.1/Zeo(+) using the following restriction sites: A3A, KpnI and XbaI (pcA3A-HA.ZEO); A3B, HindIII and XhoI (pcA3B-HA.ZEO); A3C, HindIII and XhoI (pcA3C-HA.ZEO); A3F, PmeI and PmeI (pcA3F-HA.ZEO); A3G, EcoRI and XhoI (pcA3G-HA.ZEO); and A3H, EcoRI and XhoI (pcA3H-HA.ZEO). The APOBEC3A mutants pcA3A.E72A and pcA3A.CCAA were generated by overlapping PCR using pcA3A-HA.ZEO as template. 5′ and 3′ fragments were amplified with primer sets specific for the overlap region, including the mutations and the external primers (hu3A1, 5′-GCTTGGTACCACCATGGAAGCCAGCCCAGCATCCG-3′; hu3A4, 5′-CATGCTCGAGTCAAGCGTAATCTGGAACGTC-3′). Subsequently, the mixture of both PCR products was amplified with the two external primers. The resulting fragment was cleaved with KpnI and XhoI and cloned into pcDNA3.1(+)Zeo. Sequence analysis revealed an additional mutation (M153I) in pcA3A-CCAA. The identity of the described APOBEC3 expression plasmids was confirmed by sequence analyses. Western Blot Analysis—For analysis of APOBEC3 protein expression, 2 × 105 HeLa cells were seeded and transfected with 2 μg of plasmid DNA the next day using FuGENE 6 transfection reagent (Roche Applied Science). Two days after transfection, cells were lysed using RIPA lysis buffer (25 mm Tris, pH 8.0, 137 mm NaCl, 1% glycerol, 0.5% sodium deoxycholate, 1% Nonidet P-40, 2 mm EDTA, pH 8, 0.1% SDS, and protease inhibitors), and lysates were cleared by centrifugation. Samples were boiled in Laemmli buffer and subjected to SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane. HA-APO-BEC3 proteins were detected using an anti-HA antibody (1:6,000 dilution, MMS-101P; Covance) and anti-mouse horseradish peroxidase (Amersham Biosciences). For the detection of α-tubulin, an anti-tubulin antibody (1:10,000 dilution, B5-1-2, Sigma) was applied. Signals were visualized by ECL (Amersham Biosciences). L1 Retrotransposition Reporter Assay—Retrotransposition rates were determined by applying the rapid and quantitative transient L1 retrotransposition assay described previously (61Wei W. Morrish T.A. Alisch R.S. Moran J.V. Anal. Biochem. 2000; 284: 435-438Crossref PubMed Scopus (85) Google Scholar). Briefly, for each transfection reaction, HeLa cells were seeded in 6-well tissue culture dishes at 2 × 105 cells/well. The following day, each well was cotransfected with 0.5 μg of reporter plasmid pJM101/L1RP (62Kimberland M.L. Divoky V. Prchal J. Schwahn U. Berger W. Kazazian Jr., H.H. Hum. Mol. Genet. 1999; 8: 1557-1560Crossref PubMed Scopus (135) Google Scholar) and 0.5 μg of t
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