Human Apolipoprotein B mRNA-editing Enzyme-catalytic Polypeptide-like 3G (APOBEC3G)Is Incorporated into HIV-1Virions through Interactions with Viral and Nonviral RNAs*

Received for publication,May 24,2004

Published,JBC Papers in Press,June 20,2004,DOI 10.1074/jbc.M405761200

Evguenia S.Svarovskaia?,Hongzhan Xu?,Jean L.Mbisa?,Rebekah Barr?,Robert J.Gorelick§,Akira Ono?,Eric O.Freed?,Wei-Shau Hu?,and Vinay K.Pathak??

From the ?HIV Drug Resistance Program and §AIDS Vaccine Program,Science Applications International Corporation-Frederick,Inc.,NCI-Frederick,National Institutes of Health,Frederick,Maryland 21702

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G)is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection.APOBEC3G deami-nates deoxycytidines in minus strand DNA to deoxyuri-dines,resulting in G to A hypermutation and viral inac-tivation.Human immunodeficiency virus type 1(HIV-1)virion infectivity factor counteracts the antiviral activ-ity of APOBEC3G by inducing its proteosomal degrada-tion and preventing virion incorporation.To elucidate the mechanism of viral suppression by APOBEC3G,we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1deletion mutants.Virus-like particles derived from constructs in which pol ,env ,and most of gag were deleted still contained high levels of cytidine deaminase activity;in addition,coimmunoprecipitation of APOBEC3G and HIV-1Gag in the presence and ab-sence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex.Viral particles lacking HIV-1genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30–40%of the wild-type level,indicating that interactions with vi-ral RNA are not necessary for incorporation.In addi-tion,viral particles produced from an nucleocapsid zinc finger mutant contained ?1%of the viral genomic RNA but ?30%of the cytidine deaminase activity.The reduc-tion in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions.These results indicate that interactions with viral proteins or viral genomic RNA are not essen-tial for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

Retroviral genomes,including that of HIV-1,1undergo mas-sive G to A substitutions in one or a few rounds of replication (1,

2).Because this high rate of G to A substitution (?2mutations/100nucleotides)is 1,000-fold greater than the rate of substi-tutions that arise during normal error-prone viral replication (?2?10?5mutations/nucleotides/replication),it is named hy-permutation (1).Recent studies indicate that the HIV-1Vif protein protects viral replication from a host restriction factor that induces hypermutation of the HIV-1genome (3–6).Cer-tain “restricted”cell lines and primary T cells suppress repli-cation of HIV-1genomes that do not express Vif (?Vif virions),whereas other “permissive”cell lines allow replication of the ?Vif virions (7).Human apolipoprotein B mRNA-editing en-zyme-catalytic polypeptide-like 3G (APOBEC3G),first identi-fied as CEM15(8),is a dominant acting host factor that is responsible for the restricted cell phenotype (3–6,9).APOBEC3G is a member of a family of cytidine deaminases that play a central role in generating somatic hypermutation and mRNA editing (10).Recent studies have shown that APOBEC3G expression in viral producer cells causes deamina-tion of deoxycytidines in minus strand viral DNA to deoxyuri-dines,thereby resulting in G to A hypermutation (3–6,9).APOBEC3G must be packaged into virions to exhibit antivi-ral activity because its expression in target cells does not result in hypermutation or inhibition of viral replication.HIV-1Vif overcomes the antiviral activity of APOBEC3G by inducing its rapid degradation through the proteosomal pathway,thereby preventing its incorporation into virion (11–15).It has also been reported that HIV-1Vif reduces the efficiency of transla-tion of APOBEC3G mRNA (16).We and others have shown recently that a single amino acid substitution in human APOBEC3G confers resistance to HIV-1Vif (13,17–19).

The mechanism by which APOBEC3G is incorporated into HIV-1virions as well as other diverse retroviruses such as simian immunodeficiency virus,murine leukemia virus,and equine infectious anemia virus is unknown (4,6,20).The proteins and RNA components of these viruses share few com-mon determinants that could be exploited by APOBEC3G to gain entry into viral particles.Furthermore,the high evolu-tionary potential of HIV-1and other retroviruses should allow them to modify potential binding determinants quickly to pre-vent APOBEC3G incorporation.Instead,retroviruses have evolved the elaborate mechanism of encoding Vif to induce the proteosomal degradation of APOBEC3G.On the other hand,some protein determinants,such as the Y X DD catalytic do-main of reverse transcriptases or the zinc finger motifs of NC

*This work was supported in part with Federal funds from the NCI,National Institutes of Health Contract NO1-CO-12400with Science Applications International Corporation-Frederick,Inc.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.?To whom correspondance should be addressed:HIV Drug Resist-ance Program,Center for Cancer Research,NCI-Frederick,P.O.Box B,Bldg.535,Rm.334,Frederick,MD 21702.Fax:301-846-6013;E-mail:vpathak@http://www.doczj.com/doc/2b23ce8d453610661ed9f4a0.html.1

The abbreviations used are:HIV-1,human immunodeficiency virus type 1;APOBEC3G,apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G;CA,capsid;EGFP,enhanced green fluorescent

protein;eIF3k,cytoplasmic translational initiation factor 3subunit k;MA,matrix;NC,nucleocapsid;PBMC,peripheral blood mononuclear cells;PBS,phosphate-buffered saline;?,HIV-1packaging signal;Vif,virion infectivity factor;VLP,virus-like particle;Gag,group antigen;HDV,HIV-1-derived vector.

T HE J OURNAL

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B IOLOGICAL

C HEMISTRY

Vol.279,No.34,Issue of August 20,pp.35822–35828,2004

Printed in U.S.A.

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