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Berkeley MCELLBI 110 - DNA Deamination Mediates Innate Immunity to Retroviral Infection

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Cell, Vol. 113, 803–809, June 13, 2003, Copyright 2003 by Cell PressDNA Deamination Mediates Innate Immunityto Retroviral Infectionmajor targets for HIV infection in vivo) and has beenshown to be a potent inhibitor of Vif-deficient HIV(Sheehy et al., 2002).Reuben S. Harris,1,3,4,* Kate N. Bishop,2,3Ann M. Sheehy,2Heather M. Craig,2Svend K. Petersen-Mahrt,1Ian N. Watt,1Michael S. Neuberger,1and Michael H. Malim2CEM15/APOBEC3G is a member of the APOBEC fam-ily of proteins of which the RNA editing protein APO-1Medical Research Council Laboratory ofMolecular Biology BEC1 is the founder (Teng et al., 1993; Jarmuz et al.,2002). Similarity to APOBEC1 suggested that CEM15/Hills RoadCambridge CB2 2QH APOBEC3G might also function by deaminating cyto-sine in RNA—either a host message or the viral RNAUnited Kingdom2Department of Infectious Diseases genome—although it has not been demonstrated tohave any RNA editing activity.Guy’s, King’s and Street Thomas’ School ofMedicine However, CEM15/APOBEC3G has been shown to beable to act as a DNA mutator in E. coli, with the mutationsKing’s College LondonLondon SE1 9RT attributable to dC→dU deamination (Harris et al., 2002).This led us to speculate (Harris et al., 2003) that CEM15/United KingdomAPOBEC3G triggers the block to retroviral infectivity bydeaminating dC→dU in the first (minus)-strand of theviral cDNA during a postentry step of its lifecycle. Here,Summarythis hypothesis is tested using a helper virus-dependentMLV-based assay system. We show that MLV producedCEM15/APOBEC3G is a cellular protein required forusing cells expressing CEM15/APOBEC3G has a dimin-resistance to infection by virion infectivity factor (Vif)-ished infectivity as monitored using fluorescent protein-deficient human immunodeficiency virus (HIV). Here,encoding genes embedded in viral sequence. Moreover,using a murine leukemia virus (MLV)-based system,this correlated with the introduction of a phenomenallywe provide evidence that CEM15/APOBEC3G is a DNAhigh mutation load into the viral cDNA, nearly all thedeaminase that is incorporated into virions during viralmutations being G→A substitutions as read-out on theproduction and subsequently triggers massive deami-viral plus (genomic) strand. This result is only consistentnation of deoxycytidine to deoxyuridine within the ret-with CEM15/APOBEC3G causing massive deaminationroviral minus (first)-strand cDNA, thus providing aof dC→dU in the viral minus (first cDNA) strand, thusprobable trigger for viral destruction. Furthermore, HIVestablishing CEM15/APOBEC3G as the critical compo-Vif can protect MLV from this CEM15/APOBEC3G-nent of this innate defense mechanism. Furthermore,dependent restriction. These findings imply that tar-HIV Vif expression is able to counteract the CEM15/geted DNA deamination is a major strategy of innateAPOBEC3G effect on MLV infectivity indicating that Vifimmunity to retroviruses and likely also contributesserves to overcome a general mode of innate antiretrovi-to the sequence variation observed in many virusesral defense that is mediated by DNA deamination.(including HIV).ResultsIntroductionCEM15/APOBEC3G Can Deaminate DNAProductive infection by HIV depends on the virus-We first used a biochemical assay to assess whetherencoded protein Vif (Fisher et al., 1987; Strebel et al.,CEM15/APOBEC3G was directly capable of deaminat-1987). Vif expression is necessary only in the virus-pro-ing DNA in vitro. Purified, recombinant CEM15/APO-ducing cell; in its absence, infection of a subsequentBEC3G was indeed able to deaminate dC→dU in a sin-target cell terminates at a postentry step through thegle-strand DNA oligonucleotide substrate (Figure 1), anaction of an innate antiviral mechanism (Sova and Vol-activity that has recently also been observed with othersky, 1993; von Schwedler et al., 1993; Simon and Malim,APOBEC family members, AID (Bransteitter et al., 2003;1996; Madani and Kabat, 1998; Simon et al., 1998a).Chaudhuri et al., 2003) and APOBEC1 (Figure 1A, laneHow Vif mediates HIV infectivity is not yet known and1 and Petersen-Mahrt and Neuberger, 2003).few clues are apparent in its amino acid sequence as itis highly variable amongst HIV isolates and shows littleMLV Produced in CEM15/APOBEC3GⴙCells Hassimilarity to other proteins. But, central to this infectionDiminished Infectivityrestriction mechanism is the human protein CEM15/In addition to regulating the infectivity of lentivirusesAPOBEC3G, which is expressed in human T cells (thesuch as HIV, earlier work suggested that Vif also stimu-lates infection and replication of the distantly related*Correspondence: [email protected] MLV (Simon et al., 1998b). This raised3These authors contributed equally to this work.the possibility that CEM15/APOBEC3G might also be4Present Address (beginning August 1, 2003): Department of Bio-able to impede infection by MLV. We therefore soughtchemistry, Molecular Biology and Biophysics, University of Minne-to develop a helper virus-dependent, MLV-based assaysota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN55455.system. Stocks of recombinant MLV encoding yellowCell804Figure 2. CEM15/APOBEC3G Diminishes MLV Infectivity293T-derived stocks of YFP-encoding MLV produced by cotransfec-tion with a control vector or with 1 ␮gor3␮g of pCEM15:HA wereused in challenges of Mus dunni fibroblasts across a range of inputinocula (normalized units of RT). Similar data were obtained in paral-Figure 1. DNA Deamination by CEM15/APOBEC3Glel challenges of N-3T3 cells (data not shown).(A) Deamination of dC within a single-stranded oligonucleotide bypurified His-tagged CEM15/APOBEC3G (as well as by recombinantAPOBEC1 control) was monitored by subsequent treatment of thebeen infected but with the GFP gene having been ren-oligonucleotide with uracil-DNA glycosylase (UDG)/NaOH whichdered nonfunctional by CEM15/APOBEC3G-inducedbreaks the oligonucleotide at the site of dC→dU deamination (illus-mutations. Target cells that were negative (-lo) for GFPtrated to the left of the gel image).expression (as well as their GFP-hi counterparts) were(B) Western blot of E.coli extracts expressing recombinant CEM15/therefore isolated by flow cytometry and the retroviralAPOBEC3G (upper band, lane 1) or a control plasmid (lane 2).DNA present in cell lysates amplified by PCR, cloned,and sequenced.A comparison of the GFP gene sequences recoveredfluorescent protein (YFP) were produced in 293T cellsfrom


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Berkeley MCELLBI 110 - DNA Deamination Mediates Innate Immunity to Retroviral Infection

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