BasicResiduesintheNucleocapsidDomainofGagAre ... · order to facilitate efficient assembly of HIV-1 capsids. During virion formation, 5000 HIV-15 Gag polypeptides assemble into a

Basic Residues in the Nucleocapsid Domain of Gag AreRequired for Interaction of HIV-1 Gag with ABCE1 (HP68), aCellular Protein Important for HIV-1 Capsid Assembly*

Received for publication, July 5, 2005, and in revised form, October 6, 2005 Published, JBC Papers in Press, November 7, 2005, DOI 10.1074/jbc.M507255200

Jaisri R. Lingappa1, Julia E. Dooher2, Michael A. Newman3, Patti K. Kiser3, and Kevin C. Klein4

From the Department of Pathobiology and the Department of Medicine, University of Washington, Seattle, Washington 98195

Duringhuman immunodeficiency virus, type 1 (HIV-1) assembly,Gag polypeptides multimerize into immature HIV-1 capsids. Thecellular ATP-binding protein ABCE1 (also called HP68 or RNase Linhibitor) appears to be critical for proper assembly of the HIV-1capsid. In primate cells, ABCE1 associates with Gag polypeptidespresent in immature capsid assembly intermediates. Here we dem-onstrate that the NC domain of Gag is critical for interaction withendogenous primate ABCE1, whereas other domains in Gag can bedeletedwithout eliminating the association ofGagwithABCE1.NCcontains two Cys-His boxes that form zinc finger motifs and areresponsible for encapsidation of HIV-1 genomic RNA. In addition,NC contains basic residues known to play a critical role in nonspe-cific RNA binding, Gag-Gag interactions, and particle formation.We demonstrate that basic residues in NC are needed for the Gag-ABCE1 interaction, whereas the cysteine and histidine residues inthe zinc fingers are dispensable. Constructs that fail to interact withprimate ABCE1 or interact poorly also fail to form capsids and arearrested at an early point in the immature capsid assembly pathway.Whereas others have shown that basic residues in NC bind nonspe-cifically to RNA, which in turn scaffolds or nucleates assembly, ourdata demonstrate that the same basic residues in NC act eitherdirectly or indirectly to recruit a cellular protein that also promotescapsid formation. Thus, in cells, basic residues in NC appear to actby two mechanisms, recruiting both RNA and a cellular ATPase inorder to facilitate efficient assembly of HIV-1 capsids.

During virion formation, �5000 HIV-15 Gag polypeptides assembleinto a spherical immature capsid at the cytosolic face of the plasmamembrane. Multimerization of Gag is coordinated with encapsidationof genomic RNA. Additionally, other viral and cellular proteins areincorporated into virions during capsid formation. Evidence suggeststhat in cells, capsid assembly occurs via an ATP-dependent, stepwisepathway of discrete assembly intermediates (1–4). Furthermore, HIV-1capsid assembly appears to require a host protein of 68 kDa (1, 4),referred to in previous studies as HP68 or RNase L inhibitor (1, 4–17).Recent bioinformatic analysis indicates that ABCE1 is the most appro-

priate name for this protein, which is the sole member of the ATP-binding cassette protein family E and is present in many species that donot encode RNase L (16). In this study, the term ABCE1 will be usedinstead ofHP68. ABCE1 is highly conserved and ubiquitously present ineukaryotes, including yeast, as well as archaebacteria. In a variety ofeukaryotic species, ABCE1 is critical for ribosome biogenesis (5–9). Asis the case with ribosomes, capsids are large multiprotein complexesthat contain RNA and are capable of self-assembly in vitro but probablyassemble in a regulated fashion within cells (18–21). Analogous to ribo-somes, some viral capsids may utilize ABCE1 to chaperone and coordi-nate their assembly.Depletion-reconstitution studies in a cell-free system and dominant

negativemutant studies in cells indicate that ABCE1 plays an importantrole in assembly of immature HIV-1 capsids (4). In addition, co-immu-noprecipitation with antibody to endogenous ABCE1 demonstratesthat ABCE1 is associated with Gag polypeptides present in assemblyintermediates isolated from primate cells expressing HIV-1 or otherprimate lentiviruses (1, 4). ABCE1 releases from Gag upon completionof immature capsid assembly and is therefore not present in significantamounts in HIV-1 particles released from cells (1, 4). Analysis of arecently reported crystal structure of ABCE1 suggests that the twonucleotide binding domains in ABCE1 act with a hinge region toundergo a clamp-like motion, with an estimated combined movementof �40° during the ATP-driven power stroke (15). Together these find-ings suggest a model in which ABCE1 binds post-translationally toHIV-1 Gag in capsid assembly intermediates and promotes ATP-de-pendent conformational changes important for assembly, thereby func-tioning as a chaperone during HIV-1 capsid formation. The exact man-ner in which ABCE1 acts during assembly has yet to be determined butcould include conformational changes associated either with formationof the capsid shell, packaging RNA into the capsid, or targeting assem-bling Gag to membranes.The 55-kDa HIV-1 Gag polypeptide is composed of four major

domains (fromN to C terminus): matrix (MA), capsid (CA), nucleocap-sid (NC), and p6. Initial analysis of the Gag-ABCE1 association sug-gested that NC is required for interaction of Gag with ABCE1, whereasp6 is dispensable (1, 4). These data are consistent with previous studiesdemonstrating that NC contains determinants (previously termed the Idomain) required for Gag polypeptide multimerization, whereas p6 isnot needed for capsid assembly but is required for budding (reviewed inRef. 22). NC contains two Cys-His boxes that form zinc fingers, arehighly conserved among retroviruses, and are known to be importantfor specific incorporation of HIV genomic RNA (reviewed in Ref. 23). Inaddition, NC is highly basic; 15 of its 55 amino acids are arginines andlysines dispersed throughout the domain. These basic residues havebeen shown to bind RNA nonspecifically (24, 25). Evidence suggeststhat RNA binding by these basic residues promotes Gag multimeriza-tion, with RNA acting as a nucleator or scaffold (24, 26–28). However,

* This work was supported by National Institutes of Health (NIH) Grant R01 AI048389 (toJ. R. L.). The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Pathobiology, Box 357238,University of Washington, 1959 NE Pacific St., Seattle, WA 98195. Tel.: 206-616-9305;Fax: 206-543-3873; E-mail: [email protected].

2 Recipient of a Magnuson Fellowship from the University of Washington.3 Recipient of NIH Grant T32 AI007509-08.4 Recipient of NIH Grant T32 CA09229.5 The abbreviations used are: HIV, human immunodeficiency virus; MA, matrix; CA, cap-

sid; NC, nucleocapsid; WT, wild type; RT, reverse transcriptase; GST, glutathioneS-transferase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 7, pp. 3773–3784, February 17, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

FEBRUARY 17, 2006 • VOLUME 281 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 3773

by guest on February 26, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

these basic residues could also act by other mechanisms to promotecapsid assembly.Here we demonstrate that the basic residues withinNC are necessary

to recruit endogenous ABCE1 into Gag-containing assembly interme-diates. Furthermore, we find that the cysteine and histidine residues inNC are not required for the Gag-ABCE1 interaction and that largeregions of theMAandCAdomains ofGag are also dispensable. Velocitysedimentation analyses demonstrate that two NC mutants that fail toform fully assembled immature capsids (including a mutant that fails tobind ABCE1 and one that binds ABCE1 poorly) are arrested at earlypoints in the assembly pathway in cells. Together, our findings revealthat the basic residues in NC that bind nonspecifically to RNA also areimportant for the interaction of Gag with the cellular protein ABCE1,which facilitates HIV-1 capsid formation. Thus, NC appears to act bytwo mechanisms to promote efficient HIV-1 capsid assembly in thecomplex environment of the cytoplasm.

EXPERIMENTAL PROCEDURES

Plasmids—For mammalian cell transfection plasmids, Gag muta-tions were engineered into the pSVGagRRE-R construct, which wasobtained from David Rekosh (29) and encodes Gag and the Revresponse element from the BH10 strain of HIV-1. Truncations wereengineered by introduction of two stop codons after the amino acid inGag indicated in the construct name, using site-directed mutagenesis(Stratagene). To make other constructs, a SacI site was engineered intothe parental construct immediately downstream from the Gag codingregion by site-directed mutagenesis. Gag mutations were engineeredusing standard PCRprocedures and inserted into the SacI sites on eitherside of the Gag coding region in the modified pSVGagRRE-R plasmid.TheKR10A construct was engineered in an analogousmanner by fusingthe NC-p6 domains from a template plasmid encoding the KR10Amutations (M1–2/BR (24)), obtained from Jeremy Luban, to theMA-CA domains in pSVGagRRE-R using standard PCR procedures.Plasmids encodingWTGag, Tr361, and Tr437 for in vitro transcrip-

tion have been described previously (1–3). Plasmids encoding CH1A,CH2A, and CH1/2A for in vitro transcription were engineered usingstandard PCR procedures into the previously described pSPGag plas-mid (WT Gag), which encodes SF2 Gag downstream from the SP6promoter and a Xenopus 5�-untranslated region (1–3, 30, 31). The plas-mid encoding KR10A for in vitro transcription was generated by ampli-fying theM1–2/BRGagmutant coding region described above (24) andinserting it into theNcoI and EcoRI sites of pSPGag using standard PCRmethods. Because M1–2/BR was generated from the HXB2 and BH10strains of HIV-1 (see Ref. 24 for details), a matched HXB2/BH10 pSPWT Gag plasmid was also generated and found to behave the same asthe SF2 Gag plasmid. The FLAG-tagged ABCE1 construct has beendescribed previously (1). The plasmid pcDNA-APO3G, which encodeswild-type human Apobec3G with a myc-HisA tag at the C terminus,was generated by Drs. Klaus Strebel and Sandra Kao (54) and obtainedfrom the NIH AIDS Research and Reference Reagent Program.To create the GST-ABCE1 plasmid, PCR was used to amplify the

previously described ABCE1 coding region (4). This coding region wasinserted into the EcoRI and BamHI sites of the pGEX4T-1 vector(Amersham Biosciences). Coding regions of all plasmids generated inthis study were confirmed by sequencing.

Transfections and Immunoprecipitations—COS-1 cells (from Afri-can green monkey) or human 293T cells were transfected in 60-mmdishes with 4 �l of pSVGagRRE-R encodingWT ormutant Gag and 0.5�l of pCMVRev (32, 33) using 24 �l of Lipofectamine (Invitrogen) or 15�l of Lipo 2000 (Invitrogen). Cells were harvested 45 h post-transfection

in 250 �l of Nonidet P-40 buffer (containing 0.625% Nonidet P-40, 10mM Tris acetate, pH 7.4, 50 mM potassium acetate, and 100 mM NaCl),supplemented with 10 mM EDTA and protease inhibitor mixture formammalian cells (Sigma). Cell lysates were clarified by centrifugation at1000 rpm for 10 min in a GH 3.8 rotor using an Allegra 6R centrifuge(Beckman Coulter) and centrifugation in a microcentrifuge at 18,000 �g for 30 s. Clarified lysates were divided equally and subjected to immu-noprecipitation using affinity-purified�-ABCE1, rabbit IgG, or affinity-purified �-Apobec3G, as noted, coupled to protein A immobilized onTris-acryl beads (Pierce) as described previously (1, 4). The antibody toApobec3G (34) is directed against the C-terminal 29 amino acids inApobec3G and was affinity-purified against the peptide immunogenand coupled to Tris-acryl beads in parallel with �-ABCE1. For two con-structs (�CA and �MACA), twice as much cell lysate was input intoimmunoprecipitations to compensate for reduced expression. Immu-noprecipitates were analyzed by SDS-PAGE, transferred to nitrocellu-lose (MSI), and subjected to immunoblotting using a monoclonal anti-body to the capsid domain of Gag (Dako) as previously described (1).Aliquots representing 2% of the cell lysate input were analyzed in par-allel with immunoprecipitations on all blots.

RNase A Treatment—Clarified lysates of COS-1 or 293T cells, trans-fected and harvested as above, were treated with RNase A (Qiagen) atthe indicated concentrations for 10min at 37 °C. Lysates were subjectedto centrifugation at 18,000 � g for 30 s, and equivalent aliquots of thesupernatant were used to program immunoprecipitations.

Quantitative RT-PCR—RNAwas purified from cell lysates by adding20 �l of lysate to 200 �l of RNAqueous lysis buffer (Ambion). Aftermixing, 20�l of a control lysate (murine EL4 cells; see below)was spikedinto the reaction to serve as a control for RNA purification and reversetranscription efficiency. RNA was then isolated per the manufacturer’sprotocol. Eluates were then treated with rDNase 1 (Ambion) and sub-sequently subjected to reverse transcription using random DNA prim-ers and Superscript II reverse transcriptase (�RT; Invitrogen). Quanti-tative PCR was performed using iQ SYBR green supermix (Bio-Rad).Serial dilutions of a correspondingDNA templatewere run in parallel. Astandard curve of CT versus �log[DNA] was calculated, in which the[DNA] of the highest dilution was arbitrarily set at 1, and sample valueswere extrapolated from the standard curves. Reactions minus RT(�RT) were processed in parallel, and �RT values were subtractedfrom the �RT values. Values are reported as 0 if the �RT value wasgreater than the �RT value. Sequences for the primers are 5�-gactatg-tagaccggttctat-3� (forward) and 5�-caaaactcttgccttatggccgggtcctcc-3�(reverse) for HIV-1 (Gag), 5�-cacggctgcttccagc-3� (forward) and5�-ggaaggctggaagagt-3� (reverse) for human actin, and 5�-cactgccgcatc-ctct-3� (forward) and 5�-ggaaggctggccaaga-3� (reverse) for murineactin. For EL4 cell lysate, EL4 cells were lysed in lysis buffer (RNaqueous;Ambion) at a concentration of 5 � 104 cells/�l.

Cell-free Assembly Reactions—In vitro transcription and cell-freetranslation usingwheat germ extract andTran35S-label (ICNBiochemi-cals) were performed as described previously (1, 2). Cell-free reactionswere programmed using amixture of 40% FLAG-ABCE1 transcript and60% WT or mutant Gag transcripts, as described previously (1). Cell-free translations were diluted 300-fold in Nonidet P-40 buffer and sub-jected to immunoprecipitation with FLAG antibody coupled to beads(Sigma) or mouse IgG (Sigma) with protein G beads (Pierce), asdescribed previously (1). Immunoprecipitations were analyzed by SDS-PAGE and autoradiography in parallel with aliquots of total cell-freereaction representing 5% of input.

GST-ABCE1 Pull-down Assays—Competent BL21 Escherichia coli(Novagen) were transformed with GST-ABCE1. E. coli were grown to

Basic Residues in NC Are Critical for Gag-ABCE1 Association

3774 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 7 • FEBRUARY 17, 2006


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

FIGURE 1. The Gag-ABCE1 interaction is progressively reduced upon truncation of Gag in NC. A, diagram showing NC residues in BH10 Gag, with arrows indicating the last amino acidpresent in each truncation construct. Lines demarcate where the NC domain meets the spacer regions, SP1 and SP2. Basic residues in NC are shown in gray. B, Gag truncation constructs arediagrammed on the right with major domains labeled above and Cys-His boxes (CH1 and CH2) shown in gray. Lysates of COS-1 cells transfected with the indicated constructs were subjectedto immunoprecipitation with �-ABCE1 (immune, I) or nonimmune control antibody (N) and followed by immunoblotting with an antibody to Gag. Equivalent aliquots of total input (T) areshown to indicate migration and level of expression. Immunoprecipitations were performed under either native conditions (Native) or after denaturation (Denat). For each construct, all lanesshown were taken from a single exposure. Experiments in each panel were repeated three times, and representative data are shown. C, the reduction in ABCE1 interaction relative to wild-typeGag upon progressive truncation in NC was quantitated from three repeats of this experiment (statistical significance; *, p �0.05; **, p �0.01). Indicated below the bar graph for each constructare the number of lysines and arginines in NC (#KR) and the presence or absence of the first and second Cys-His boxes (CH1/CH2).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

FIGURE 2. Altering Lys and Arg residues but not Cys and His residues in NC eliminates the Gag-ABCE1 interaction. Lysates from COS-1 cells expressing the indicated mutantswere subjected to immunoprecipitation (IP) under native conditions with �-ABCE1 (immune, I) or nonimmune control antibody (N) and followed by immunoblotting with anantibody to Gag. Constructs examined include mutants containing Cys-His box deletions (A) and amino acid substitutions (B). Equivalent aliquots of total input (T) are shown toindicate migration and level of expression. All lanes were taken from a single exposure. Diagrams in A show amino acids in NC for each construct, with major domains labeled above,lysines and arginines in gray, cysteines and histidines outlined, and substituted amino acids indicated with black dots. Experiments in each panel were repeated three times, andrepresentative data are shown. C, the reduction in ABCE1 interaction relative to wild-type Gag for each construct was quantitated from three repeats of each experiment (statisticalsignificance; *, p � 0.05; **, p � 0.01). Indicated below the bar graph for each construct are the number of lysines and arginines in NC (#KR) and presence or absence of first and secondCys-His boxes (CH1/CH2).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

late log phase, induced with 0.5 mM isopropyl 1-thio-�-D-galactopy-ranoside (Sigma) for 2 h, centrifuged at 6000 rpm for 10 min in aSLA-1500 rotor (Sorvall), and resuspended in 8 ml of cold phos-phate-buffered saline. Phenylmethylsulfonyl fluoride was added to100 mM to the resuspended cells, and six pulses (30 s each) with asonicator were used to lyse the bacterial cells. Bacterial lysate wasclarified by centrifugation in an SS-34 rotor (Sorvall) at 12,000 rpmfor 10 min, and 1.5 ml of the resulting supernatant was incubatedwith 140 �l of packed glutathione-Sepharose beads (Amersham Bio-sciences) for 45 min at room temperature. Before incubating withcell lysate, GST-ABCE1-bound glutathione-Sepharose was washedthree times with 1 ml of phosphate-buffered saline plus 1% Sarcosyland then twice with 1 ml of phosphate-buffered saline alone. COS-1cell lysates were harvested in Nonidet P-40 buffer supplementedwith 4 mM magnesium acetate and incubated with the washed GST-ABCE1 glutathione-Sepharose columns for 1 h at 4 °C (300 �l oflysate/column). After incubation with COS-1 cell lysate, glutathi-one-Sepharose was washed five times with 1 ml of Nonidet P-40wash buffer (0.625% Nonidet P-40, 10 mM Tris acetate, 50 mM potas-sium acetate, 100 mM NaCl, adjusted to pH 8.5) and twice in 1 ml ofthe same buffer lacking detergent. Three 1-ml elutions were carriedout using buffer containing 10 mM Tris acetate, pH 7.4, 50 mM potas-sium acetate, 100 mM NaCl, and 40 mM glutathione.

Velocity Sedimentation—Calibration of gradients with markers andfor calculation of approximate S values has been described previously(2). For analysis of immature released capsids, medium was collected45 h post-transfection and passed through a 0.22-�m filter. NonidetP-40 was added to a final concentration of 1% to the filtered media, and100 �l was layered onto discontinuous step gradients containing 500 �leach of 20, 40, 50, and 75% sucrose inNonidet P-40 buffer supplementedwith 4 mM MgAc and subjected to velocity sedimentation in a TLS55rotor at 45,000 rpm for 45 min at 4 °C (Beckman Coulter). Fractions(200�l) were serially collected from the top of the gradient, precipitatedwith trichloroacetic acid, and analyzed by immunoblotting as describedabove.For analysis of cellular complexes, 100 �l of cell lysates were layered

onto step gradients containing either 500 �l each of 20, 40, 50, and 75%sucrose (for separation of 10/80 S complexes from 500/750 S com-plexes) or 400�l each of 5, 10, 15, 30, and 40% sucrose (for separation of10 S complexes from 80 S complexes) in Nonidet P-40 buffer withoutMgAc. Gradients were subjected to velocity sedimentation under thesame conditions as above. Fractions (150 �l) were collected seriallyfrom the top of the gradient, and equivalent amounts of gradient frac-tions were analyzed by immunoblotting.

Quantitation—Quantitation was performed by digitizing immunob-lots using an Agfa Duoscan T1200 scanner and Photoshop 5.5 software(Adobe Systems Inc.). Mean band densities were determined andadjusted for band size and background. Dilution standards were per-formed to ensure that immunoblots were in the linear range for semi-quantitative analysis.

RESULTS

Progressive Truncation in NC Leads to a Corresponding Reduction inthe Gag-ABCE1 Interaction—Previously, we demonstrated that an anti-serum directed against endogenous primate ABCE1 (�-ABCE1) co-im-munoprecipitates wild-typeHIV-1Gag fromprimate cells infectedwithHIV-1 or transfected with an HIV-1 proviral construct. Abundant cel-lular proteins such as tubulin and actin are not co-immunoprecipitatedby �-ABCE1 (4). Since earlier findings suggested that the NC domain ofGag is important for the Gag-ABCE1 interaction in mammalian cells

(4), we wanted to identify the features in NC that are required for theGag-ABCE1 interaction. Therefore, we engineered HIV-1 BH10 Gagmutants that contained progressive truncations in NC (Fig. 1A) intopreviously described transfection constructs that allow expression ofthe HIV-1 Gag precursor in the absence of other HIV-1 gene productsbesides Rev (33). In COS-1 cells, transfection with these constructsresults in production and release of virion-like particles containingimmatureHIV-1 capsids (1, 2, 4, 33).Whereasmost truncationmutantsexpressed at levels comparable with wild-type Gag in COS-1 cells,mutants that were truncated within Cys-His boxes expressed poorly inCOS-1 cells and were not examined further (data not shown).Truncation constructs were expressed in COS-1 cells, and lysates

were subjected to immunoprecipitation with �-ABCE1 under nativeconditions. Co-immunoprecipitation of Gag was assessed by immuno-blotting (Fig. 1B, left panels, Native). The two longest truncation con-structs (Tr437 and Tr427) associated with ABCE1 as well as wild-typeGag. Four constructs of intermediate length (Tr412, Tr410, Tr409, andTr405) interacted with ABCE1 to a moderate degree but not as well aswild-type Gag. Finally, the two constructs truncated most proximally(Tr388 and Tr361) failed to associate with ABCE1 (Fig. 1B, left panels,Native), even when long exposures were examined (data not shown).Quantitation revealed that these differences in ABCE1 interaction weresignificant (Fig. 1C). When lysates were denatured to disrupt protein-protein interactions, �-ABCE1 immunoprecipitated endogenousABCE1 present in COS-1 cells in all cases (data not shown) but failed toco-immunoprecipitate WT Gag or any of the Gag truncation mutants(Fig. 1B, right panels,Denat). This control confirms that native interac-tions are required for association of HIV-1 Gag with ABCE1, consistentwith previous findings (1, 4). Together, these data demonstrate that lossof NC residues correlates with a reduction in association of Gag withABCE1.Analysis of features in these truncated constructs revealed that the

two constructs that interacted with ABCE1 as well as wild-type (Tr427and Tr437) contained both Cys-His boxes and 14 or 15 basic residues.The four constructs that interacted to an intermediate extent (Tr405,Tr409, Tr410, and Tr412) contained one Cys-His box and 7–11 basicresidues. Finally, the two constructs that failed to interact with ABCE1(Tr361 and Tr388) contained no Cys-His boxes and four or fewer basicresidues. The shortest truncation mutant from our series that associ-ated with ABCE1, Tr405, contained one Cys-His box and seven basicresidues in NC (see Fig. 1, A and C (bottom)).

Substitution of Lysines andArginines butNotCysteines andHistidinesin NC Eliminates ABCE1 Association—Since both the zinc fingers andthe basic residues in NCwere altered upon truncation of NC, themuta-tions described above failed to distinguish whether only one of thesefeatures was critical for the Gag-ABCE1 interaction. Therefore, weengineered additional mutations to determine whether the Cys-Hisboxes or the dispersed basic residues in NC govern association withABCE1. First we assessed the effect of deleting the Cys-His boxes. Gagmutants containing deletions of either the first Cys-His box or the sec-ond Cys-His box (Gag�CH1 versusGag�CH2) associated with ABCE1,as indicated by immunoprecipitation of COS-1 lysates with �-ABCE1(Fig. 2A). Deletion of both Cys-His boxes (Gag�CH1/2) reduced theGag-ABCE1 interaction to very low but detectable levels (Fig. 2A).Each Cys-His box in NC contains not only the cysteine and histidine

residues that are critical for zinc finger formation but also a few basicresidues. Thus, our deletion of both Cys-His boxes (Gag�CH1/2)resulted in the loss of five of the 15 lysines and arginines present in NC(see diagrams in Fig. 2A). Consequently, results obtained using theGag�CH1/2 construct did not allow us to precisely define the contri-




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

bution of the Cys-His boxes versus the basic residues. Therefore, weengineered point mutants that would allow us to completely dissociatethe contribution of cysteines and histidines from that of lysines andarginines inNC. In constructs CH1A andCH2A, all of the cysteines andhistidines in either the first or second Cys-His box were replaced withalanines, whereas CH1/2A contained the same substitutions in bothCys-His boxes. In all of these constructs, the lysines and arginines pres-ent in NC were unaltered. As shown in Fig. 2B, when expressed inCOS-1 cells, all three constructs were co-immunoprecipitated by�-ABCE1, indicating that the Gag-ABCE1 interaction was maintainedeven upon substitution of all the cysteines and histidines in NC. Co-immunoprecipitation of CH1A and CH2A by �-ABCE1 was similar to

wild-type Gag. In contrast, substitution of all of the cysteines and histi-dines inNC (CH1/2A) resulted in reduced associationwith endogenousABCE1, but the association was consistently detectable (Fig. 2C).Having established that substitution of cysteine and histidine resi-

dues critical for zinc finger formation did not eliminate the Gag-ABCE1interaction, we next addressed the contribution of basic charge substi-tutions. Previously, Cimarelli et al. (24) found that mutation of the 10arginine and lysine residues located in and between the Cys-His boxesresulted in loss of viral replication, virion production, and Gag-Gaginteractions. When we substituted the same 10 lysines and arginines inNC with alanines (KR10A), complete elimination of the Gag-ABCE1interaction was observed, despite expression levels similar to wild-type

FIGURE 3. The Gag-ABCE1 interaction is not eliminated upon substitution of four or fewer basic residues in NC and deletion of other domains in Gag. Cell lysates weresubjected to immunoprecipitation under native conditions with �-ABCE1 (immune, I) or nonimmune control antibody (N) and immunoblotted with an antibody to Gag. Constructsexamined include amino acid substitutions in NC (A) and deletions of regions of MA and CA (B), as diagrammed to the right, as well as constructs described previously (see Figs. 1 and2). Equivalent aliquots of total input (T) are shown to indicate migration and level of expression. All lanes were taken from a single exposure. Experiments in each panel were repeatedthree times, and representative data are shown. Diagrams in A show amino acids in NC for each construct, with lysines and arginines in gray, cysteines and histidines outlined,substituted amino acids indicated with black dots, and major domains labeled above. Diagrams in B show Cys-His boxes (CH1 and CH2) in gray and deleted regions with a dashed line.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Gag (Fig. 2B, T lanes). Even very long exposures of immunoblots failedto detect co-immunoprecipitation of KR10A by �-ABCE1. Theseresults were confirmed by quantitation (Fig. 2C). Together, these dataindicate that the association of HIV-1 Gag with ABCE1 is dependent onbasic residues in NC and does not require intact zinc fingers in NC.To evaluate the number of basic charge substitutions that are

required to abolish the interaction of Gag with ABCE1, we engineeredGag constructs containing substitutions of fewer than 10 basic residues.Because initial experiments revealed that alanine substitution of threeor four basic residues in the region between the two Cys-His boxes onlyhad small effects on the ABCE1 interaction (data not shown), we engi-neered a more extreme charge disruption by substituting 3 or 4 lysinesand arginines in this linker region of NCwith glutamic acids (KR3E andKR4E, respectively). Upon expression of these constructs in COS-1cells, association with endogenous ABCE1 was observed by co-immu-noprecipitation, albeit at reduced levels (Fig. 3A). These data suggestthat between 5 and 10 basic residues in NC need to be mutated (leaving5–10 basic residues intact) to completely eliminate association of full-length Gag with endogenous ABCE1. The results are similar to findingsobtained using truncationmutants in Fig. 1, in which constructs encod-ing seven or more basic residues were found to associate with ABCE1.Since the exact number of residues required for loss of the Gag-ABCE1interaction is likely to vary with the exact position of the mutated resi-dues within NC, the choice of substituting amino acid, the length of theGag construct used, and the exact coding sequence of theGag strain, wedid not attempt finer resolution mapping of basic residues.

Deletion of Large Regions of MA and CA Does Not Eliminate theGag-ABCE1 Interaction—Others have reported that deletions of largeregions of MA and CA do not have significant effects on infectivity, aslong as themyristoylation signal and associated charged residues inMAare left intact (35, 36). Consistent with this, we found that deletion of alarge region of CA (residues 143–276 in Gag; �CA) or deletion of mostof MA and CA (residues 12–282 in Gag; �MACA) reduced but did noteliminate the ability of Gag to associate with ABCE1 (Fig. 3B). In addi-

tion, deletion of the major homology region (residues 285–304 in Gag;�MHR) within CA had little effect on the Gag-ABCE1 interaction (Fig.3B). Together, these findings indicate that most of MA and CA are notessential for the Gag-ABCE1 interaction. However, the reduction inassociation observed with the large deletions raises the possibility thatthese domains may contain residues that modulate the Gag-ABCE1interaction.

Confirmation of the Role of Basic Charge in NC for Gag-ABCE1 Inter-action Using Other Approaches—The interaction of ABCE1 with Gagwas initially identified by biochemical dissection of a cell-free systemthat supports de novo assembly of capsids closely resembling immatureHIV-1 capsids produced in cells (1, 2, 4, 37). Immunodepletion-recon-stitution experiments in this system were also used to demonstrate thecritical role of ABCE1 in post-translational events in HIV-1 capsid for-mation (4). To validate findings obtained using �-ABCE1 in COS-1cells, we examined the co-immunoprecipitation of HIV-1 Gag mutantsin this cell-free HIV-1 capsid assembly system. FLAG-tagged humanABCE1 was co-translated either with HIV-1 wild-type Gag or withselected Gag mutants in parallel reactions and then subjected to immu-noprecipitation under native conditions with antibody to FLAG(�-FLAG). Consistent with our previous observations, �-FLAG co-im-munoprecipitated wild-type Gag and Tr437 but not Tr361 (Fig. 4A). Inaddition, �-FLAG co-immunoprecipitated CH1A, CH2A, and CH1/2Ato varying extents (Fig. 4A) but did not immunoprecipitate the KR10Amutant (Fig. 4B). Thus, these NCmutations have comparable effects onthe Gag-ABCE1 interaction when assessed in different cellular contexts(primate cells versus a cell-free system programmed with wheat germextract) or using different antibodies (�-ABCE1 to detect endogenousABCE1 versus �-FLAG to detect epitope-tagged ABCE1).We also examined the interaction of WT and mutant Gag with a

recombinant GST-ABCE1 fusion protein (encoding human ABCE1)produced in E. coli using a pull-down assay. Lysates of COS-1 cellsexpressing either wild-type or mutant Gag constructs were incubatedwith GST-ABCE1 purified from E. coli and bound to glutathione beads.

FIGURE 4. Other approaches validate the finding that basic charge in NC is critical for the Gag-ABCE1 interaction. A and B, the indicated Gag constructs and FLAG-taggedhuman ABCE1 (f-ABCE1) were co-translated in a cell-free translation and assembly system containing [35S]methionine. Reactions were subjected to immunoprecipitation with�-FLAG (immune, I) or nonimmune control antibody (N). Migrations of WT Gag and Gag mutants are indicated to the left and the right. Equivalent aliquots of total input (T) are shownto indicate migration and level of expression. Lower bands represent radiolabeled polypeptides that terminated early or initiated late. Asterisks indicate the predicted migration ofeach Gag construct in the immune lane. All lanes were taken from a single exposure. C, recombinant GST-ABCE1 fusion protein from E. coli was bound to glutathione-agarose columns.Lysates of COS-1 cells expressing WT Gag, KR10A, CH2A, or CH1/2A were applied to GST-ABCE1 columns in parallel. Columns were washed, and three elutions (E1, E2, and E3) wereperformed using buffer containing glutathione. Equivalent amounts of the last wash (W), E1, E2, and E3 were analyzed for the presence of GST-ABCE1 bound to WT or mutant Gagfrom the cell lysate by immunoblotting (WB) with �-ABCE1 (top panels) and antibody to Gag (bottom panels). The first lane of each panel (T) shows an aliquot of total input cell lysateto indicate migration and level of expression. Note that the cell lysate does not contain GST-ABCE1, which is derived only from the column. Experiments in each panel were repeatedthree times, and representative data are shown.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

GST-ABCE1 and proteins bound to GST-ABCE1 were eluted from thebeads using glutathione, and the presence of Gag and ABCE1 in eluateswas assessed by immunoblotting. Following incubation of lysates con-taining WT Gag, final washes contained no Gag or ABCE1, whereasglutathione eluted both GST-ABCE1 and Gag, indicating that WT Gagwas bound to GST-ABCE1 (Fig. 4C). In contrast, after incubation withlysates expressing the negative control Tr361 (Gag truncated proximalto NC; see Fig. 1A), recombinant GST-ABCE1 was detected in eluates,but Tr361 was not (data not shown). KR10A expressed in COS-1 celllysates also failed to bind to GST-ABCE1, whereas CH1A, CH2A, andCH1/2A from cell lysates bound to intermediate levels (Fig. 4C). Thus,comparable results were obtainedwhen constructs that separately abol-ish zinc finger motifs versus basic residues in NC were examined bythree different approaches, including co-immunoprecipitation ofendogenous ABCE1 in COS-1 cells, co-immunoprecipitation ofepitope-tagged human ABCE1 expressed in a cell-free assembly system

and pull-down from COS-1 cells with recombinant GST-ABCE1.Therefore, we conclude that the basic residues in theNCdomain of Gagare critical for association of Gag with ABCE1.

The Interaction between Gag and ABCE1 Is Resistant to RNase ATreatment—Many studies have shown that the basic residues in theNCdomain of Gag are responsible for binding to nonspecific RNA(reviewed in Ref. 23). Association of Gag with other proteins, such asApobec3G, via an RNA bridge has been demonstrated (e.g. see Refs. 38and 39). Because ABCE1 may have RNA binding elements (6, 15), it ispossible that the charged residues in NC allow Gag to associate withABCE1 by an RNA bridge.To address this, we examined the effect of RNase A on the Gag-

ABCE1 interaction, using the Gag-Apobec3G interaction as a controlfor RNase sensitivity. Cellular lysates expressing the nearly completeHIV genome (HIV-1�env) and Apobec3G were treated in parallel withdifferent concentrations of RNase A and then subjected to immunopre-

FIGURE 5. The ABCE1-Gag interaction is insensitive to concentrations of RNase A that degrade HIV-1 RNA and cellular RNA. 293T cells were transfected to express eithergenomic HIV-1 and Apobec3G constructs or Gag constructs alone, as indicated. A, lysates were harvested, and equivalent aliquots were treated with RNase A (final concentrations of0 –1000 �g/ml, as indicated). Treated lysates were subjected to immunoprecipitation with either �-ABCE1 (I), nonimmune control antibody (N), or �-Apobec3G (A), as noted.Immunoblotting was performed using an antibody to Gag. Equivalent aliquots of total input (T) are shown to indicate migration of cleaved or uncleaved Gag bands and level ofexpression. All lanes for each construct were taken from a single exposure. The experiment was repeated three times, and representative data are shown. B, real time quantitativeRT-PCR was used to determine the relative amounts of HIV-1 (Gag) and human (Hu) actin RNA for selected lysates shown in A. An aliquot of untreated murine lysate was added to eachtransfected cell lysate after RNase treatment, and the amount of murine (Mu) actin was also determined as a control for RNA isolation and RT efficiency.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

cipitation using antibody to endogenous ABCE1, a nonimmune controlantiserum, or antibody to Apobec3G (Fig. 5, lanes I, N, and A, respec-tively). Immunoblotting was used to assess coimmunoprecipitation ofGag by these antibodies. The association of Gag with Apobec3G waslost upon treatmentwith increasing concentrations of RNaseA (Fig. 5A,top two panels, A lanes), consistent with previous reports (e.g. see Refs.38 and 39). In contrast, treatment with RNase A had no effect on theassociation of Gag with ABCE1 (Fig. 5A, top two panels, I lanes). Similarresults were obtained in the presence or absence of Vif (HIV-1�env�vif;Fig. 5A, compare top two panels), which is known to neutralize theactivity of Apobec3G (40).We also transfected cells to expresswild-typeGag alone, the CH1/2�A Gag mutant, or the �CH1/2 Gag mutant.These two mutants were chosen, since both bind to ABCE1 at some-what reduced levels (see Fig. 2). Even in the absence of other HIV-1proteins, the Gag-ABCE1 interaction was resistant to RNase A (Fig. 5A,bottom panels). These data reveal that the Gag-ABCE1 interaction isresistant to RNase A treatment when compared with the RNase A-sen-sitive Gag-Apobec interaction.To further confirm that RNase A was effective in these experiments,

we subjected an aliquot of selected lysates used for immunoprecipita-tion in Fig. 5A to reverse transcription followed by quantitative PCR.HIV-1-specific RNAs and actin mRNA were virtually eliminated upontreatment with even 1�g/ml RNaseA (Fig. 5B), indicating that RNaseAwas indeed active. Murine actin, from untreated murine cell lysate thatwas mixed in after RNase treatment to serve as a positive control forRNA extraction and RT efficiency (see “Experimental Procedures”),remained relatively constant between all samples (Fig. 5B). Thus, theGag-ABCE1 interaction is maintained even when lysates are treatedwith concentrations of RNase in excess of that required to reduce cel-lular and HIV-1 RNA to undetectable levels.

Gag Mutants That Fail to Release Completed Capsids Also Fail toProgress through the Capsid Assembly Pathway—Studies by Cimarelli etal. (24) showed that Gagmutants encoding the KR10A substitutions donot support Gag-Gag interactions. Furthermore, they demonstratedthat cells expressing these mutants in the context of the complete HIVgenome do not release significant numbers of virions. The few virionsreleased have abnormal cores by electron microscopy (24). To deter-mine whether mutants that exhibited reduced ABCE1 binding wereable to release immature capsids, we examined the media from cellsexpressing wild-type andmutant constructs. Immunoblotting of lysatesfrom transfectedCOS-1 cells revealed that theNCmutants expressed tosimilar levels (Fig. 6A). Media from these cells were treated with deter-gent to remove the envelopes of released viral particles and then sub-jected to velocity sedimentation as previously described (1, 2, 4). Asexpected, wild-type immature capsids were detected in the fractionscorresponding to�750 S by immunoblotting with antibody toGag (Fig.6B). Since theHIV-1 proteasewas not expressed, only immature capsidswere released. Cells expressing the negative control construct Tr361 didnot release immature capsids. Cells expressing the KR10A constructalso failed to release immature capsids into the medium as judged byvelocity sedimentation, although KR10A contains an intact p6 domainrequired for budding. Both CH1A and CH2A released �750 S capsidsinto themedium. In contrast, CH1/2A did not release immature capsidsinto the medium (Fig. 6B).Immature capsid release by CH1A and CH2A is consistent with the

findings of others showing that mutations in only one Cys-His box haveminimal effects on particle release (41). In addition, capsid release bythese constructs fits with our observation that CH1A and CH2A inter-act well with ABCE1 (Fig. 2, B andC). Conversely, the absence of capsidrelease by CH1/2A and KR10A, which interacted with ABCE1 poorly or

not at all (Fig. 2, B andC), suggests defects in intracellular events duringimmature capsid formation. Previously, we have demonstrated thatwild-type Gag and assembly-competent Gag mutants progress throughthe entire assembly pathway, progressively forming the �10, �80,�150, and �500 S intracellular assembly intermediates before formingcompleted �750 S capsids (2). In contrast, assembly-defective Gagmutants are arrested at different points along the immature capsidassembly pathway with accumulation of assembly intermediates thatprecede the point of blockade (1–3, 30). To determine why KR10A andCH1/2A fail to produce virions, we examined intracellular capsidassembly intermediates formedbyKR10AandCH1/2. Lysates ofCOS-1cells expressing WT and mutant Gag constructs were analyzed usingvelocity sedimentation gradients in which early assembly intermediates(�10 and �80 S) migrate at the top of the gradient, and late (�500 and�750 S) assembly intermediates migrate at the bottom. Analysis of theassembly-competent WTGag and CH2A constructs revealed the pres-ence of both early and late assembly intermediates at steady state (Fig.7A, left panels). Similar results were obtained for CH1A (data notshown). In contrast, the assembly-defective KR10A and CH1/2A con-structs formed only early assembly intermediates and accumulated

FIGURE 6. Velocity sedimentation analysis reveals that capsids are not releasedfrom cells expressing the KR10A and CH1/2. A, COS-1 cell lysates of wild-type andindicated mutant Gag constructs were harvested, and equivalent aliquots were analyzedby immunoblotting to demonstrate level of expression of each construct in cells. B, at thesame time, medium from each plate was collected, treated with detergent to removeenvelopes from viral particles, and analyzed by velocity sedimentation. Immunoblottingwas performed on each fraction to detect Gag. Gradients are shown with fractions fromthe top of the gradient, representing smaller S values at the left. The position of �750 Simmature capsids is shown by a dark bar. The experiment was repeated twice, and rep-resentative data are shown.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

these in large amounts (Fig. 7A, left panels). These data indicate thatboth KR10A and CH1/2A fail to produce completed capsids becausethey are arrested at early points in the immature capsid assemblypathway.Since KR10A fails to interact with ABCE1, whereas CH1/2A inter-

acts with ABCE1 poorly, it is likely that they exhibit different defectsduring early events in immature capsid formation. We have previ-ously shown that the �10 S complex does not contain ABCE1,whereas the �80 S complex represents the first assembly intermedi-ate in which ABCE1 binds to Gag (1, 4). Moreover, the �80 S assem-bly intermediate is a critical one, since depletion of ATP causesarrest of Gag in the assembly pathway, with accumulation of the �80and �150 S assembly intermediates (2). Thus, formation of and/orexit from the �80/150 S assembly intermediates is likely to be rate-limiting and critical for assembly.To distinguish between early assembly intermediates, cell lysates

were analyzed by velocity sedimentation using a gradient that com-pletely separates the first two assembly intermediates (�10 S from

�80 S). WT Gag and CH2A, both of which are assembly-competent,contained clear peaks corresponding to both the �10 and �80 Sassembly intermediates at steady state (Fig. 7B). In contrast, theKR10A mutant formed only the �10 S complex. The failure ofKR10A to form the �80 S assembly intermediate (Fig. 7B) is consist-ent with its inability to interact with ABCE1 (Fig. 2, B and C).CH1/2A formed a small but reproducible peak in the �80 S region ofthe gradient (Fig. 7B), superimposed on a trail of the �10 S assemblyintermediate. The significantly reduced binding of CH1/2A toABCE1 (Fig. 2, B and C) with abnormal or inadequate formation ofthe �80 S assembly intermediate (in which Gag associates withABCE1) could explain the failure of CH1/2A to progress beyond the�80 S stage of the assembly pathway. Thus, these data suggest thatalthough the cysteines and histidines in the zinc fingers are notrequired for minimal binding of ABCE1, the presence of at least oneintact Cys-His box in NC may be important for reaching thresholdlevels of ABCE1 binding and consequently for proper ABCE1 func-tion in the intracellular capsid assembly pathway.

FIGURE 7. Velocity sedimentation analysisreveals that KR10A and CH1/2 are arrested inthe form of early assembly intermediates. A,COS-1 cell lysates of wild type and the indicatedmutant Gag constructs were analyzed by velocitysedimentation on gradients that separate earlyfrom late assembly intermediates. Fractions wereimmunoblotted, and the amount of Gag in eachlane was quantitated and graphed as percentageof total Gag. Positions of early (�10 to �80 S) andlate (�500 to �750 S) assembly intermediates areshown. B, the same lysates were analyzed as in Abut using a different velocity sedimentation gradi-ent that separates the �10 S assembly intermedi-ate from the �80 S assembly intermediate, as indi-cated. Positions of early �10 and �80 S assemblyintermediates are shown. Experiment wasrepeated twice, and representative data areshown.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

DISCUSSION

Studies presented here demonstrate that basic residues in the NCdomain of Gag are critical for the association of the cellular ATP-bind-ing protein ABCE1 (previously known asHP68 or RLI) withHIV-1Gag.Substitution of 10 of 15 lysines and arginines in NC completely abol-ished the interaction of Gag with endogenous ABCE1 present in COS-1cells. In contrast, substitution of all of the cysteines and histidines inNCdid not eliminate the Gag-ABCE1 interaction, indicating that the Gag-ABCE1 interaction can occur in the absence of intact zinc fingers. Largeregions of MA and CA are also not essential for the Gag-ABCE1 asso-ciation per se. Taken together with our previous finding that the p6domain of Gag is dispensable for this interaction (1, 4), these data indi-cate that basic residues in NC are important determinants of the Gag-ABCE1 association in primate cells. More detailed mapping revealedthat Gag constructs containing a minimum of 6–10 lysines and argin-ines in NC were able to interact with endogenous ABCE1 in primatecells, albeit at reduced levels.Whereas basic charge inNCappears to be critical for theGag-ABCE1

interaction, one caveat to note is that other motifs and regions of Gagcould modulate this interaction and could also be needed for ABCE1 tofunction properly during assembly. Notably, the data presented here donot address whether the N terminus of MA plays a role in Gag-ABCE1binding. Furthermore, the reduction in the Gag-ABCE1 interactionseen with deletions in MA and CA raise the possibility that residues inthese regions influence recruitment and function of ABCE1 during cap-sid formation. Similarly, substitution of as few as four basic residueswith glutamic acids (KR4E) and substitution of all the cysteines andhistidines (CH1/2A) resulted in significant reduction in ABCE1 bind-ing. In the case of CH1/2A, assembly is arrested at the �80 S assemblyintermediate, raising the possibility that reduced ABCE1 binding pre-vents ABCE1 from functioning properly during capsid assembly. Fur-ther investigation of this possibility will require a better understandingof how ABCE1 acts to promote capsid formation.NC from HIV-1 and other retroviruses is known to interact nonspe-

cifically with RNA as well as in a highly specific manner with genomicRNA (reviewed in Ref. 23). The nonspecific RNA interaction is impor-tant for promoting Gag multimerization and capsid formation andappears to be governed by basic charge in NC (24, 25, 42–45). In con-trast, packaging of the genome of HIV-1 and many other retrovirusesrequires intact zinc fingers within NC (46–51). These and other find-ings support a widely accepted model in which association of basicresidues with RNA promotes multimerization of the �5000 Gagpolypeptides that are needed to form a single immature capsid, whereastheCys-His boxes govern encapsidation of genomic RNA (24, 26, 27, 52,53). However, the finding that RNAplays a critical role in assembly doesnot exclude the possibility that other factors may also play an importantrole in promoting proper assembly. Recruitment of proteins that pro-mote capsid assembly could be important in cells, where the concentra-tion of Gag is typically quite low, barriers to efficient assembly are likelyto exist, and efficiency of virion production is critical for propagation.Although it remains to be determined whether primate ABCE1 has

RNA-binding properties, our data suggest that the Gag-ABCE1 inter-action is not dependent on an RNA bridge. We have previously foundthat the Gag-ABCE1 interaction is relatively resistant to 1 �g/ml RNaseA (4). Here we demonstrate that the interaction is resistant at to RNaseA at 1000 �g/ml, which greatly exceeds the concentration of RNase Arequired to fully degrade cellular and HIV-1 RNA and disrupt anotherknown RNase-sensitive interaction in the same extract. One explana-tion for these findings is that the Gag NC domain may be associatedwith ABCE1 largely through protein-protein interactions. An alternate

possibility is that RNA binding by NC may promote Gag-Gag interac-tions that in turn alter the conformation of Gag, thereby exposing abinding site for ABCE1 elsewhere in Gag. In this model, after ABCE1 isbound to Gag, RNA is no longer required to maintain the altered con-formation that exposes the ABCE1 binding site. Further investigationwill be required to distinguish between these models in which the basiccharge in NC acts either directly or indirectly to promote the Gag-ABCE1 interaction. Note that other primate lentiviral Gag proteinsinteract with ABCE1 during assembly (1). Studies suggest that HIV-2and SIV Gag proteins also utilize basic residues to recruit endogenousABCE1 in primate cells, although the exact residues involved have notbeen mapped (1, 30).In summary, our data suggest the following model. Whereas a wide

variety of unrelated viruses bind nonspecifically to RNA to promotecapsid formation, HIV-1 and other primate lentiviruses appear to haveevolved a mechanism in which the same RNA-binding residues also actdirectly or indirectly to recruit ABCE1, a cellular protein that furtherfacilitates the capsid assembly process. By promoting efficient capsidformation in cells, ABCE1 may act as a molecular chaperone in concertwith RNA to ensure Gag multimerization under circumstances whereassembly is not favored. Since ABCE1 is involved in promoting ribo-some assembly (5–9), it would be present in an ideal location to associ-ate with and act on newly synthesized Gag polypeptides.

Acknowledgments—We thank David Rekosh for the pSVGagRRE-R andpCMV Rev plasmids and Jeremy Luban for the M1–2/BR plasmid, LorneWalker and Sherri Dellos for technical support, LorneWalker for comments onthe manuscript, and Mark Orr and Jamie Schoenborn for help with real timePCR and for providing EL4 cells. We also thank the NIH AIDS Research andReference Regent Program, Division of AIDS, NIAID, for pcDNA-APO36 fromDrs. Klaus Strebel and Sandra Kao. JRL is a cofounder of Prosetta Corp.

REFERENCES1. Dooher, J. E., and Lingappa, J. R. (2004) J. Virol. 78, 1645–16562. Lingappa, J. R., Hill, R. L., Wong, M. L., and Hegde, R. S. (1997) J. Cell Biol. 136,

567–5813. Singh, A. R., Hill, R. L., and Lingappa, J. R. (2001) Virology 279, 257–2704. Zimmerman, C., Klein, K. C., Kiser, P. K., Singh, A. R. S., Firestein, B. L., Riba, S. C.,

and Lingappa, J. R. (2002) Nature 415, 88–925. Kispal, G., Sipos, K., Lange, H., Fekete, Z., Bedekovics, T., Janaky, T., Bassler, J.,

Aguilar Netz, D. J., Balk, J., Rotte, C., and Lill, R. (2005) EMBO J. 24, 589–5986. Yarunin, A., Panse, V. G., Petfalski, E., Dez, C., Tollervey, D., and Hurt, E. C. (2005)

EMBO J. 24, 580–5887. Zhao, Z., Fang, L. L., Johnsen, R., and Baillie, D. L. (2004) Biochem. Biophys. Res.

Commun. 323, 104–1118. Dong, J., Lai, R., Nielsen, K., Fekete, C. A., Qiu, H., and Hinnebusch, A. G. (2004)

J. Biol. Chem. 279, 42157–421689. Estevez, A. M., Haile, S., Steinbuchel, M., Quijada, L., and Clayton, C. (2004) Mol.

Biochem. Parasitol. 133, 137–14110. Bisbal, C.,Martinand, C., Silhol,M., Lebleu, B., and Salehzada, T. (1995) J. Biol. Chem.

270, 13308–1331711. Bisbal, C., Silhol, M., Laubenthal, H., Kaluza, T., Carnac, G., Milligan, L., Le Roy, F.,

and Salehzada, T. (2000)Mol. Cell Biol. 20, 4959–496912. Martinand, C., Salehzada, T., Silhol, M., Lebleu, B., and Bisbal, C. (1998) Eur. J. Bio-

chem. 254, 248–25513. Martinand, C., Montavon, C., Salehzada, T., Silhol, M., Lebleu, B., and Bisbal, C.

(1999) J. Virol. 73, 290–29614. Martinand, C., Salehzada, T., Silhol, M., Lebleu, B., and Bisbal, C. (1998) J. Interferon

Cytokine Res. 18, 1031–103815. Karcher, A., Buttner, K.,Martens, B., Jansen, R. P., andHopfner, K. P. (2005) Structure

(Camb.) 13, 649–65916. Kerr, I. D. (2004) Biochem. Biophys. Res. Commun. 315, 166–17317. Le Roy, F., Laskowska, A., Silhol, M., Salehzada, T., and Bisbal, C. (2000) J. Interferon

Cytokine Res. 20, 635–64418. Hage, A. E., and Alix, J. H. (2004)Mol. Microbiol. 51, 189–20119. El Hage, A., Sbai, M., and Alix, J. H. (2001)Mol. Gen. Genet. 264, 796–80820. Lingappa, V. R., and Lingappa, J. R. (2005)Mt. Sinai. J. Med. 72, 141–160




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

21. Kushner, D. J. (1969) Bacteriol. Rev. 33, 302–34522. Freed, E. O. (1998) Virology 251, 1–1523. Berkowitz, R., Fisher, J., and Goff, S. P. (1996) Curr. Top. Microbiol. Immunol. 214,

177–21824. Cimarelli, A., Sandin, S., Hoglund, S., and Luban, J. (2000) J. Virol. 74, 3046–305725. Schmalzbauer, E., Strack, B., Dannull, J., Guehmann, S., and Moelling, K. (1996)

J. Virol. 70, 771–77726. Campbell, S., and Vogt, V. M. (1995) J. Virol. 69, 6487–649727. Campbell, S., and Rein, A. (1999) J. Virol. 73, 2270–227928. Muriaux, D., Costes, S., Nagashima, K., Mirro, J., Cho, E., Lockett, S., and Rein, A.

(2004) J. Virol. 78, 12378–1238529. Smith, A. J., Srinivasakumar, N., Hammarskjold, M. L., and Rekosh, D. (1993) J. Virol.

67, 2266–227530. Dooher, J. E., and Lingappa, J. R. (2004) J. Med. Primatol. 33, 272–28031. Dooher, J. E., Pineda, M. J., Overbaugh, J., and Lingappa, J. R. (2004) J. Med. Primatol.

33, 262–27132. Lewis, N., Williams, J., Rekosh, D., and Hammarskjold, M. L. (1990) J. Virol. 64,

1690–169733. Smith, A. J., Cho, M. I., Hammarskjold, M. L., and Rekosh, D. (1990) J. Virol. 64,

2743–275034. Newman, E.N., Holmes, R. K., Craig, H.M., Klein, K. C., Lingappa, J. R.,Malim,M.H.,

and Sheehy, A. M. (2005) Curr. Biol. 15, 166–17035. Accola, M. A., Strack, B., and Gottlinger, H. G. (2000) J. Virol. 74, 5395–540236. Borsetti, A., Ohagen, A., and Gottlinger, H. G. (1998) J. Virol. 72, 9313–931737. Lingappa, J. R., Newman, M. A., Klein, K. C., and Dooher, J. E. (2005) Virology 333,

114–12338. Schafer, A., Bogerd, H. P., and Cullen, B. R. (2004) Virology 328, 163–168

39. Zennou, V., Perez-Caballero, D., Gottlinger, H., and Bieniasz, P. D. (2004) J. Virol. 78,12058–12061

40. Sheehy, A. M., Gaddis, N. C., Choi, J. D., and Malim, M. H. (2002) Nature 418,646–650

41. Dorfman, T., Luban, J., Goff, S. P., Haseltine, W. A., and Gottlinger, H. G. (1993)J. Virol. 67, 6159–6169

42. Dawson, L., and Yu, X. F. (1998) Virology 251, 141–15743. Bowzard, J. B., Bennett, R. P., Krishna, N. K., Ernst, S. M., Rein, A., and Wills, J. W.

(1998) J. Virol. 72, 9034–904444. Sandefur, S., Smith, R. M., Varthakavi, V., and Spearman, P. (2000) J. Virol. 74,

7238–724945. Zhang, Y., and Barklis, E. (1997) J. Virol. 71, 6765–677646. Berkowitz, R. D., Ohagen, A., Hoglund, S., and Goff, S. P. (1995) J. Virol. 69,

6445–645647. Gorelick, R. J., Henderson, L. E., Hanser, J. P., and Rein, A. (1988) Proc. Natl. Acad. Sci.

U. S. A. 85, 8420–842448. Gorelick, R. J., Nigida, S. M., Jr., Bess, J. W., Jr., Arthur, L. O., Henderson, L. E., and

Rein, A. (1990) J. Virol. 64, 3207–321149. Lee, E. G., Alidina, A., May, C., and Linial, M. L. (2003) J. Virol. 77, 2010–202050. Meric, C., and Goff, S. P. (1989) J. Virol. 63, 1558–156851. Zhang, Y., and Barklis, E. (1995) J. Virol. 69, 5716–572252. Muriaux,D.,Mirro, J., Harvin,D., andRein, A. (2001)Proc. Natl. Acad. Sci. U. S. A.98,

5246–525153. Sandefur, S., Varthakavi, V., and Spearman, P. (1998) J. Virol. 72, 2723–273254. Kao, S., Khan, M. A., Miyagi, E., Plishka, R., Buckler-White, A., and Strebel, K. (2003)

J. Virol 77, 11398–11407




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

KleinJaisri R. Lingappa, Julia E. Dooher, Michael A. Newman, Patti K. Kiser and Kevin C.

AssemblyHIV-1 Gag with ABCE1 (HP68), a Cellular Protein Important for HIV-1 Capsid

Basic Residues in the Nucleocapsid Domain of Gag Are Required for Interaction of

doi: 10.1074/jbc.M507255200 originally published online November 7, 20052006, 281:3773-3784.J. Biol. Chem.

10.1074/jbc.M507255200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/281/7/3773.full.html#ref-list-1

This article cites 54 references, 33 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M507255200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;281/7/3773&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/281/7/3773

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=281/7/3773&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/281/7/3773

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/281/7/3773.full.html#ref-list-1

http://www.jbc.org/

Documents

BasicResiduesintheNucleocapsidDomainofGagAre ... · order to facilitate efficient assembly of HIV-1 capsids. During virion formation, 5000 HIV-15 Gag polypeptides assemble into a