Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus...

2006, 80(16):7939. DOI: 10.1128/JVI.00355-06. J. Virol.

Anjali Joshi, Kunio Nagashima and Eric O. Freed Virion MaturationInteractions, Gag-Membrane Binding, and Capsid Disrupts Virus Assembly, Gag-GagHuman Immunodeficiency Virus Type 1 Mutation of Dileucine-Like Motifs in the

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JOURNAL OF VIROLOGY, Aug. 2006, p. 7939–7951 Vol. 80, No. 160022-538X/06/$08.00�0 doi:10.1128/JVI.00355-06

Mutation of Dileucine-Like Motifs in the Human ImmunodeficiencyVirus Type 1 Capsid Disrupts Virus Assembly, Gag-Gag Interactions,

Gag-Membrane Binding, and Virion MaturationAnjali Joshi,1 Kunio Nagashima,2 and Eric O. Freed1*

Virus-Cell Interaction Section, HIV Drug Resistance Program,1 and Image Analysis Laboratory, Research Technology Program,SAIC-Frederick,2 National Cancer Institute at Frederick, Frederick, Maryland

Received 20 February 2006/Accepted 11 May 2006

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55Gag drives the assembly andrelease of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role inthese processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CAcontains two dileucine-like motifs (L189/L190 and I201/L202) implicated in regulating the localization of Gag tomultivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimerinterface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactionsduring core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1replication, we introduced a series of mutations into these motifs in the context of a full-length, infectiousHIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virusparticle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization,and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting,the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages.Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-likemotifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported hereprovide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CAdimerization, particle maturation, and virion infectivity.

Human immunodeficiency virus type 1 (HIV-1) assemblyand release are driven by the Gag polyprotein precursorPr55Gag, whose expression is sufficient for the formation ofvirus-like particles (VLPs) (13, 45). Pr55Gag is composed offour major domains—matrix (MA), capsid (CA), nucleocapsid(NC), and p6—and two small spacer peptides (SP1 and SP2)located C terminal to CA and NC, respectively. Each of thesedomains plays an important role in VLP production. MA con-tains the determinants in Gag that interact directly with thelipid bilayer and thus regulate the binding of Gag to mem-brane. The CA domain is composed of two structural units, i.e.,the N-terminal domain (NTD), which plays a crucial role invirion maturation (see below), and the C-terminal domain(CTD), which bears residues necessary for Gag-Gag oligomer-ization (17, 18). Specifically, the CA CTD induces Gag dimer-ization and mutations in residues that form the CA CTD dimerinterface severely disrupt Gag assembly and virus particle pro-duction (19, 49). NC also promotes Gag oligomerization dur-ing assembly, largely because of the ability of basic residueswithin NC to interact with viral RNA enabling the nucleic acidto serve as a scaffold along which Gag molecules can align andmultimerize (2, 3, 5, 7, 30). CA alone can assemble into tubularstructures in vitro without NC, and mutations in the CTDdimer interface disrupt this assembly reaction (4, 12, 18, 19, 21,22, 48). These findings suggest that the CA CTD is critical for

mediating the initial Gag-Gag contacts during assembly. Fi-nally, p6 bears a “late” domain that promotes particle buddingand release by interacting with cellular endosomal sorting ma-chinery, most notably the endosomal sorting complex requiredfor transport 1 (ESCRT-I) component Tsg101 (9, 29).

In most commonly studied cell types (e.g., HeLa, 293T,COS, and T-cell lines), HIV-1 assembly occurs predominantlyon the plasma membrane. However, in other cell types, par-ticularly the physiologically relevant primary monocyte-derivedmacrophage (MDM), assembly and budding take place in anintracellular compartment identified as the multivesicular body(MVB) or late endosome (31, 36, 40, 43). The itinerary thatGag follows to reach its destination in the cell remains illdefined; it has been suggested that even in cell types in whichassembly and budding occur predominantly at the plasmamembrane, Gag traffics though an endosomal or endocyticcompartment before reaching the cell surface (10, 20, 32). Ahighly basic domain in MA and a region of MA referred to as the“80’s domain” play an important role in Gag targeting to theplasma membrane. Mutations in these sequences lead to retar-geting of virus assembly from the plasma membrane to the MVBin HeLa cells and in T-cell lines, thereby recapitulating the phe-notype observed for wild-type (WT) Gag in MDMs (16, 36, 38).

During or immediately after virus release from the infectedcell, the viral protease (PR) cleaves Pr55Gag at sites betweenthe major Gag domains to generate the mature Gag proteinsMA, CA, NC, and p6. Cleavage of Gag by PR leads to amorphological transition in the virus particle, a process knownas maturation, which is essential for virus infectivity (13). Dur-ing maturation, the CA protein undergoes a conformational

* Corresponding author. Mailing address: Virus-Cell Interaction Sec-tion, HIV Drug Resistance Program, NCI-Frederick, Bldg. 535/Rm. 108,Sultan Street, Frederick, MD 21702-1201. Phone: (301) 846-6223. Fax:(301) 846-6777. E-mail: [email protected].

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transformation and reassembles into helical arrays in which theCA NTD forms hexameric rings that are linked into a contin-uous lattice by the CTD (25, 51). Mutations have been de-scribed throughout CA that interfere with the formation ofconical cores and hence block virus infectivity (49).

Lindwasser and Resh (27) observed that fusing a large re-gion of HIV-1 Gag encompassing the CA CTD, SP1, and NCto a cytoplasmic tail deletion-containing derivative of CD4resulted in rapid internalization of the CD4-Gag chimera.These results suggested that the CA CTD-SP1-NC region ofGag might contain a motif(s) responsible for the association ofGag with cellular endocytic machinery. Inspection of this re-gion of Gag revealed two di-Leu-like motifs (L189/L190 andI201/L202) within the CA CTD. Although the role of thesedi-Leu-like motifs in the internalization of CD4-Gag chimeraswas not tested, mutation of these motifs severely disruptedVLP production and reduced MVB localization of Gag in COScells treated with the drug U18666A, which induces the redis-tribution of cholesterol from the plasma membrane to MVBs.The authors proposed that these motifs, particularly I201/L202,might be responsible for the association of Gag with MVBs(27). However, these residues also lie in the vicinity of the CACTD dimer interface, raising the possibility that mutations atthese positions might disrupt virus particle production by im-peding CA-mediated Gag-Gag interactions.

To investigate the role of the CA di-Leu-like motifs in mul-tiple aspects of HIV-1 replication, we introduced both single-and double-amino-acid substitutions into these motifs in thecontext of a full-length, infectious HIV-1 molecular clone. Weexamined the effects of the mutations on virus assembly, Gagtargeting, membrane binding and multimerization, virus mat-uration, replication, and infectivity. Our results indicate thatmutation of the CA di-Leu-like motifs severely inhibits virusparticle production for a number of widely varied reasons,including impaired Gag-membrane binding, disrupted Gag oli-gomerization, and altered CA folding. While mutations in thedi-Leu-like motifs markedly inhibited virus assembly and re-lease, they did not affect targeting of Gag to the MVB in eitherHeLa cells or primary human MDMs.

MATERIALS AND METHODS

Cells, transfections, and infections. HeLa and TZM-bl cells (the latter wereobtained from J. Kappes through the NIH AIDS Research and ReferenceReagent Program) (42) were cultured in Dulbecco modified Eagle medium asdescribed previously (24). Jurkat and MT-4 T-cell lines were maintained aspreviously described (24). Monocyte-derived macrophages were prepared andcultured as previously described (14). Plasmid DNA was purified with the plas-mid purification maxi prep kit (QIAGEN, Valencia, CA), and transfections wereperformed with the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) byfollowing the manufacturer’s protocol. Jurkat and MT-4 T cells were transfectedby the DEAE-dextran transfection protocol as previously reported (24). Forinfection assays, virus stocks were prepared by HeLa cell transfection as de-scribed above. Transfected-cell supernatants were passed through a 0.45-�m-pore-size filter, normalized for reverse transcriptase (RT) activity, and used ininfections as indicated. TZM-bl cells were infected in the presence of DEAE-dextran at a final concentration of 20 �g/ml.

Mutagenesis and DNA cloning. CA mutants were constructed in the pNL4-3backbone with the Quick Change site-directed mutagenesis kit (Stratagene, LaJolla, CA). Primer sequences will be made available on request. The PR�

versions of the above mutants were constructed by introducing the ApaI (pNL4-3nucleotide [nt] 2006)-to-EcoRI (pNL4-3 nt 5743) fragment from pNL4-3/PR�

(23) into the CA mutant pNL4-3 derivatives. The 1GA/FLAG-tagged derivativeswere constructed by inserting the Sph1 (pNL4-3 nt 1443)-to-Apa1 (pNL4-3 nt

2006) fragment of the CA single and double mutants into pNL4-3/1GA/FLAG(35, 38).

Radioimmunoprecipitation and Western blot analysis. Methods used for met-abolic labeling of HeLa cells, preparation of cell and virion lysates, and immu-noprecipitation of viral proteins have been described in detail previously (15, 50).Briefly, transfected HeLa cells were metabolically labeled with [35S]Met/Cys andvirions were pelleted by ultracentrifugation. Cell and virus lysates were immu-noprecipitated with HIV immunoglobulin (HIV-Ig) obtained from NABI Bio-pharmaceuticals and the National Heart Blood and Lung Institute through theNIH AIDS Research and Reference Reagent Program and subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed byfluorography. For Western blotting, proteins were separated by SDS-PAGE(10% gel) and transferred to polyvinylidene difluoride membranes (Millipore).To detect hemagglutinin (HA)- and FLAG-tagged proteins, membranes wereincubated with anti-HA7 (Sigma, St. Louis, MO) or anti-FLAGM2 (Sigma, St.Louis, MO) antibodies (Abs), followed by horseradish peroxidase (HRP)-con-jugated anti-mouse Ig (Amersham Biosciences Uppsala, Sweden). The CA-related proteins in cells were detected with HIV-1 anti-p25/p24 SF2 antiserum(obtained from the Division of AIDS through the NIH AIDS Research andReference Reagent Program), followed by HRP-conjugated anti-rabbit Ig (Am-ersham Biosciences, Uppsala, Sweden). To detect HIV-1 proteins in cells, mem-branes were incubated with HIV-Ig, followed by HRP-conjugated anti-human Ig(Amersham Biosciences, Uppsala, Sweden). Antigens recognized by Abs weredetected with enhanced chemiluminescence reagents (Perkin-Elmer, Boston,MA). Quantification of bands was performed with the Alpha Innotech digitalimager.

Gag membrane binding and multimerization assays. Assays for Gag mem-brane binding and higher-order Gag multimerization were conducted as previ-ously described (35, 39). Briefly, HeLa cells were transfected with the pNL4-3/PR� clones expressing either WT or mutant CA. Transfected cells were pulsedwith [35S]Met/Cys for 5 min and chased for 15 min. Cells were then washed withcold phosphate-buffered saline, scraped, and sonicated on ice to achieve disrup-tion of 90% of the cells. Postnuclear supernatants of cell homogenates weresubjected to equilibrium flotation centrifugation at 35,000 rpm for 16 h with85.5%, 65%, and 10% (wt/vol) sucrose gradients. Membrane-bound and non-membrane-bound fractions were then collected and lysed with 2� radioimmu-noprecipitation assay buffer (280 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4,2% NP-40, 1% sodium deoxycholate, 0.1% SDS, 20 mM iodoacetamide, pro-tease inhibitors). Both membrane and nonmembrane fractions were then eitherdenatured by boiling in the presence of 0.057 volume of 2� sample buffer (125mM Tris-HCl [pH 6.8] containing 6% SDS, 10% 2-mercaptoethanol, and 20%glycerol) or left nondenatured. Immunoprecipitations were then performed onboth denatured and nondenatured fractions with HIV-Ig as already described.

Fluorescence microscopy and electron microscopy (EM). Fluorescence mi-croscopy of transfected cells cultured in chamber slides (Nunc) was conducted aspreviously described (38). Briefly, cells were fixed 24 to 48 h posttransfection with3.7% formaldehyde in 100 mM sodium phosphate buffer (pH 7.2) for 20 min,permeabilized with phosphate-buffered saline containing 0.1% Triton X-100, andincubated with anti-p17 monoclonal Ab (Advanced Biotechnologies Inc., Co-lumbia, MD), followed by Texas Red-conjugated anti-mouse IgG. Cells weremounted with Aqua Poly Mount (Polysciences Inc., Warrington, PA) and exam-ined with a Delta Vision RT microscope. When double staining for Gag andCD63 with two different mouse monoclonal Abs, Abs were fluorescently labeledwith the Zenon One Alexa 488 or 594 IgG1 labeling kit (Molecular Probes)according to the manufacturer’s protocol. To avoid dissociation and rebinding ofZenon One reagents, cells were incubated in the fixation buffer after incubationwith each Ab. Fluorescence microscopy in primary MDMs was performed aspreviously described (33). Briefly, virus stocks prepared by transfecting HeLacells with WT or IL-201,202AA mutant pNL4-3, pCMVNLGagPolRRE (37),and pHCMV-G (52) were used to infect MDMs. Cells were fixed postinfection,costained with anti-p17 and anti-CD63 Abs, and examined with a DeltaVisionRT microscope. Ten Gag-positive cells were analyzed, and costaining betweenGag and CD63 in both HeLa cells and MDMs was determined with the soft-WoRx colocalization module. This tool generates a product image of two chan-nels after subtracting a threshold value for each. A scatter plot is then created onthe basis of the two intensities on a pixel-by-pixel basis. The Pearson coefficientof correlation indicates how closely the two intensities are colocalized (fullcolocalization is 1.0) and is defined as the sum of products of the standard scoresof two measures divided by the degrees of freedom. Fixation of cells, preparationof samples, and EM were performed as described previously (16).

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RESULTS

Mutation of the HIV-1 CA di-Leu-like motifs leads to severedefects in virus assembly and release. To define the role of theCA di-Leu-like motifs in HIV-1 assembly and release, we in-troduced mutations into these motifs in the context of thefull-length molecular clone pNL4-3 (Fig. 1). Initially, we sub-stituted Leu189/Leu190 and Ile201/Leu202 for alanines to gener-ate mutants LL189,190AA and IL201,202AA, respectively. Asa control, we also included the assembly-deficient WM184,185AAmutant (39), which contains substitutions in two key residues ofthe CA CTD dimer interface (49). HeLa cells were transfected inparallel with WT or mutant molecular clones and were metabol-ically labeled with [35S]Met/Cys. Levels of cell-associated andvirion-associated HIV-1 proteins were determined by radioimmu-noprecipitation of cell and virion lysates with HIV-Ig (see Mate-rials and Methods). The LL189,190AA and IL201,202AA muta-tions reduced virus particle production approximately 10-fold and5-fold, respectively (Fig. 2A). As previously reported (39), theWM184,185AA mutant also displayed severely impaired virusparticle production. In addition to defective particle production,the CA mutants showed abnormal processing of the Pr55Gag

precursor to the Gag processing intermediate p41Gag and themature CA protein p24 (Fig. 2A). Specifically, the mutantsshowed elevated levels of p41Gag relative to Pr55Gag, and theLL189,190AA mutant displayed an increased ratio of Pr55Gag top24 (CA). Interestingly, the IL201,202AA mutations also resultedin the appearance of an additional protein product smaller than24 kDa (Fig. 2A).

To evaluate further the roles of Leu189, Leu190, Ile201, andLeu202 in virus assembly and release, we individually mutatedeach of these residues to Ala. As depicted in Fig. 2B, the CAsingle mutants also showed various degrees of defects in virus

assembly and release, with the L190A mutant being the mostcompromised. Interestingly, the CA single mutants also exhib-ited defects in Gag processing. In addition, the smaller proteinproduct associated with the CA double mutant IL201,202AA(Fig. 2A) could also be seen with the L202A mutant (Fig. 2B).These data thus indicate that both the CA double and singlemutants exhibit various degrees of defects in HIV assemblyand release, with the double mutants being affected more se-verely than the single mutants.

The IL201,202AA and L202A mutants display aberrant CAprocessing. As shown in Fig. 2A and B, a protein productsmaller than 24 kDa was associated with the CA double mutantIL201,202AA and the single mutant L202A. A similar pheno-type has been observed previously upon mutation of buriedhydrophobic residues in the CA NTD (e.g., F40A) (47). It ispossible that mutation of CA202 induces protein misfolding,thereby exposing normally buried sites within CA to cleavageby PR. To determine whether the �24-kDa protein product isCA related, lysates from cells transfected with theLL189,190AA, IL201,202AA, and WM184,185AA double mu-tants or the L202A single mutant were subjected to SDS-PAGE and probed with either anti-p24 Ab or HIV-Ig. In bothinstances, a protein product of approximately 20 kDa was vis-ible only for the double mutant IL201,202AA and the singlemutant L202A (data not shown), indicating that this species isindeed CA related. To further address the issue of abnormalprocessing of CA by PR, we generated PR� versions of the CAdouble and single mutants and performed immunoprecipita-tions following transfection of HeLa cells with these PR�

clones. The PR� versions of the IL201,202AA and L202Amutants no longer showed the presence of the �20-kDa CA-related product (data not shown). These data support thehypothesis that mutation of CA202 induces CA misfolding andaberrant processing by PR. VLP release in the context of thePR� clones was also severely inhibited by the LL189,190AA,IL201,202AA, and WM184,185AA double mutants (data notshown).

Since the IL201,202AA and L202A mutants appeared toshow defects in CA protein folding, we sought to examinewhether these mutants would release non-particle-associatedCA from virus-expressing cells. To examine this possibility,HeLa cells were transfected with the LL189,190AA,IL201,202AA, and WM184,185AA double mutants or theL202A single mutant. Following metabolic radiolabeling, re-leased virus was pelleted by ultracentrifugation and both thevirus pellet lysate and the postvirus supernatant were immu-noprecipitated with HIV-Ig (Fig. 3). Cells transfected with WTpNL4-3 or the LL189,190AA or WM184,185AA double mu-tant released barely detectable levels of non-particle-associ-ated CA. In contrast and interestingly, the IL201,202AA mu-tant released large amounts of non-particle-associated p24(CA). The L202A mutant released lower, but detectable, levelsof free CA. Overall, these findings suggest that the severeassembly defects observed for the IL201,202AA mutant couldbe a consequence of CA misfolding and/or instability.

Replication kinetics of the CA mutants in T-cell lines; iso-lation of viral revertants. To characterize further the pheno-types of the CA mutants, virus replication kinetics were eval-uated in T-cell lines. This analysis revealed that neither of theCA double mutants showed detectable replication, even when

FIG. 1. Sequence and structure of the HIV-1 CA CTD. The aminoacid sequence of the CA CTD is shown at the top. The CTD dimerinterface residues W184/M185 and the putative di-Leu-like motifs L189/L190 and I201/L202 are indicated in red. At the bottom is shown astructural model of a dimer of the HIV-1 CA CTD (provided by S. R.Durell) with the locations of L189, L190, I201, and L202 indicated by thered, orange, yellow, and green balls, respectively. Residue L189 lies atthe CA dimer interface; residues L190 and L202 are buried, whileresidue I201 is exposed. The structure was constructed with data from1A43.pdb (51).

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FIG. 2. The CA mutants LL189,190AA and IL201,202AA are severely defective in virus assembly and release. HeLa cells were transiently transfected withWT pNL4-3 (WT) or derivatives containing the indicated (A) CA double or (B) CA single mutations. Transfected HeLa cells were metabolically labeled with[35S]Met/Cys, and virions were pelleted by ultracentrifugation. Cell and virion lysates were immunoprecipitated with HIV-Ig and analyzed by SDS-PAGE,followed by phosphorimager analysis. The positions of the Env precursor gp160, the mature surface Env glycoprotein gp120, the Gag precursor Pr55Gag, theGag processing intermediate p41Gag, and p24 (CA) are shown. The �20-kDa protein product detected with the IL201,202AA and L202A mutants is indicatedby an unmarked arrow. Virus release efficiency was calculated from at least three independent experiments as the amount of radiolabeled, virion-associated Gagas a fraction of the total (cell plus virion) radiolabeled Gag protein detected. Error bars show standard deviations.


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transfected cultures were maintained for 3 months (data notshown). We also observed that none of the single mutants wasable to replicate in Jurkat T cells (data not shown). In contrast,in the more permissive MT-4 T-cell line, the L189A, L190A,and I201A single mutants all replicated, albeit with markedlydelayed kinetics. The L202A mutant did not replicate in MT-4cells (Fig. 4A).

To determine whether the delayed replication observed forthe L189A, L190A, and I201A single mutants represented theemergence of viral revertants, virus stocks were collected dur-ing the peak of RT activity and used to reinfect both Jurkat andMT-4 cells. The CA single mutants that were previously unableto grow in Jurkat cells now replicated with kinetics comparableto those of the WT (Fig. 4B). In MT-4 cells, the repassagedviruses replicated with WT kinetics (Fig. 4C). These resultssuggested that the CA single mutants reverted during theirpassage in MT-4 cells. To confirm this prediction and to char-acterize potential revertants, viral DNA was isolated from in-fected Jurkat and MT-4 cells. The Gag coding region was thenamplified by PCR and sequenced. The sequencing data indi-cated that the L189A mutant had reverted back to the WTcodon (Leu), whereas the L190A and I201A mutants acquireda Val codon in place of the original mutant (Ala) codon (Fig.4D). The same changes were identified upon sequencing ofviral DNAs derived from both Jurkat and MT-4 cells (data notshown). Viruses carrying the L190V or I201V mutation were

assembled and released as efficiently as the WT and alsoshowed WT replication kinetics in Jurkat T cells (data notshown). Single-cycle infectivity data indicated that the CA dou-ble mutants and the L189A, L190A, I201A, and L202A mu-tants were severely defective while the L190V and I201V mu-tants displayed WT infectivity (data not shown). These findingsindicate that the L189L190 and I201L202 motifs are not requiredfor full Gag function in that L190 and I201 can be replaced withVal with no adverse effects on assembly, release, or virus rep-lication.

Immunofluorescence microscopy and EM reveal distinct de-fects for the IL201,202AA and LL189,190AA mutants. To morerigorously elucidate the mechanism by which mutation of CAresidues 189, 190, 201, and 202 induced defects in virus pro-duction and replication, HeLa cells were transfected in parallelwith WT or mutant molecular clones and stained with anti-p17(MA) Ab. Immunofluorescence analysis revealed that cellstransfected with WT pNL4-3 or the IL201,202AA mutantshowed cell surface punctate staining typical of WT Gag (Fig. 5).In contrast, the LL189,190AA mutant showed an accumulationof cytosolic, non-plasma membrane-associated Gag with a lackof cell surface puncta. This pattern of Gag staining was sug-gestive of a defect in Gag membrane binding and/or multimer-ization. For comparison, we analyzed the CA CTD dimer in-terface mutant WM184,185AA. Mutation of CA residue 184 or185 has previously been shown to induce a severe defect in

FIG. 3. Mutation of L202 leads to the release of free CA and CA-derived product. HeLa cells transfected with WT pNL4-3 or the indicated CAmutants were metabolically labeled with [35S]Met/Cys, and virions were pelleted by ultracentrifugation. Cell- and virion-associated material andthe supernatant obtained from virus pelleting (postvirus supernatant) were immunoprecipitated with HIV-Ig and analyzed by SDS-PAGE,followed by phosphorimager quantification. Results from one representative experiment are shown; similar results were obtained in an indepen-dently repeated experiment.


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CA-CA and Gag-Gag interactions (18, 49) and also to impairmembrane binding (39). The WM184,185AA mutant showed apattern of Gag staining essentially indistinguishable from thatof LL189,190AA (Fig. 5). We also determined the Gag local-ization pattern of the single mutants. The L189A and L190Amutants showed diffuse cytosolic staining similar to that of theLL189,190AA double mutant and the presence of some cellsurface punctate staining. The I201A and L202A mutants ex-hibited a WT localization pattern, with a considerable amountof cell surface punctate Gag staining (Fig. 5). Overall, theimmunofluorescence data suggested that mutation of CA189

and CA190 induced defects in Gag multimerization and/ormembrane binding.

To examine the effects of the CA mutations on virus particlemorphology, we transfected HeLa cells with WT pNL4-3 or theCA mutant derivatives and performed thin-section transmis-sion EM. pNL4-3-transfected cultures showed large numbersof released virus particles, many of which contained conical,condensed cores and which were �100 to 120 nm in diameter(Fig. 6). In contrast, virus particles produced by theIL201,202AA mutant were highly distorted, were morpholog-ically immature or possessed acentric cores, and were abnor-mally large (approximately 300 to 400 nm in diameter). Nobudding or released virus particles were observed for the

LL189,190AA mutant. EM analysis of the CA single mutantsalso showed gross defects in particle morphology and a failureto form conical cores (Fig. 6).

Assembly and release of the IL201,202AA but not theLL189,190AA mutant can be efficiently rescued by coexpres-sion with WT Gag. The immunofluorescence analysis pre-sented above suggested that the LL189,190AA mutant may bedefective for Gag multimerization. To investigate this possibil-ity directly, we used a previously described assay (2, 34) toevaluate the ability of CA mutant Gag to be rescued uponcoexpression with WT Gag. In this assay, Gag mutants areanalyzed in the context of a C-terminally epitope-tagged, non-myristylated (1GA) (16) pNL4-3 derivative. Because of theelimination of the N-terminal myristylation signal, these Gagsare unable to bind membrane and assemble when expressedalone. However, upon coexpression with WT Gag bearing adifferent C-terminal epitope tag, the nonmyristylated Gag pro-tein is incorporated into virions via coassembly with WT Gag.The extent to which the nonmyristylated mutant Gag protein isrecovered in VLP fractions provides a measurement of theefficiency with which it can engage in Gag-Gag interactions. Toperform this analysis, FLAG-tagged versions of pNL4-3/1GAexpressing either WT or mutant CA were cotransfected with apNL4-3 derivative encoding HA-tagged WT Gag. Rescue of

FIG. 4. Replication kinetics of CA mutants and isolation of viral revertants. (A) MT-4 T cells were transfected with either WT pNL4-3 or theindicated CA single mutants. Cells were split every 2 days; supernatant was reserved at each time point for RT analysis. Repassage of CA singlemutants and isolation of viral revertants. (B) Jurkat and (C) MT-4 T-cell lines were infected with WT or CA mutant virus (normalized for RTactivity) obtained from the MT-4 replication experiment shown in panel A. Culture supernatant was reserved every 2 days for RT analysis. Cellpellets were also harvested for DNA isolation, PCR amplification, and sequencing of the gag gene. P2 after the mutant name denotes passage 2.(D) Sequence profile of revertant viruses obtained from repassage of CA single mutants in Jurkat and MT-4 cells. The codons found in WTpNL4-3, the mutant derivatives, and the PCR-amplified viral DNA are indicated; the encoded amino acid is indicated in parentheses. For L202,no revertants were obtained (none).


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the mutant (FLAG-tagged) Gag protein by the WT (HA-tagged) Gag protein was then monitored by quantitative im-munoblotting with anti-HA and anti-FLAG Abs. The resultsindicated that no particle-associated, FLAG-tagged Pr55Gag

was released upon single expression of 1GA Gag (Fig. 7, lane8). However, coexpression with HA-tagged WT Gag led toefficient rescue of FLAG-tagged 1GA Gag in the virion-asso-ciated fraction (Fig. 7, lane 9). Similarly, the 1GA/IL201,202AA mutant could be rescued almost as efficiently asthe 1GA Gag protein containing WT CA (Fig. 7, lane 10). Incontrast, the 1GA/LL189,190AA and 1GA/WM184,185AAmutants displayed three- to fivefold reductions in the ability tocoassemble with WT Gag and be recovered in virion-associ-ated fractions (Fig. 7, lanes 11 and 12). These data reinforceour immunofluorescence findings and confirm that theLL189,190AA mutant is defective in Gag-Gag interactions toan extent similar to that of the WM184,185AA CTD dimerinterface mutant. Interestingly, all of the CA single mutantscould be efficiently rescued (data not shown), consistent withtheir less severe phenotype relative to the double mutants.

The LL189,190AA mutant is defective in membrane binding.We previously demonstrated that the WM184,185AA dimerinterface mutant displays a defect in membrane binding that ismost likely linked to its inability to multimerize efficiently (39).

The similarities observed here between the WM184,185AAand LL189,190AA mutants in Gag localization and Gag mul-timerization suggested that the LL189,190AA mutant was alsodefective in membrane binding, as reported previously (27). Toevaluate the ability of the CA double mutants to bind mem-brane, we performed membrane flotation assays with sucrosegradients (Fig. 8A). This analysis indicated that while theIL201,202AA mutant Gag protein showed membrane bindingability similar to that of the WT, the amount of LL189,190AAGag recovered in membrane fractions was approximately halfthat of WT Gag. The membrane binding ability of theWM184,185AA mutant was likewise reduced approximatelytwofold (Fig. 8A).

We previously described an assay that measures higher-or-der, in vivo Gag multimerization (39). This assay is based onthe observation that membrane-bound HIV-1 Gag is poorlydetected by immunoprecipitation prior to denaturation. Thus,comparison of the amount of Gag detected with versus withoutdenaturation (i.e., the amount of Gag that is “epitope ex-posed”) provides a measure of higher-order Gag multimeriza-tion. As part of our membrane flotation analysis presentedabove, we also determined the degree of epitope exposure forthe CA double mutants. Consistent with the VLP rescue assayshown in Fig. 7, we observed that the IL201,202AA mutant

FIG. 5. Subcellular localization of CA-mutant Gag. HeLa cells were transfected with WT pNL4-3 or the indicated CA-mutant derivatives. Cellswere fixed 24 h posttransfection and stained with anti-HIV-1 p17 (MA) Ab followed by fluorescence microscopy with a DeltaVision RTmicroscope.


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displayed a level of epitope masking (i.e., higher-order Gagmultimerization) similar to that of WT in the membrane frac-tion (Fig. 8A). In contrast, the LL189,190AA double mutantshowed significantly increased epitope exposure in the mem-brane fraction, confirming its defect in Gag multimerization.Consistent with our earlier data (39) and similar to theLL189,190AA double mutant, a high percentage ofWM184,185AA mutant Gag could be immunoprecipitatedfrom the membrane-bound fractions without denaturation(Fig. 8A).

We also applied the membrane flotation and epitope expo-sure assays to the CA single mutants. While the I201A andL202A mutants did not show statistically significant defects,the L189A mutant displayed a significant reduction in mem-brane binding (Fig. 8B) and a small, but significant, increase inepitope exposure. The L190A mutant exhibited an intermedi-ate phenotype, with a small reduction in membrane binding

(Fig. 8B). Altogether, the membrane flotation and epitopemasking data presented in Fig. 8 reinforce the results of ourimmunofluorescence microscopy and Gag rescue assays andindicate that the CA mutant LL189,190AA but notIL201,202AA is defective in Gag multimerization and mem-brane binding.

The IL-201,202AA mutant does not disrupt MVB targeting.The immunofluorescence data presented in Fig. 6 indicate thatthe pattern of Gag localization for IL201,202AA is indistin-guishable from that of the WT, suggesting that this mutant isnot defective in Gag trafficking. However, since it was sug-gested that the I201/L202 di-Leu-like motif may play an impor-tant role in the association of Gag with endocytic machinery(27), we sought to determine whether the IL201,202AA mu-tation would affect the MVB localization of Gag. As men-tioned in the introduction, we have previously demonstratedthat mutations in the highly basic domain of MA (e.g., 29KT/

FIG. 6. EM analysis of CA-mutant virions. HeLa cells were transfected with WT pNL4-3 or the indicated CA-mutant derivatives. Cells werefixed at 2 days posttransfection and observed by transmission EM. The bar under each panel represents a 100-nm scale.


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31KT) induce the relocalization of Gag to a CD63� late en-dosomal or MVB compartment (36, 38). If the I201/L202 di-Leu-like motif were important for the association of Gag withMVB, we would predict that the IL201,202AA mutation woulddisrupt MVB localization in the context of 29KT/31KT-mutantMA. To test this hypothesis, we constructed a pNL4-3 derivativecontaining both the MA (29KT/31KT) and CA (IL201,202AA)mutations. HeLa cells were transfected with this molecularclone or with the 29KT/31KT MA mutant clone. Transfectedcells were fixed and costained with anti-p17 (MA) and anti-CD63 Abs (Fig. 9A). As we reported previously (36), the29KT/31KT MA-mutant Gag protein largely localized to theMVB (R � 0.82 � 0.05) (Fig. 9A, top). Interestingly, the CAIL201,202AA mutation did not significantly alter Gag localiza-tion to the MVB (R � 0.80 � 0.04) (Fig. 9A, bottom). We alsointroduced the CA LL189,190AA mutation into the 29KT/31KT MA mutant clone. This mutant showed a cytosolicdistribution of Gag (data not shown) similar to that seenwith the LL189,190AA mutant (Fig. 5), consistent with the

defect in membrane binding imposed by disruption of theL189/L190 motif.

To extend these findings to a cell type in which WT Gag ispredominantly MVB targeted, we examined the localization ofWT Gag or the IL201,202AA mutant in primary humanMDMs (Fig. 9B). Again, we found no difference in the CD63colocalization of the IL201,202AA mutant relative to that ofthe WT (R � 0.7028 � 0.17 for WT and 0.6826 � 0.102 forIL201,202AA). Together, these findings indicate that disrup-tion of the I201/L202 di-Leu-like motif does not prevent thetargeting of Gag to the late endosome or MVB.

DISCUSSION

In this study, we demonstrate that mutations in the putativedi-Leu-like motifs in the HIV-1 CA CTD lead to severe defectsin virus assembly and release, virion maturation, and infectiv-ity. Several of the single-amino-acid mutants reverted in cul-ture; analysis of these revertants indicated that Leu-to-Val

FIG. 7. Analysis of the rescue of CA mutant Gag into VLPs upon coexpression with WT Gag. HeLa cells were transfected with FLAG-taggedversions of either 1GA/pNL4-3 or 1GA/pNL4-3 derivatives containing the indicated CA double mutations. Rescue of the nonmyristylated (1GA)Gag protein into VLPs was measured by cotransfection with WT HA-tagged Gag. Cell and virus lysates were subjected to SDS-PAGE, followedby immunoblotting with anti-HA or anti-FLAG Ab. Quantification of Pr55Gag bands was performed with the Alpha Innotech digital imagingsystem. Virus release efficiency was calculated as the percent release of FLAG-tagged 1GA Gag, with the amount of FLAG-tagged 1GA Gagcontaining WT CA rescued into VLPs by HA-tagged Gag being given a relative value of 100%. Lane numbers under the graph correspond to therespective lanes in the anti-FLAG Western blot. In the graph, 1GA-F, 1GA/IL-F, 1GA/LL-F, and 1GA/WM-F refer to FLAG-tagged 1GA Gagproteins containing WT, IL201,202AA, LL189,190AA, and WM184,185AA CA, respectively. 55HA denotes HA-tagged WT Gag. Data representaverages from at least three independent experiments � the standard deviations.


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substitutions at both residues 190 and 201 were well tolerated,despite the fact that these mutations disrupt the integrity of thedi-Leu-like motifs. Finally, the IL201,202AA mutant efficientlylocalized to the MVB in both HeLa cells and MDMs, thusdemonstrating that the I201/L202 di-Leu-like motif is not re-quired for MVB targeting.

The defects induced by the mutations described here em-phasize the role of the CA CTD dimer interface in both virusparticle production and virion maturation. Structural modelingrevealed that while residue I201 is surface exposed, L202 isburied (Fig. 1). We therefore speculate that mutation of theI201/L202 motif disrupted normal CA folding, making a previ-ously buried site(s) accessible for cleavage by PR. Interestingly,the L202A mutation did not significantly affect virus particle

production but impaired virion maturation, virus replication,and infectivity, suggesting that mutation of L202 predominantlyaffected CA-CA interactions following particle release, ratherthan Gag-Gag interactions during assembly. We note that ab-errant cleavage of CA by PR has also been observed uponmutation of buried residues in the CA NTD (47). The releaseof free CA observed with mutants containing substitutions inCA residue 202 (Fig. 3) may result from a loss of virion stabilityfollowing aberrant cleavage of the mutant CA by PR.

The double mutant LL189,190AA showed clear defects inGag membrane binding and multimerization, consistent withprevious results (27). Residue L189 lies in the CA dimer inter-face (Fig. 1), a domain whose function in promoting CA-CAinteractions both in vivo and in vitro is well established (8, 11,

FIG. 8. Effects of CA mutations on membrane binding and Gag multimerization. HeLa cells were transfected with pNL4-3/PR� versions of the(A) CA double or (B) CA single mutants. Transfected cells were pulse-chase labeled with [35S]Met/Cys and sonicated, and postnuclearsupernatants were subjected to membrane flotation centrifugation on sucrose gradients (see Materials and Methods). The membrane (M) andnonmembrane (NM) fractions were isolated and immunoprecipitated with HIV-Ig either with (�) or without (�) prior denaturation. Pr55Gag

bands were quantified by phosphorimager analysis. The graphs in the lower left of panels A and B indicate membrane binding efficiency. Percentepitope exposure (lower right), which is a measurement of higher-order Gag multimerization (39), was determined by calculating the percentageof Gag recovered by immunoprecipitation from nondenatured relative to denatured fractions. Note that epitope masking is observed predomi-nantly with membrane-associated Gag. P values represent significant differences between the WT and mutant Gag proteins as determined by theStudent t test.


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18, 19, 28, 49). The dimer interface forms a hydrophobic corethat includes residues Val181, Trp184, Met185, and Leu189 (18,19, 49). Consistent with these observations, the L189A singlemutant showed more-severe defects in membrane binding thandid the L190A mutant (Fig. 9B). These defects can be attrib-uted to the fact that the CTD dimer interface links the hex-americ rings formed by the CA NTD into a continuous latticeand that disruption of residues in this domain impair CA-CAinteractions (25). Our data obtained with both theLL189,190AA and WM184,185AA mutants support the pro-posal of von Schwedler and coworkers (49) that the CA CTDdimer interface is important not only for CA-CA interactionsduring core maturation but also for Gag-Gag interactions dur-ing particle assembly.

The role of Gag multimerization in Gag-membrane bindingremains to be fully elucidated. Several studies comparing full-length and C-terminally truncated Gag constructs suggestedthat Gag multimerization mediated by NC may enhance mem-brane binding (41, 44). By using membrane flotation centrifu-gation techniques that separate membrane-associated Gagfrom non-membrane-bound Gag oligomers, we observed that aGag truncation mutant containing only MA and the CA NTDdisplayed efficient membrane binding at steady state (34) but

reduced membrane binding kinetics (37). Mutations in SP1that inhibit Gag multimerization have also been reported toreduce membrane binding (26), and dimerization of the Roussarcoma virus MA increased its capacity to associate withmembrane in vitro (6). Structural studies with HIV-1 MA havedemonstrated that low-order multimerization leads to greaterexposure of the myristate moiety, thereby presumably increas-ing membrane binding ability (46). We recently reported thata severely assembly-deficient mutant lacking all of the basicresidues in NC displayed WT levels of membrane bindingwhereas the WM184,195AA CA dimer interface mutant wasmembrane binding defective (39). The data presented herethat were obtained with the LL189,190AA mutant support andextend our proposal that Gag-Gag contacts mediated by theCA CTD, but not higher-order Gag assembly mediated by thebasic residues in NC, are required for efficient and stablebinding of Gag to membrane.

As mentioned in the introduction, Lindwasser and Resh (27)observed that the IL201,202AA mutation severely disruptedVLP production and reduced MVB localization of Gag in COScells treated with the drug U18666A. They proposed that thedefect in particle production exhibited by this mutant Gagresulted from its failure to traffic to the MVB. Alternatively,

FIG. 9. MVB localization of the IL201,202AA CA mutant. (A) HeLa cells were transfected with pNL4-3/29KT/31KT (top) or with thederivative containing the IL201,202AA CA mutation (bottom). (B) WT NL4-3 or the IL201,202AA CA-mutant derivative was expressed in primaryhuman MDMs (Materials and Methods). Cells were fixed 24 h posttransfection, permeabilized, and stained with anti-HIV-1 p17 (MA) (green) andanti-CD63 (red) Abs, followed by microscopy with a DeltaVision RT microscope. The softWoRx colocalization module was used to determinecostaining between Gag and CD63 on an average of 8 to 10 cells. One representative field for each is depicted. When data were compiled andaveraged for all of the cells analyzed, the Gag/CD63 colocalization in the context of WT CA was R � 0.82 � 0.05 in HeLa cells and R � 0.7028 �0.17 in MDMs; for the IL201,202AA CA mutant, it was R � 0.80 � 0.04 in HeLa cells and 0.6826 � 0.102 in MDMs. R � Pearson coefficient ofcorrelation.


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they suggested that the effect of the IL201,202AA mutation onvirus assembly and release might be distinct from a role in Gagtrafficking. Our data support the latter hypothesis. The resultspresented here suggest that the impaired virus particle produc-tion induced by mutations in the L189/L190 and I201/L202 motifsis due to a diversity of defects in Gag-membrane binding,multimerization, and folding. We also find that MVB localiza-tion of IL201,202AA Gag is not affected in HeLa cells in thecontext of a MA mutation that disrupts plasma membranetargeting (Fig. 9A) or in MDMs in the context of otherwiseWT Gag (Fig. 9B). Finally, the fact that Leu-to-Val substitu-tions at both residues L190 and I201 are well tolerated arguesagainst a requirement in HIV-1 replication for di-Leu-likemotifs at these positions (1).

Our results do not exclude the possibility that the CA CTDdi-Leu-like motifs could promote the internalization of CD4-Gag chimeras, nor do they contradict the observation thatthese motifs affect Gag endocytosis in cells treated withU18666A (27). However, our data suggest that these motifs donot play a major role in Gag trafficking under physiologicalconditions. Ongoing studies will attempt to define further theviral determinants involved in Gag targeting and elucidate thecellular machinery that regulates the localization of HIV-1assembly.

ACKNOWLEDGMENTS

We thank S. Ablan for expert technical assistance, Ferri Soheilianfor EM support, and Vineet KewalRamani, Judith Levin, Akira Ono,and members of the Freed lab for critical reviews of the manuscript.We also thank S. R. Durell for providing the CA CTD model shownin Fig. 1.

The HIV-Ig and anti-p24 Abs were obtained through the AIDSResearch and Reference Reagent Program, Division of AIDS, NIAID,NIH. This research was supported by the Intramural Research Pro-gram of the Center for Cancer Research, National Cancer Institute,NIH, and by the Intramural AIDS Targeted Antiviral Program. Thisproject was funded in part with federal funds from the National CancerInstitute, NIH, under contract N01-CO-12400.

The content of this publication does not necessarily reflect the viewsor policies of the Department of Health and Human Services, nor doesmention of trade names, commercial products, or organizations implyendorsement by the U.S. Government.

REFERENCES

1. Bonifacino, J. S., and L. M. Traub. 2003. Signals for sorting of transmem-brane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395–447.

2. Burniston, M. T., A. Cimarelli, J. Colgan, S. P. Curtis, and J. Luban. 1999.Human immunodeficiency virus type 1 Gag polyprotein multimerizationrequires the nucleocapsid domain and RNA and is promoted by the capsid-dimer interface and the basic region of matrix protein. J. Virol. 73:8527–8540.

3. Campbell, S., and A. Rein. 1999. In vitro assembly properties of humanimmunodeficiency virus type 1 Gag protein lacking the p6 domain. J. Virol.73:2270–2279.

4. Campbell, S., and V. M. Vogt. 1995. Self-assembly in vitro of purified CA-NCproteins from Rous sarcoma virus and human immunodeficiency virus type 1.J. Virol. 69:6487–6497.

5. Cimarelli, A., S. Sandin, S. Hoglund, and J. Luban. 2000. Basic residues inhuman immunodeficiency virus type 1 nucleocapsid promote virion assemblyvia interaction with RNA. J. Virol. 74:3046–3057.

6. Dalton, A. K., P. S. Murray, D. Murray, and V. M. Vogt. 2005. Biochemicalcharacterization of Rous sarcoma virus MA protein interaction with mem-branes. J. Virol. 79:6227–6238.

7. Dawson, L., and X. F. Yu. 1998. The role of nucleocapsid of HIV-1 in virusassembly. Virology 251:141–157.

8. del Alamo, M., J. L. Neira, and M. G. Mateu. 2003. Thermodynamic dissec-tion of a low affinity protein-protein interface involved in human immuno-deficiency virus assembly. J. Biol. Chem. 278:27923–27929.

9. Demirov, D. G., and E. O. Freed. 2004. Retrovirus budding. Virus Res.106:87–102.

10. Dong, X., H. Li, A. Derdowski, L. Ding, A. Burnett, X. Chen, T. R. Peters,T. S. Dermody, E. Woodruff, J. J. Wang, and P. Spearman. 2005. AP-3directs the intracellular trafficking of HIV-1 Gag and plays a key role inparticle assembly. Cell 120:663–674.

11. Dorfman, T., A. Bukovsky, A. Ohagen, S. Hoglund, and H. G. Gottlinger.1994. Functional domains of the capsid protein of human immunodeficiencyvirus type 1. J. Virol. 68:8180–8187.

12. Ehrlich, L. S., B. E. Agresta, and C. A. Carter. 1992. Assembly of recombi-nant human immunodeficiency virus type 1 capsid protein in vitro. J. Virol.66:4874–4883.

13. Freed, E. O. 1998. HIV-1 Gag proteins: diverse functions in the virus lifecycle. Virology 251:1–15.

14. Freed, E. O., G. Englund, and M. A. Martin. 1995. Role of the basic domainof human immunodeficiency virus type 1 matrix in macrophage infection.J. Virol. 69:3949–3954.

15. Freed, E. O., and M. A. Martin. 1994. Evidence for a functional interactionbetween the V1/V2 and C4 domains of human immunodeficiency virus type1 envelope glycoprotein gp120. J. Virol. 68:2503–2512.

16. Freed, E. O., J. M. Orenstein, A. J. Buckler-White, and M. A. Martin. 1994.Single amino acid changes in the human immunodeficiency virus type 1matrix protein block virus particle production. J. Virol. 68:5311–5320.

17. Gamble, T. R., F. F. Vajdos, S. Yoo, D. K. Worthylake, M. Houseweart, W. I.Sundquist, and C. P. Hill. 1996. Crystal structure of human cyclophilin Abound to the amino-terminal domain of HIV-1 capsid. Cell 87:1285–1294.

18. Gamble, T. R., S. Yoo, F. F. Vajdos, U. K. von Schwedler, D. K. Worthylake,H. Wang, J. P. McCutcheon, W. I. Sundquist, and C. P. Hill. 1997. Structureof the carboxyl-terminal dimerization domain of the HIV-1 capsid protein.Science 278:849–853.

19. Ganser-Pornillos, B. K., U. K. von Schwedler, K. M. Stray, C. Aiken, andW. I. Sundquist. 2004. Assembly properties of the human immunodeficiencyvirus type 1 CA protein. J. Virol. 78:2545–2552.

20. Gould, S. J., A. M. Booth, and J. E. Hildreth. 2003. The Trojan exosomehypothesis. Proc. Natl. Acad. Sci. USA 100:10592–10597.

21. Gross, I., H. Hohenberg, C. Huckhagel, and H. G. Krausslich. 1998. N-terminal extension of human immunodeficiency virus capsid protein convertsthe in vitro assembly phenotype from tubular to spherical particles. J. Virol.72:4798–4810.

22. Gross, I., H. Hohenberg, and H. G. Krausslich. 1997. In vitro assemblyproperties of purified bacterially expressed capsid proteins of human immu-nodeficiency virus. Eur. J. Biochem. 249:592–600.

23. Huang, M., J. M. Orenstein, M. A. Martin, and E. O. Freed. 1995. p6Gag isrequired for particle production from full-length human immunodeficiencyvirus type 1 molecular clones expressing protease. J. Virol. 69:6810–6818.

24. Kiernan, R. E., A. Ono, G. Englund, and E. O. Freed. 1998. Role of matrixin an early postentry step in the human immunodeficiency virus type 1 lifecycle. J. Virol. 72:4116–4126.

25. Li, S., C. P. Hill, W. I. Sundquist, and J. T. Finch. 2000. Image reconstruc-tions of helical assemblies of the HIV-1 CA protein. Nature 407:409–413.

26. Liang, C., J. Hu, J. B. Whitney, L. Kleiman, and M. A. Wainberg. 2003. Astructurally disordered region at the C terminus of capsid plays essentialroles in multimerization and membrane binding of the Gag protein of humanimmunodeficiency virus type 1. J. Virol. 77:1772–1783.

27. Lindwasser, O. W., and M. D. Resh. 2004. Human immunodeficiency virustype 1 Gag contains a dileucine-like motif that regulates association withmultivesicular bodies. J. Virol. 78:6013–6023.

28. Melamed, D., M. Mark-Danieli, M. Kenan-Eichler, O. Kraus, A. Castiel, N.Laham, T. Pupko, F. Glaser, N. Ben-Tal, and E. Bacharach. 2004. Theconserved carboxy terminus of the capsid domain of human immunodefi-ciency virus type 1 Gag protein is important for virion assembly and release.J. Virol. 78:9675–9688.

29. Morita, E., and W. I. Sundquist. 2004. Retrovirus budding. Annu. Rev. CellDev. Biol. 20:395–425.

30. Muriaux, D., J. Mirro, D. Harvin, and A. Rein. 2001. RNA is a structuralelement in retrovirus particles. Proc. Natl. Acad. Sci. USA 98:5246–5251.

31. Nguyen, D. G., A. Booth, S. J. Gould, and J. E. Hildreth. 2003. Evidence thatHIV budding in primary macrophages occurs through the exosome releasepathway. J. Biol. Chem. 278:52347–52354.

32. Nydegger, S., M. Foti, A. Derdowski, P. Spearman, and M. Thali. 2003.HIV-1 egress is gated through late endosomal membranes. Traffic 4:902–910.

33. Ono, A., S. D. Ablan, S. J. Lockett, K. Nagashima, and E. O. Freed. 2004.Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting tothe plasma membrane. Proc. Natl. Acad. Sci. USA 101:14889–14894.

34. Ono, A., D. Demirov, and E. O. Freed. 2000. Relationship between humanimmunodeficiency virus type 1 Gag multimerization and membrane binding.J. Virol. 74:5142–5150.

35. Ono, A., and E. O. Freed. 1999. Binding of human immunodeficiency virustype 1 Gag to membrane: role of the matrix amino terminus. J. Virol.73:4136–4144.

36. Ono, A., and E. O. Freed. 2004. Cell-type-dependent targeting of human


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

immunodeficiency virus type 1 assembly to the plasma membrane and themultivesicular body. J. Virol. 78:1552–1563.

37. Ono, A., and E. O. Freed. 2001. Plasma membrane rafts play a critical role inHIV-1 assembly and release. Proc. Natl. Acad. Sci. USA 98:13925–13930.

38. Ono, A., J. M. Orenstein, and E. O. Freed. 2000. Role of the Gag matrixdomain in targeting human immunodeficiency virus type 1 assembly. J. Virol.74:2855–2866.

39. Ono, A., A. A. Waheed, A. Joshi, and E. O. Freed. 2005. Association ofhuman immunodeficiency virus type 1 Gag with membrane does not requirehighly basic sequences in the nucleocapsid: use of a novel Gag multimeriza-tion assay. J. Virol. 79:14131–14140.

40. Pelchen-Matthews, A., B. Kramer, and M. Marsh. 2003. Infectious HIV-1assembles in late endosomes in primary macrophages. J. Cell Biol. 162:443–455.

41. Platt, E. J., and O. K. Haffar. 1994. Characterization of human immunode-ficiency virus type 1 Pr55gag membrane association in a cell-free system:requirement for a C-terminal domain. Proc. Natl. Acad. Sci. USA 91:4594–4598.

42. Platt, E. J., K. Wehrly, S. E. Kuhmann, B. Chesebro, and D. Kabat. 1998.Effects of CCR5 and CD4 cell surface concentrations on infections by macro-phagetropic isolates of human immunodeficiency virus type 1. J. Virol. 72:2855–2864.

43. Raposo, G., M. Moore, D. Innes, R. Leijendekker, A. Leigh-Brown, P. Bena-roch, and H. Geuze. 2002. Human macrophages accumulate HIV-1 particlesin MHC II compartments. Traffic 3:718–729.

44. Sandefur, S., V. Varthakavi, and P. Spearman. 1998. The I domain is re-quired for efficient plasma membrane binding of human immunodeficiencyvirus type 1 Pr55Gag. J. Virol. 72:2723–2732.

45. Swanstrom, R., and J. W. Wills. 1997. Synthesis, assembly, and processing ofviral proteins, p. 263–334. In J. M. Coffin, S. H. Hughes, and H. E. Varmus(ed.), Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, N.Y.

46. Tang, C., E. Loeliger, P. Luncsford, I. Kinde, D. Beckett, and M. F. Summers.2004. Entropic switch regulates myristate exposure in the HIV-1 matrixprotein. Proc. Natl. Acad. Sci. USA 101:517–522.

47. Tang, S., T. Murakami, B. E. Agresta, S. Campbell, E. O. Freed, and J. G.Levin. 2001. Human immunodeficiency virus type 1 N-terminal capsid mu-tants that exhibit aberrant core morphology and are blocked in initiation ofreverse transcription in infected cells. J. Virol. 75:9357–9366.

48. von Schwedler, U. K., T. L. Stemmler, V. Y. Klishko, S. Li, K. H. Albertine,D. R. Davis, and W. I. Sundquist. 1998. Proteolytic refolding of the HIV-1capsid protein amino-terminus facilitates viral core assembly. EMBO J.17:1555–1568.

49. von Schwedler, U. K., K. M. Stray, J. E. Garrus, and W. I. Sundquist. 2003.Functional surfaces of the human immunodeficiency virus type 1 capsidprotein. J. Virol. 77:5439–5450.

50. Willey, R. L., J. S. Bonifacino, B. J. Potts, M. A. Martin, and R. D. Klausner.1988. Biosynthesis, cleavage, and degradation of the human immunodefi-ciency virus 1 envelope glycoprotein gp160. Proc. Natl. Acad. Sci. USA85:9580–9584.

51. Worthylake, D. K., H. Wang, S. Yoo, W. I. Sundquist, and C. P. Hill. 1999.Structures of the HIV-1 capsid protein dimerization domain at 2.6 Å reso-lution. Acta Crystallogr. D Biol. Crystallogr. 55(Pt. 1):85–92.

52. Yee, J. K., T. Friedmann, and J. C. Burns. 1994. Generation of high-titerpseudotyped retroviral vectors with very broad host range. Methods CellBiol. 43(Pt. A):99–112.


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Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus...

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