identification of a conserved residue of foamy virus gag required

JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.15.6857–6864.2001

Aug. 2001, p. 6857–6864 Vol. 75, No. 15

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Identification of a Conserved Residue of Foamy Virus GagRequired for Intracellular Capsid Assembly

SCOTT W. EASTMAN AND MAXINE L. LINIAL*

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, andDepartment of Microbiology, University of Washington, Seattle, Washington 98195

Received 27 December 2000/Accepted 3 May 2001

In contrast to all retroviruses but similar to the hepatitis B virus, foamy viruses (FV) require expression ofthe envelope protein for budding of intracellular capsids from the cell, suggesting a specific interaction betweenthe Gag and Env proteins. Capsid assembly occurs in the cytoplasm of infected cells in a manner similar to thatfor the B- and D-type viruses; however, in contrast to these retroviruses, FV Gag lacks an N-terminalmyristylation signal and capsids are not targeted to the plasma membrane (PM). We have found that mutationof an absolutely conserved arginine (Arg) residue at position 50 to alanine (R50A) of the simian foamy virusSFV cpz(hu) inhibits proper capsid assembly and abolishes viral budding even in the presence of the envelope(Env) glycoproteins. Particle assembly and extracellular release of virus can be restored to this mutant withthe addition of an N-terminal Src myristylation signal (Myr-R50A), presumably by providing an alternate sitefor assembly to occur at the PM. In addition, the strict requirement of Env expression for capsid budding canbe bypassed by addition of a PM-targeting signal to Gag. These results suggest that intracellular capsidassembly may be mediated by a signal akin to the cytoplasmic targeting and retention signal CTRS found inMason-Pfizer monkey virus and that FV Gag has the inherent ability to assemble capsids at multiple sites likeconventional retroviruses. The necessity of Env expression for particle egress is most probably due to the lackof a membrane-targeting signal within FV Gag to direct capsids to the PM for release and indicates thatGag-Env interactions are essential to drive particle budding.

Foamy viruses (FV), classified in the Spumavirus genus ofthe Retroviridae family, are unique viruses sharing morpho-genic features found among many diverse types of envelopedviruses, including the human hepatitis B virus. Although FVscause substantial cytopathic effects in tissue culture (hence thename “foamy”, referring to the highly vacuolated appearanceof infected cells), an asymptomatic and persistent infection isseen in nature in a wide variety of organisms including non-human primates, cats, cattle, and horses (22, 27, 42, 46). Thegenomic organization of the FVs, including the prototype mo-lecular cloned simian foamy virus SFVcpz(hu), is similar tothat of other complex retroviruses, with several additionalopen reading frames located 39 of the canonical gag, pol, andenv genes, including the transcriptional transactivator gene, tas(23, 29, 36). Unlike in retroviruses, however, the Pol protein isexpressed from a unique spliced mRNA, not as a Gag-Polfusion, and therefore must be specifically incorporated intonewly forming capsids (14, 28, 47). In addition, reverse tran-scription of the genome is initiated early, during budding ofcapsids, viral egress, or prior to infection of new cells, suggest-ing a novel coordination of morphogenesis (50).

The main FV structural protein, Gag, is also different fromthat of other retroviruses because of the lack of proteolyticprocessing into the MA, CA, and NC domains, and, corre-spondingly, extracellular viral particles possess an immaturemorphology (9). The majority of viral protease-specific Gagcleavage is limited to a single event near the C terminus of the

protein, releasing an approximately 3-kDa protein from the71-kDa precursor peptide (13, 16, 35), although it has beenproposed that additional cleavage occurs on infection of newcells during viral uncoating (16, 34, 39). In addition, FV Gaglacks the major homology region found in the capsid proteinsof all other retroviruses, as well as the signature Cys-His boxmotifs found in all retroviral Gag NC proteins (43a). Instead,FV Gag possesses three glycine-arginine rich domains, termedGR boxes I to III, situated at the C terminus of the protein,which are involved in nucleic acid binding and nuclear local-ization (41, 48) and possibly particle density (4). By 24 hpostinfection, a strong nuclear Gag signal is seen in all celltypes infected with FVs (the serological hallmark of FV infec-tion), although transport of Gag to the nucleus is not essentialfor the production of infectious virus and the role of Gagnuclear localization is not known (22, 41, 48).

FV particle assembly occurs in the cytosolic compartment (8,10). Similar to the intracisternal A-type particles but distinctfrom all other retroviruses, FV capsids bud through the endo-plasmic reticulum (ER) membrane. The FV envelope (Env)protein is also retained in the ER by means of a trilysine motiflocated at the C-terminal cytoplasmic tail of Env (18, 19). FVmorphogenesis requires the presence of the Env protein toallow release of virus from the cell, a mechanism also em-ployed by hepatitis B virus, such that capsid budding is com-pletely inhibited in the absence of Env expression (2, 5, 15). Incontrast, all other retroviruses have the ability to assemble andbud capsids from a variety of cell types on the sole expressionof the gag gene (3; Wills and Craven, Editorial).

While the mechanism of intracellular capsid assembly is notfully understood for the B- and D-type retroviruses, recentexperiments with the D-type retrovirus Mason-Pfizer monkey

* Corresponding author. Mailing address: Division of Basic Sci-ences, Fred Hutchinson Cancer Research Center, A3-015, 1100 Fair-view Ave. N., Seattle, WA 98109-1024. Phone: (206) 667-4442. Fax:(206) 667-5939. E-mail: [email protected].

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virus (MPMV) have show that an 18-amino-acid domain nearthe N terminus of Gag, termed the cytoplasmic targeting andretention signal (CTRS) and centered on a highly conservedarginine (Arg) residue at position 55, is required to direct thecytoplasmic assembly of capsids (7, 32, 38, 40). Mutation ofArg55 of Gag to tryptophan (R55W) results in a switch ofmorphology to the default type C capsid assembly at theplasma membrane (PM), a process dependent on the N-ter-minal myristylation signal (37, 38). Remarkably, fusion of theCTRS to green fluorescent protein (GFP) resulted in discretestaining at cytoplasmic sites, and, furthermore, addition of theCTRS to the C-type retrovirus Moloney murine leukemia virusGag protein conferred intracellular assembly (7).

All sequenced FV Gag proteins have a high level of conser-vation near the N terminus, including an absolutely conservedarginine at amino acid 50. Comparison of this region of FVGag with the CTRS of MPMV reveals a number of identicalresidues, comprising a domain of FV Gag [GXWGX3RX7L(Q/V)D], centered on the conserved Arg (7, 42). We predictedthat if cytoplasmic assembly of FV capsids is mediated by se-quences in this region, mutation of conserved residues mightblock assembly altogether, since the FV Gag protein is notmyristylated and is not known to possess a membrane-target-ing signal. We found that alanine or tryptophan substitution atposition 50 (R50A/W) of SFVcpz(hu) severely disrupts capsidassembly in transfected cells and completely abolishes the re-lease of virus from the cell even in the presence of Env. Capsidassembly and budding of the R50A mutant was restored if Gagwas given a targeting signal via the addition of an N-terminalSrc myristylation signal (33, 43, 44). Extracellular particleswere produced at wild-type levels but were not infectious, andGag cleavage was completely blocked. Surprisingly, addition ofa myristylation signal bypassed the strict requirement for Envin particle budding, suggesting that Gag does not possess aninherent targeting signal and therefore relies on interactionwith Env for particle egress.

MATERIALS AND METHODS

Recombinant plasmid DNAs. The infectious molecular clone of SFVcpz(hu),designated pHSRV13, was used for all experiments described below (30). Pro-viral mutants R50A and R50W were created by PCR mutagenesis using theMorph kit (5 Prime33 Prime) with a modified version of FV subclone 1 (2)renamed G3 (AvrII and EcoRV sites deleted from the polylinker, consisting of a2,885-bp fragment of pHSRV13 from EagI to SwaI) as a template and using theoligonucleotides primers MAR1A (59-GGACAAATT GAGGCATTTCAGATGG-39) and MAR1W (59-GTGGGGACAAATTGAGTGGTTTC AGATGGTACG-39) to change the Arg residue (AGA) to Ala (GCA) and Trp (TGG),respectively. The Src myristylation signal (GSSKSKPKD) was introduced intothe G3 subclone by inverse PCR with the oligonucleotides SRC1 (59-GGCTCATCGAAGAGCAAGCCTAAGGACGAACTTGATGTTGAAGC-39) andSRC2 (59-GTCCTTAGGCTTGCTCTTCGATGAGCCCATTGTCTATTGGCTTT-39) to create the Myr and Myr-R50A proviruses. The Env deletion mutantswere cloned with a 2,536-bp fragment (PacI [position 4644] to BlpI [position9193]) from the provirus DMN (deleted from MroI [BspEI] [position 6957] toNdeI [position 8970] provided by Martin Loechelt) (2).

All 293T cell transfections were conducted with proviruses containing theimmediate-early promoter from the cytomegalovirus (CMV) in place of the U5region of the 59 long terminal repeat (LTR) sequence of pHSRV13, such thatexpression of the viral RNA is initiated from the same nucleotide as for thewild-type RNA transcribed from U3 (D. Baldwin and M. Linial, unpublisheddata). Briefly, the CMV immediate-early promoter was PCR amplified frompCR3.0 (Invitrogen) with oligonucleotide primers containing EagI and XhoI sites(59 and 39, respectively) to clone into a modified pHSRV13 subclone (sub1)described previously (2) containing a linker with an additional XhoI site between

EagI and XhoI (Baldwin and Linial, unpublished). The R50A and Myr-R50Amutants were introduced into CMVsub1 with restriction sites AvrII and PflMI.

Cells and transfections. FAB indicator cells, BHK cells containing an inte-grated copy of the b-galactosidase gene under the control of the SFVcpz(hu)LTR (49), were maintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 5% fetal bovine serum. Human embryonic lung cells (HEL),thymocytes (CF3TH), and 293 T cells were maintained in Dulbecco’s modifiedEagle’s medium with 10% fetal bovine serum. FAB cell transient transfectionswere conducted using the Lipofectamine reagent (Life Technologies, Inc.) aspreviously described (2) with 5 mg of proviral DNA, as well as the Fugenereagent (Roche Molecular Biochemicals) in which 10 mg of proviral DNA wasadded to 250 ml of DMEM containing 25 ml of Fugene, mixed and incubated atroom temperature for 15 min, added to cells in 10 ml of medium, and rinsed after24 h. 293 T cell transient transfections were conducted using a modified calciumphosphate method (6) in which 8 mg of proviral DNA plus 2 mg of LTR-GFPreporter plasmid were combined with 0.5 ml of 0.25 M CaCl2, then added to 0.5ml of 23 BES buffered solution mixed and incubated at room temperature for 20min, and added for 18 to 20 h to 10-cm-diameter dishes containing cells atapproximately 75 to 85% confluency in 10 ml of medium.

Western blotting. Proteins were analyzed by Western blotting of cell lysatesand viral supernatants with the anti-Gag polyclonal antiserum as previouslydescribed (2), with the following modifications. FAB and 293 T cells werescraped in phosphate-buffered saline (PBS) 40 to 42 h posttransfection, pelleted,and rinsed three times with PBS. Cell pellets were lysed in 1 ml of antibody buffer(20 mM Tris [pH 7.5], 50 mM NaCl, 0.5% NP-40, 0.5% sodium dodecyl sulfate[SDS], 0.5% deoxycholate, 0.5% aprotinin, supplemented with 100 mg of phe-nylmethylsulfonyl fluoride per ml and 1 mg of leupeptin per ml), passed througha 23-gauge syringe to shear chromosomal DNA, cleared of cell debris by cen-trifugation in the microcentrifuge, mixed with SDS protein sample buffer, boiled,and loaded on SDS-polyacrylamide gel electrophoresis (PAGE) minigels (10%polyacrylamide). Culture supernatants were passed through 0.45-mm-pore-sizesyringe filters and pelleted through a 20% sucrose cushion (2 ml) at 24,000 rpmand 4°C for 2 h in a total volume of 17.5 ml (SW28 rotor; Beckman). Viral pelletswere resuspended with protein sample buffer and loaded onto SDS-PAGE mini-gels (10% polyacrylamide). After separation, proteins were transferred to Im-mobilon-P membranes (Millipore), blocked in 5% nonfat milk in PBS, andincubated with anti-Gag antiserum at 1:2,000 overnight. The membranes werewashed three times in PBS containing 0.1% Tween 20 (PBS-T) and incubatedwith horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Amer-sham) antibody at 1:7,500 dilution for 1 h. They were then washed four times for20 min in PBS-T and visualized by enhanced chemiluminescence (Amersham).

Linear-velocity sedimentation gradients. Transiently transfected 293 T cellswere washed with PBS and lysed in NP-40 lysis buffer (1% NP-40, 50 mM NaCl,10 mM Tris-Cl [pH 7.4], 5 mM EDTA) for 30 min on ice. Lysates were clearedwith an initial low-speed centrifugation at 4,000 3 g for 10 minutes followed bycentrifugation at 15,000 3 g for 5 min in a microcentrifuge, and the resultingsupernatants were pelleted through 750 ml of 20% sucrose in TNE (20 mM Tris[pH 7.5], 150 mM NaCl, 1 mM EDTA) at 25,000 rpm for 2 h and 4°C in a totalvolume of 5 ml (Ti55 rotor; Beckman). The pellets were resuspended in 300 mlof TNE, placed onto 5 ml of NP-40 lysis buffer containing sucrose step gradientsconsisting of 1.2 ml of 20%, 2 ml of 40%, and 1.5 ml of 66% sucrose, andultracentrifuged at 30,000 rpm for 1 h (Ti55 rotor; Beckman) at 4°C, and eight635-ml fractions were collected from the top, as well as the pellet fraction(resuspended in NP-40 lysis buffer) (26). Fractions were precipitated for 1 h at220°C with trichloroacetic acid (TCA) (consisting of a final concentration of25%) and 10 mg of yeast tRNA, centrifuged at 16,000 3 g for 20 min in amicrocentrifuge, washed with 10% TCA and then with 100% acetone, air dried,and resuspended in 13 protein sample buffer. Fractions were subjected to SDS-PAGE (10% polyacrylamide) and Western blotting. Extracellular virus was re-covered from culture supernatants by pelleting through 20% sucrose and ana-lyzed on these gradients after removal of viral envelopes by treatment with 1%NP-40 in TNE.

Indirect immunofluorescence (IFA). Transiently transfected FAB cells onglass coverslips were rinsed with PBS and fixed for 5 min at room temperaturewith 4% paraformaldehyde at 36 to 40 h posttransfection. The cells were per-meabilized with 1% Triton-X in PBS for 5 min, washed with PBS, and blockedin 5% heat-inactivated bovine serum albumin for at least 30 min at 4°C. Thecoverslips were then rinsed and incubated with anti-Gag serum (1:2,000) for 1 hat 37°C, rinsed with PBS for 15 min three times, incubated with anti-rabbitfluorescein isothiocyanate (FITC)-conjugated antibody (1:1,000) for 45 min at25°C, and rinsed with PBS as before. They were then stained with 49,6-diamidino-2-phenylindole (DAPI; 0.2, mg/ml) for 5 min in double-distilled H2O washed in

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double-distilled H2O, and mounted in Vectashield (Vector Laboratories). Im-aging was performed using a Nikon TE300 microscope and Metamorph software.

RESULTS

Point mutation of the arginine at position 50 (R50) of FVGag inhibits particle assembly and blocks viral budding. Anal-ysis of the Gag amino acid sequence of all FV isolates shows anabsolute conservation of the arginine at position 50 (R50) fromthe N terminus (Fig. 1A). The sequence of SFVcpz(hu) in thevicinity of this conserved R50 is reminiscent of the CTRS ofthe D-type retrovirus MPMV (Fig. 1B) (7, 38), including anumber of identical residues (7, 38), comprising a domain ofFV Gag [GXWGX3RX7L(Q/V)D] (42). To determinewhether R50 of SFVcpz(hu) is involved in intracytoplasmicassembly, we made substitutions to alanine (R50A) and tryp-

tophan (R50W) (Fig. 1C). Proviral mutants under the controlof the CMV immediate-early promoter were transfected into293T cells along with the wild-type (wt) molecular clone ofSFVcpz(hu), HSRV. Intracellular Gag proteins and extracel-lular virus that was purified from culture supernatants werethen analyzed by Western blotting. The cellular Gag expres-sion levels (Fig. 2A) of R50A and R50W were comparable tothose of wt HSRV. Extracellular release of virus (Fig. 2B),however, was completely abolished with these mutant provi-ruses compared to the budding competent virus. wt HSRVcannot spread in this cell type and therefore provides a propercontrol for a single-cycle infection. We performed the sametransient-transfection experiments with FAB cells, which arepermissive for replication and viral spread, to determinewhether the block to assembly and viral release was cell typespecific, and we observed identical results (data not shown).

Next, we tested the hypothesis that the lack of particle bud-ding observed with the R50A/W mutants is caused by theinhibition of intracellular capsid assembly. We first used astandard equilibrium density centrifugation method to detectassembled retroviral particles in the cell. Transiently trans-fected FAB cell lysates were placed on gradients by previouslydescribed methods (1), and Gag protein was found to band ata density of approximately 1.14 g/ml for both the wt and R50Amutant proviruses on Western blots (data not shown). Thisfinding was both inconsistent with our prediction of FV intra-cellular assembly occurring via a CTRS-type domain and puz-zling since we observed a complete lack of extracellular virusproduced from the R50A mutant provirus. However, previousexperiments performed in the laboratory using proviral mu-tants lacking gag (DGag) indicated that viral polymerase pro-teins exhibited banding characteristics that are indistinguish-able from banding seen with wt virus on Western blots offractions collected from identical density equilibrium gradients(Baldwin and Linial, unpublished). Thus, the equilibrium cen-trifugation does not appear to distinguish between unas-sembled viral protein complexes and assembled viral capsidsunder these conditions. Therefore, we used the linear-velocity

FIG. 1. Schematic representation of FV Gag sequence alignments,genomic structure map, and location of proviral mutants. (A) Se-quence alignment of the N-terminal portion of the Gag protein fromall characterized FV molecular clones including the amino acid posi-tion number. p, absolute conservation; /, conservative change. (B) Se-quence alignment (Clustal W) of the N-terminal portion of SFVcpz(hu) Gag with the CTRS domain from the D-type retrovirus MPMV,with the absolutely conserved Arg residue highlighted in bold. (C)Genomic structure of SFVcpz(hu) gag including the 59 region of CMV-driven HSRV proviral constructs containing the CMV immediate-early promoter in place of the 59 LTR for initiation of viral transcrip-tion. The location of all proviral mutant sequences is shown in relationto wt HSRV. The Myr mutant contains the 10-amino-acid N-terminalSrc myristylation signal (MGSSKSKPKD) in place of the first 10 aminoacids of Gag. R50A and R50W have alanine and tryptophan substitu-tions, respectively, of the conserved arginine residue at position 50.DEnv proviruses have a 2-kb deletion in env (2).

FIG. 2. R50A/W mutation blocks the release of virus from FV-transfected cells. Western blot analysis of transiently transfected 293Tcells expressing proteins from wt and mutant CMV-driven provirusesis shown. (A) Cellular lysates prepared 40 h posttransfection from cellstransfected with HSRV or the conserved Arg substitution mutantsR50A and R50W, as well as a mock-transfected negative control. (B)Extracellular virus isolated from the same culture supernatants by 20%sucrose cushion sedimentation, including wt HSRV, the conserved Argsubstitution mutants R50A and R50W, and the mock-transfected neg-ative control. Viral proteins were visualized using the anti-Gag sera.MW, molecular weight in thousands.

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sedimentation techniques that were previously used to exam-ine the human immunodeficiency virus (HIV) capsid assemblypathway (26). We used a modified lysis procedure with 1%NP-40 in an attempt to discriminate bona fide intracellularcapsids from unassembled Gag monomers and protein aggre-gates. Cell lysates were centrifuged through 20% sucrose, andthe pelleted material was placed on linear-velocity sedimenta-tion gradients made from 20 to 66% sucrose containing 1%NP-40 and centrifuged under conditions found to band HIVcapsids (approximately 750S) in the middle of the gradient(fraction 5) (26). We found that whereas cellular expressionlevels of HSRV and R50A mutant Gag were identical (Fig.3A), the R50A mutant gradient was devoid of Gag proteincompared with the wt gradient containing Gag capsids in frac-tion 6 (Fig. 3B). This indicated that the initial 20% sucrose spinseparated actual capsids (HSRV) from protein aggregates(R50A).

Plasma membrane targeting of R50A restores capsid assem-bly and extracellular release of virus. We next tested whetherthe R50A mutant, lacking a putative intracellular signalingdomain, could synthesize particles if supplied with an alternatetargeting signal. There is precedent for this type of experiment,since studies with the mouse intracisternal A-type particle ret-rovirus, which assembles and buds capsids into the ER thatremain within the ER lumen, have shown that redirection ofGag to the PM by myristylation allows extracellular release ofvirus (43). We substituted the Src targeting signal, a 10-amino-acid N-terminal peptide previously reported to act as a domi-nant plasma membrane-targeting domain dependent on myri-stylation and the presence of 3 lysine residues, for the first 10

amino acids at the N terminus of the R50A mutant Gag pro-tein to produce the Myr-R50A provirus (Fig. 1B) (44, 45).CMV-driven proviral mutants or HSRV were transiently trans-fected into 293 T cells, and cellular lysates and viral super-natants were analyzed. We first checked the ability of themyristylated form of the R50A mutant (Myr-R50A) to formintracellular capsids by using the intracellular assembly assaydescribed above. Cellular lysates from HSRV- or Myr-R50A-transfected cells (Fig. 4A) were pelleted through 20% sucrose,placed on linear-velocity gradients, and fractionated. Remark-ably, these results indicated that the Myr-R50A mutant Gagwas able to pellet through 20% sucrose and was found topossess Gag sedimentation characteristics indistinguishablefrom those of the wt on the linear-velocity gradients (Fig. 4B).

Next, we determined whether myristylation of R50A Gagcould restore viral budding. Again, 293 T cells were transfectedwith the proviral constructs and Western blot analyses wereperformed from whole-cell lysates. The results show similarexpression levels of cellular Gag (Fig. 5A) for HSRV, R50A,and Myr-R50A, although proteolytic processing of Gag wasinhibited. Analysis of purified culture supernatants (Fig. 5B),however, revealed a rescue of viral release on addition ofthe PM-targeting signal (Myr-R50A), at levels comparable tothose for HSRV, whereas the R50A mutant did not releasevirus. We consistently observed the absence of Gag cleavagewith the extracellular Myr-R50A viruses. Identical results wereobserved using FAB cells (data not shown). In addition, extra-cellular virus produced from cells transfected with HSRV orthe Myr-R50A mutant proviruses (Fig. 5C, cell lysate) wasanalyzed on linear-velocity sedimentation gradients after re-moval of the viral envelope with 1% NP-40. The results showthat the Myr-R50A mutant exhibited sedimentation charac-teristics identical to those of wt HSRV, with Gag banding infraction 6 (Fig. 5C). The infectivity of the Myr-R50A wasassessed using the FAB assay (49), and the virus was found tobe noninfectious, containing less than 1 infectious unit (IU)per ml of culture supernatant, compared with 105 to 106 IU/mlfor wt virus (data not shown).

Subcellular localization of mutant Gag proteins and assem-bled capsid structures. Subcellular localization of the mutantviral proteins was analyzed by indirect immunofluorescence(IFA) of transiently transfected FAB cells using the anti-Gag

FIG. 3. R50A mutation inhibits capsid assembly. Western blotanalysis of transiently transfected 293T cells expressing proteins fromwt and mutant CMV-driven proviruses is shown. (A) Total-cell lysates.(B) Cell lysates from HSRV and the R50A mutant. These lysates werepelleted through 20% sucrose, resuspended in TNE, and layered onto20, 40, and 66% sucrose linear velocity sedimentation gradients. Frac-tions were collected from the top of the gradients (lane 1) and TCAprecipitated. Viral proteins were visualized using the anti-Gag sera.The arrow indicates the location of HIV ;750S particles analyzed onparallel gradients.

FIG. 4. Plasma membrane targeting of R50A mutant with the Src-myristylation signal restores intracellular capsid assembly. Westernblot analysis of transiently transfected 293T cells expressing proteinsfrom wt and mutant CMV-driven proviruses is shown. (A) Total-celllysates. (B) Linear-velocity sedimentation gradient analysis of cell ly-sates from HSRV (top) and Myr-R50A mutants (bottom). Viral pro-teins were visualized using the anti-Gag sera.



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serum. Expected distinctive nuclear fluorescence was seen incells transfected with HSRV (Fig. 6B), as well as the R50Amutant (Fig. 6C) (41). Addition of the Src membrane-bindingdomain to wt and R50A mutant Gag significantly altered pro-tein localization. Cell nuclei appeared dark with a correspond-ing punctate staining of cytoplasmic regions including PM sites(Fig. 6D and E). Comparison of the Myr mutants with a mu-tant lacking the NLS in GR box II, DNLS (50) (Fig. 6F),revealed that whereas nuclei were not stained in either case,the Myr-tagged proteins appeared to accumulate in brightlystaining regions within the cytoplasm and at the PM, as op-posed to the diffuse cytoplasmic staining seen in cells trans-fected with the DNLS mutant. These staining patterns arereminiscent of HIV Gag localization with and without N-ter-minal myristylation, respectively (31).

Redirection of Gag to the plasma membrane allows FVparticle budding in the absence of Env. We next examinedwhether the intracellular assembly mutant virus containing thePM-targeting signal, Myr-R50A, required Env glycoproteinsynthesis for extracellular release. Our previous finding thatparticle budding is dependent on the presence of Env suggeststhat FV capsids do not possess an inherent membrane-target-ing signal and that specific Gag-Env interactions are requiredfor capsids to be released from the cell. We hypothesized that

if Gag was given a specific mechanism for localization, such asthe Src plasma membrane-targeting signal, the Env require-ment could be bypassed and capsids would bud from the PM inthe absence of Env expression. We deleted most of the envgene from each provirus to create mutants (DEnv) that do notexpress Env but do express all other proteins at wt levels andconducted transfection experiments as before (2). Westernblot analysis of cellular lysates from 293 T cells transientlytransfected with the mutant proviral clones showed compara-ble levels of intracellular Gag (Fig. 7A). Analysis of extracel-lular virus purified from these culture supernatants (Fig. 7B),however, showed that in the absence of Env expression, theMyr/DEnv (lane 8) and Myr-R50A/DEnv (lane 9) mutants re-leased virus at levels at or surpassing those of budding-com-petent noninfectious control viruses D936I (integrase mutant,lane 2), and HSRV-D/A (protease active site mutant, lane 3).In contrast, HSRV/DEnv (lane 7) was completely inhibited inparticle release, similar to the R50A mutant (lane 4) and mocktransfected cells (lane 1).

DISCUSSION

In this study, we have shown that a single substitution of anabsolutely conserved residue in the N-terminal region of FVGag severely inhibits intracellular capsid assembly and ablatesviral particle release. This residue (R50) is situated in a regionof high conservation among all characterized FVs and hasmoderate homology to the recently defined CTRS signal fromthe D-type retrovirus MPMV (7). Nuclear magnetic resonanceanalysis of the MA domain of MPMV Gag suggests that the18-amino-acid CTRS region exists on an exposed loop of theprotein, not found in the matrix domain of Gag from C-typeviruses (11, 12). The CTRS is proposed to target and retainGag molecules at a specific site within the cell, allowing forlocalized increases in protein concentrations such that Gag-Gag interactions and assembly may proceed (7). It is also likelythat a cellular factor(s) is involved in the targeting process, ahypothesis supported by the recent report of the discovery ofan insect cell line defective in the transport of assembled cap-sids to the PM (32). We propose that FV assembly occurs freeof membranes in a fashion similar to the B- and D-type retro-viruses and that capsid formation is mediated by a signal akinto the CTRS domain.

Targeting domains for all characterized retroviral Gag pro-teins reside at the amino-terminal MA domain (24). There isno obvious membrane-targeting signal on FV Gag, and, cor-respondingly, we found that a disruption of the cytoplasmicassembly signal blocked capsid formation altogether. If a PMtargeting signal was provided, however, in the form of the Srcmyristylation signal, as in Myr or Myr-R50A, capsid assemblyproceeded and particle release was restored to wt levels. Aswitch to C-type assembly at the PM was not detected withthese mutants when transiently transfected cells were analyzedby electron microscopy, but this mechanism of capsid forma-tion cannot be ruled out. Indeed, we found that the totalamounts of intracellular Myr-R50A mutant Gag were alwayssignificantly reduced compared to HSRV (Fig. 5A) in cellstransfected with similar levels of provirus as detected by co-transfection of an LTR-GFP plasmid. Extracellular Myr-R50Avirus, however, was found to be present at wt levels (Fig. 5B),

FIG. 5. Plasma membrane targeting of R50A mutant restores viralbudding. Western blot analysis of transiently transfected 293T cellsexpressing proteins from wt and mutant CMV-driven proviruses isshown. (A) Cell lysates prepared 41 h posttransfection includingHSRV, the R50A mutant, and the myristylated R50A mutant, Myr-R50A, as well as a mock-transfected negative control. (B) Extracellularvirus isolated from culture supernatants of the same cell transfectionsby 20% sucrose cushion centrifugation. (C) Linear-velocity sedimen-tation gradients of extracellular virus (treated with 1% NP-40 to re-move viral envelope) produced from cells transfected with the HSRV(top) and Myr-R50A (bottom) proviruses (whole-cell lysates shown atleft). Viral proteins were visualized using anti-Gag sera. MW, molec-ular weight in thousands.



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FIG. 6. Subcellular Gag localization using IFA. Transiently transfected FAB cells (phase-contrast images [left panel]) were fixed 36 to 40 h post-transfection, incubated with anti-Gag sera, and visualized with FITC (center panel). Nuclei are stained with DAPI (right panel). The proviruses aremock (A) HSRV (B), the R50A mutant (C), Myr (D), and Myr-R50A (E), as well as the control mutant lacking the NLS in GR box II, DNLS (F) (48).



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indicating that PM targeting of Gag may be increasing theefficiency of capsid assembly and budding.

Of considerable interest is the fact that myristylated Gagproteins created capsids that budded from cells in the absenceof Env expression. FV capsids are capable of budding from thePM rather than the ER. SFVcpz(hu) mutant viruses, whichlack the Env ER retention signal, will bud at wt levels from thePM, where Env is localized (17, 18). Interestingly, the equineand bovine FV possess sequences homologous to the intracel-lular assembly domain (Fig. 1A) but lack the ER retentionsignal on the Env protein and, correspondingly, bud virussolely from the PM (21, 42). Recently, it has been reported thatthe N terminus of the leader peptide of SFVcpz(hu) Env,termed the budding domain, is required for efficient particlerelease (25). We propose that intracellular budding of FVcapsids is a process normally driven by a Gag-Env interactionbut that if capsids are given an alternate localization signal,such as the N-terminal Src membrane-targeting signal on Gag,budding may occur from the PM in the absence of Env.

Also of interest is the observation that extracellular virusproduced from the Myr-R50A mutant exhibited reduced Gagcleavage and was noninfectious. While many FV mutationscause slight alterations in intracellular Gag cleavage, virus re-leased from cells containing the Myr-R50A Gag mutant pro-virus appeared to be devoid of any proteolytic processing (Fig.

5B; compare lanes 6 and 8; Fig. 7B, compare lanes 2 and 6),whereas the provirus with only the Src-targeting signal (Myr)produced virus with an intermediate cleavage phenotype (Fig.7B, compare lanes 2, 5, and 6). Several possibilities couldaccount for this deficiency in Gag cleavage. Addition of the Srcsignal to Gag may alter the proper folding and conformation ofthe protein such that processing is prevented. Also, it is con-ceivable that these mutant viral particles lack a cellular factorrequired for protease activation or that these mutant virusparticles have reduced levels of Pol. If capsid formation ishighly site specific, perhaps a specific mechanism exists totarget Pol into assembling particles; thus, any perturbation ofthe site of assembly will affect Pol incorporation. Alternatively,these particles may be devoid of viral nucleic acid, which mayalso influence the levels of Pol in virus. A recent study analyz-ing the region upstream of the primer-binding site of SFVcpz(hu) indicates that deletion of a 29-nucleotide domain in theU5 region of the 59 LTR, while having no effect on packagingof nucleic acid, inhibits Gag cleavage in extracellular virus (20).Perhaps assembly of RNA and Pol into capsids is a highlycoordinated event occurring at a discrete location and subtlemutations in Gag have a substantial impact on this process.

The block to infection by the Myr-R50A virus could be dueto the lack of cleavage, Pol and/or genome packaging, or fail-ure to incorporate Env. Previous experiments by others haveshown that C-terminal Gag cleavage is essential for infectivity(13, 51). However, Myr-R50A viruses, which appear to budfrom the plasma membrane, are also likely to be missing Env.Lack of Env would block the Myr-R50A viruses from enteringnew cells. The presence of Pol, Env, and RNA in these virusesis currently under investigation. Our data indicate that the siteof assembly of FV capsids is critical for the formation of aninfectious viral particle. Although FV capsids have the inher-ent ability to assemble at different sites within the cell includingthe PM, assembly at the CTRS-mediated intracellular site ap-pears to be essential for proper incorporation of all viral com-ponents.

ACKNOWLEDGMENTS

S.W.E. was supported by Viral Oncology Training Grant T32 0229from the National Cancer Institute. This investigation was also sup-ported by grant CA18282 from the National Cancer Institute to M.L.L.

We thank Jaisri Lingappa (University of Washington, Pathobiology)for assistance with linear-velocity sedimentation gradients as well asinvaluable suggestions and discussion, and we thank Michael Emer-man (FHCRC) for critical review of the manuscript.

REFERENCES

1. Baldwin, D. N., and M. L. Linial. 1999. Proteolytic activity, the carboxyterminus of Gag, and the primer binding site are not required for Polincorporation into foamy virus particles. J. Virol. 73:6387–6393.

2. Baldwin, D. N., and M. L. Linial. 1998. The roles of Pol and Env in theassembly pathway of human foamy virus. J. Virol. 72:3658–3665.

3. Boulanger, P., and I. Jones. 1996. Use of heterologous expression systems tostudy retroviral morphogenesis. Curr. Top. Microbiol. Immunol. 214:237–260.

4. Bowzard, J. B., R. P. Bennett, N. K. Krishna, S. M. Ernst, A. Rein, and J. W.Wills. 1998. Importance of basic residues in the nucleocapsid sequence forretrovirus Gag assembly and complementation rescue. J. Virol. 72:9034–9044.

5. Bruss, V., and D. Ganem. 1991. The role of envelope proteins in hepatitis Bvirus assembly. Proc. Natl. Acad. Sci. USA 88:1059–1063.

6. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mam-malian cells by plasmid DNA. Mol. Cell. Biol. 7:2745–2752.

7. Choi, G., S. Park, B. Choi, S. Hong, J. Lee, E. Hunter, and S. S. Rhee. 1999.Identification of a cytoplasmic targeting/retention signal in a retroviral Gagpolyprotein. J. Virol. 73:5431–5437.

FIG. 7. Myr-R50A mutant virus budding in the absence of Env.Western blot analysis of CMV-driven mutant proviruses transfectedinto 293 T cells is shown (A) Cell lysates prepared 41 h posttransfec-tion, including the integrase mutant, D936I (lane 2), and the proteaseactive-site mutant, HSRV-D/A (lane 3), R50A (lane 4), Myr (lane 5),Myr-R50A (lane 6), the HSRV envelope deletion, HSRV/DEnv (lane7), Myr/DEnv (lane 8), and Myr-R50A/DEnv (lane 9), as well as mock-transfected cells (lane 1). (B) Extracellular virus purified from thesame culture supernatants by 20% sucrose cushion centrifugation, withthe same lane designations. Viral proteins were visualized using anti-Gag sera.



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Dow

nloaded from

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8. Chopra, H. C., J. J. Hooks, M. J. Walling, and C. J. Gibbs, Jr. 1972.Morphology of simian foamy viruses, with particular reference to virus iso-lated from spontaneous tumor of a rhesus monkey. J. Natl. Cancer Inst.48:451–463.

9. Clarke, J. K., and J. T. Attridge. 1968. The morphology of simian foamyagents. J. Gen. Virol. 3:185–190.

10. Clarke, J. K., F. W. Gay, and J. J. Attridge. 1969. Replication of simianfoamy virus in monkey kidney cells. J. Virol. 3:358–362.

11. Conte, M. R., M. Klikova, E. Hunter, T. Rumi, and S. Matthews. 1997. Thethree-dimensional solution structure of the matrix protein from the type Dretrovirus, the Mason-Pfizer monkey virus, and implications for the mor-phology of retroviral assembly. EMBO J. 16:5819–5826.

12. Conte, M. R., and S. Matthews. 1998. Retroviral matrix proteins: a structuralperspective. Virology 246:191–198.

13. Enssle, J., N. Fischer, A. Moebes, B. Mauer, U. Smola, and A. Rethwilm.1997. Carboxy-terminal cleavage of the human foamy virus Gag precursormolecule is an essential step in the viral life cycle. J. Virol. 71:7312–7317.

14. Enssle, J., I. Jordan, B. Mauer, and A. Rethwilm. 1996. Foamy virus reversetranscriptase is expressed independently from the Gag protein. Proc. Natl.Acad. Sci. USA 93:4137–4141.

15. Fischer, N., M. Heinkelein, D. Lindemann, J. Enssle, C. Baum, E. Werder,H. Zentgraf, J. G. Muller, and A. Rethwilm. 1998. Foamy virus particleformation. J. Virol. 72:1610–1615.

16. Giron, M. L., S. Colas, J. Wybier, F. Rozain, and R. Emanoil-Ravier. 1997.Expression and maturation of human foamy virus Gag precursor polypep-tides. J. Virol. 71:1635–1639.

17. Goepfert, P. A., K. Shaw, G. Wang, A. Bansal, B. H. Edwards, and M. J.Mulligan. 1999. An endoplasmic reticulum retrieval signal partitions humanfoamy virus maturation to intracytoplasmic membranes. J. Virol. 73:7210–7217.

18. Goepfert, P. A., K. L. Shaw, G. D. Ritter, Jr., and M. J. Mulligan. 1997. Asorting motif localizes the foamy virus glycoprotein to the endoplasmicreticulum. J. Virol. 71:778–784.

19. Goepfert, P. A., G. Wang, and M. J. Mulligan. 1995. Identification of an ERretrieval signal in a retroviral glycoprotein. Cell 82:543–544.

20. Heinkelein, M., J. Thurow, M. Dressler, H. Imrich, D. Neumann-Haefelin,M. O. McClure, and A. Rethwilm. 2000. Complex effects of deletions in the59 untranslated region of primate foamy virus on viral gene expression andRNA packaging. J. Virol. 74:3141–3148.

21. Holzschu, D. L., M. A. Delaney, R. W. Renshaw, and J. W. Casey. 1998. Thenucleotide sequence and spliced pol mRNA levels of the nonprimate spu-mavirus bovine foamy virus. J. Virol. 72:2177–2182.

22. Hooks, J. J., and C. J. Gibbs, Jr. 1975. The foamy viruses. Bacteriol. Rev.39:169–185.

23. Keller, A., K. M. Partin, M. Lochelt, H. Bannert, R. M. Flugel, and B. R.Cullen. 1991. Characterization of the transcriptional trans activator of hu-man foamy retrovirus. J. Virol. 65:2589–2594.

24. Krausslich, H. G., and R. Welker. 1996. Intracellular transport of retroviralcapsid components. Curr. Top. Microbiol. Immunol. 214:25–63.

25. Lindemann, D., T. Pietschmann, M. Picard-Maureau, A. Berg, M. Heinke-lein, and P. K. J. Thurow, H. Zentgraf, and A. Rethwilm. 2001. A particle-associated glycoprotein signal peptide essential for virus maturation andinfectivity. J. Virol. 75:5762–5771.

26. Lingappa, J. R., R. L. Hill, M. L. Wong, and R. S. Hegde. 1997. A multistep,ATP-dependent pathway for assembly of human immunodeficiency viruscapsids in a cell-free system. J. Cell Biol. 136:567–581.

27. Linial, M. L. 1999. Foamy viruses are unconventional retroviruses. J. Virol.73:1747–1755.

28. Lochelt, M., and R. M. Flugel. 1996. The human foamy virus pol gene isexpressed as a Pro-Pol polyprotein and not as a Gag-Pol fusion protein.J. Virol. 70:1033–1040.

29. Lochelt, M., W. Muranyi, and R. M. Flugel. 1993. Human foamy virusgenome possesses an internal, Bel-1-dependent and functional promoter.Proc. Natl. Acad. Sci. USA 90:7317–7321.

30. Lochelt, M., H. Zentgraf, and R. M. Flugel. 1991. Construction of an infec-tious DNA clone of the full-length human spumaretrovirus genome andmutagenesis of the bel 1 gene. Virology 184:43–54.

31. Ono, A., D. Demirov, and E. O. Freed. 2000. Relationship between human

immunodeficiency virus type 1 Gag multimerization and membrane binding.J. Virol. 74:5142–5150.

32. Parker, S. D., and E. Hunter. 2000. A cell-line-specific defect in the intra-cellular transport and release of assembled retroviral capsids. J. Virol. 74:784–795.

33. Pellman, D., E. A. Garber, F. R. Cross, and H. Hanafusa. 1985. An N-terminal peptide from p60src can direct myristylation and plasma membranelocalization when fused to heterologous proteins. Nature 314:374–377.

34. Pfrepper, K. I., M. Lochelt, H. R. Rackwitz, M. Schnolzer, H. Heid, andR. M. Flugel. 1999. Molecular characterization of proteolytic processing ofthe Gag proteins of human spumavirus. J. Virol. 73:7907–7911.

35. Pfrepper, K. I., M. Lochelt, M. Schnolzer, and R. M. Flugel. 1997. Expres-sion and molecular characterization of an enzymatically active recombinanthuman spumaretrovirus protease. Biochem. Biophys. Res. Commun. 237:548–553.

36. Rethwilm, A., O. Erlwein, G. Baunach, B. Maurer, and V. ter Meulen. 1991.The transcriptional transactivator of human foamy virus maps to the bel 1genomic region. Proc. Natl. Acad. Sci. USA 88:941–945.

37. Rhee, S. S., and E. Hunter. 1987. Myristylation is required for intracellulartransport but not for assembly of D-type retrovirus capsids. J. Virol. 61:1045–1053.

38. Rhee, S. S., and E. Hunter. 1990. A single amino acid substitution within thematrix protein of a type D retrovirus converts its morphogenesis to that of atype C retrovirus. Cell 63:77–86.

39. Saib, A., F. Puvion-Dutilleul, M. Schmid, J. Peries, and H. de The. 1997.Nuclear targeting of incoming human foamy virus Gag proteins involves acentriolar step. J. Virol. 71:1155–1161.

40. Sakalian, M., and E. Hunter. 1999. Separate assembly and transport do-mains within the Gag precursor of Mason-Pfizer monkey virus. J. Virol.73:8073–8082.

41. Schliephake, A. W., and A. Rethwilm. 1994. Nuclear localization of foamyvirus Gag precursor protein. J. Virol. 68:4946–4954.

42. Tobaly-Tapiero, J., P. Bittoun, M. Neves, M. C. Guillemin, C. H. Lecellier,F. Puvion-Dutilleul, B. Gicquel, S. Zientara, M. L. Giron, H. de The, and A.Saib. 2000. Isolation and characterization of an equine foamy virus. J. Virol.74:4064–4073.

43. Welker, R., A. Janetzko, and H. G. Krausslich. 1997. Plasma membranetargeting of chimeric intracisternal A-type particle polyproteins leads toparticle release and specific activation of the viral proteinase. J. Virol. 71:5209–5217.

43a.Wills, J. W., and R. C. Craven. 1991. Form, function, and use of retroviralGag proteins AIDS 5:639–654.

44. Wills, J. W., R. C. Craven, and J. A. Achacoso. 1989. Creation and expressionof myristylated forms of Rous sarcoma virus gag protein in mammalian cells.J. Virol. 63:4331–4343.

45. Wills, J. W., R. C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C. R. Erdie.1991. Suppression of retroviral MA deletions by the amino-terminal mem-brane-binding domain of p60src. J. Virol. 65:3804–3812.

46. Winkler, I., J. Bodem, L. Haas, M. Zemba, H. Delius, R. Flower, R. M.Flugel, and M. Lochelt. 1997. Characterization of the genome of felinefoamy virus and its proteins shows distinct features different from those ofprimate spumaviruses. J. Virol. 71:6727–6741.

47. Yu, S. F., D. N. Baldwin, S. R. Gwynn, S. Yendapalli, and M. L. Linial. 1996.Human foamy virus replication: a pathway distinct from that of retrovirusesand hepadnaviruses. Science 271:1579–1582.

48. Yu, S. F., K. Edelmann, R. K. Strong, A. Moebes, A. Rethwilm, and M. L.Linial. 1996. The carboxyl terminus of the human foamy virus Gag proteincontains separable nucleic acid binding and nuclear transport domains. J. Vi-rol. 70:8255–8262.

49. Yu, S. F., and M. L. Linial. 1993. Analysis of the role of the bel and bet openreading frames of human foamy virus by using a new quantitative assay.J. Virol. 67:6618–6624.

50. Yu, S. F., M. D. Sullivan, and M. L. Linial. 1999. Evidence that the humanfoamy virus genome is DNA. J. Virol. 73:1565–1572.

51. Zemba, M., T. Wilk, T. Rutten, A. Wagner, R. M. Flugel, and M. Lochelt.1998. The carboxy-terminal p3Gag domain of the human foamy virus Gagprecursor is required for efficient virus infectivity. Virology 247:7–13.



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identification of a conserved residue of foamy virus gag required

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