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Journal of Virology, July 2008, p. 6109-6119, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00503-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutations in the Amino Terminus of Foamy Virus Gag Disrupt Morphology and Infectivity but Do Not Target Assembly{triangledown}

Rachel B. Life,1,2 Eun-Gyung Lee,1 Scott W. Eastman,3 and Maxine L. Linial1,2*

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109,1 Program of Pathobiology, Department of Global Health, University of Washington, Seattle, Washington 98195,2 Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York 100163

Received 6 March 2008/ Accepted 11 April 2008


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ABSTRACT
 
Foamy viruses (FVs) assemble using pathways distinct from those of orthoretroviruses. FV capsid assembly takes place near the host microtubule-organizing center (MTOC). Assembled capsids then migrate by an unknown mechanism to the trans-Golgi network to colocalize with the FV glycoprotein, Env. Interaction with Env is required for FV capsid egress from cells; the amino terminus of FV Gag contains a cytoplasmic targeting/retention signal that is responsible for targeting assembly to the MTOC. A mutant Gag was constructed by addition of a myristylation (M) signal in an attempt to target assembly to the plasma membrane and potentially overcome the dependence upon Env for budding (S. W. Eastman and M. L. Linial, J. Virol. 75:6857-6864, 2001). Using this and additional mutants, we now show that assembly is not redirected to the plasma membrane. Addition of an M signal leads to gross morphological defects. The aberrant particles still assemble near the MTOC but do not produce infectious virus. Although extracellular Gag can be detected in a pelletable form in the absence of Env, the mutant particles contain very little genomic RNA and are less dense. Our analyses indicate that the amino terminus of Gag contains an Env interaction domain that is critical for bona fide egress of assembled capsids.


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INTRODUCTION
 
Foamy viruses (FVs) are ancient viruses (37) that are the only members of the retroviral subfamily Spumaretrovirinae. They differ in many ways from viruses in the other retroviral subfamily, the Orthoretrovirinae. FVs naturally infect all nonhuman primates and are both highly prevalent and highly transmissible. However, it is noteworthy that FVs appear to not cause any disease in any naturally or accidentally infected host. The lack of pathogenicity makes FVs an attractive vehicle for gene therapy (2, 30, 39). On a molecular level, FVs share aspects of their life cycle with the orthoretroviruses, but they also employ mechanisms that are more closely related to those of the pararetroviral hepadnaviruses such as hepatitis B virus (17). They are complex retroviruses, containing several accessory genes in addition to the canonical gag, pol, and env genes.

Like orthoretroviral Gag, FV Gag is responsible for the formation of viral capsids. However, none of the known functional domains within orthoretroviral Gag are present in FV Gag. There are major differences between orthoretroviral and FV assembly. FV Gag is not cleaved into the matrix, capsid, and nucleocapsid subunits that are characteristic of orthoretroviral Gag. This results in mature FV virions that lack a condensed core and resemble immature orthoretroviruses. Another difference is that FV Gag alone is not sufficient for particle release, which is completely reliant on the glycoprotein Env (1, 10, 23). Without Env, FV Gag can assemble into capsids, but the particles cannot bud. Further, other glycoproteins cannot replace FV Env in trans (23). This suggests that there are FV-specific interactions between Gag and Env that are required for normal budding. FV Env contains a third protein in addition to the surface and transmembrane proteins. This protein, the leader peptide, contains a domain that interacts with Gag (11, 16), possibly at the N terminus of Gag, but the precise Gag domain has yet to be identified. FV Env is also unique among retroviruses because it buds from cells and forms subviral particles (SVPs) on its own (33). Finally, in addition to requiring Env, FV particles incorporate the Pol enzymatic protein in an unusual manner. FV Pol is expressed from an individually spliced mRNA, so it does not coassemble as a Gag-Pol fusion protein as in orthoretroviruses. Pol encapsidation sequences have been located in genomic RNA, and sites within the Pol protein itself are believed to be critical for encapsidation (22, 29).

Less is known about the specifics of FV assembly than about those of orthoretroviruses. The assembly of orthoretroviral Gag monomers into capsids follows one of two distinct pathways. The C-type pathway, employed by human immunodeficiency virus (HIV) and the majority of retroviruses, takes place at a host membrane. Targeting of C-type retroviral Gag has been shown to utilize basic amino acids and a myristylation signal at the extreme N terminus of the protein (13, 46). This bipartite signal, called the membrane signal or M, has high affinity for the inner leaflet of the host cell plasma membrane. Targeting is not passive, because the tertiary structure of the full-length Gag protein controls whether or not the M signal, including the critical 14-carbon myristic acid, is exposed or not. This "myristyl switch" mechanism allows Gag to assume two conformations. The N terminus can be exposed to allow plasma membrane targeting or sequestered within the Gag protein (26). The second orthoretroviral assembly pathway is the B/D type, as exemplified by Mason-Pfizer monkey virus (MPMV). This pathway is characterized by formation of capsids in the cytoplasm of the host cell, independent of membranes. Once assembled, capsids then traffic to a cell membrane from which budding occurs. As expected, MPMV Gag contains two distinct targeting signals: an M signal as in C-type retroviruses and a cytoplasmic targeting/retention signal (CTRS) located within the N terminus of the Gag protein. The CTRS consists of approximately 16 amino acids centered on a critical arginine at position 55 (4). The CTRS is dominant over the M signal and directs MPMV Gag by an unknown mechanism to a pericentriolar region of the host cell where assembly takes place (32). The pericentriolar region of the cell is the site of the microtubule-organizing center (MTOC), the center of the host's cytoskeletal system. Only after capsids are formed does the M signal act to target the structures to a membrane for budding. For MPMV, this biphasic assembly process appears to be redundant, since abrogation of the CTRS by mutating the central arginine to either alanine or tryptophan results in assembly of infectious C-type particles at the plasma membrane (4, 27, 28). While MPMV Gag is like all other orthoretroviral Gag proteins in that it is capable of producing extracellular particles on its own, cognate expression of MPMV Env greatly improves particle release, most likely by facilitating the egress of capsids from the assembly site (31, 34).

The MTOC is targeted by other viruses as well. For example, targeting to the MTOC is thought to maximize assembly efficiency for African swine fever virus (ASFV) and herpes simplex virus (HSV) types 1 and 2 (40, 41). High assembly efficiency appears to be mediated by concentrating viral proteins, viral genomes, and specific host factors in the pericentriolar assembly site. For both ASFV and HSV, the pericentriolar assembly sites are enriched for specific host factors such as monomeric heat shock protein (Hsp) chaperones and mitochondria. The mechanism by which these host factors are recruited is not known, but at least in the case of ASFV, the assembly sites closely resemble cellular aggresomes. These cellular structures are thought to occur as part of the normal misfolded protein response, and they are characterized by enrichment of monomeric Hsp chaperones and mitochondria and are sequestered from the rest of the cytoplasm by a vimentin cage. ASFV viral factories are characterized by vimentin cages (14), while those of HSV are not (19). In the case of MPMV, the molecular chaperonin TCP-1 ring complex, which is normally enriched around the MTOC and plays a critical role in microtubule assembly (7, 35), has been shown to interact with MPMV Gag (15).

FV capsid assembly most closely resembles that of MPMV. Although FV Gag does not contain an M signal, it does contain a CTRS homologous to that of MPMV (9). The FV CTRS directs Gag assembly to a pericentriolar location, as in MPMV (32, 44). However, unlike in MPMV, a single amino acid substitution in the FV CTRS abrogates infectious particle production. This is equally apparent when the central arginine at position 50 is mutated to either alanine (R50A) or tryptophan (R50W) (9). Previous work suggested that artificial addition of an M signal could compensate for this mutation by targeting FV assembly to the plasma membrane. Replacement of the first 10 amino acids of the CTRS– mutant FV Gag with an exogenous myristylation signal was shown to restore its extracellular release but not infectivity (9). Since FV particle release is dependent on Env, traditional mutational studies to identify functional domains with the FV Gag protein have been confounded by the fact that any mutation might also disrupt the Gag-Env interaction. The above-described M+ CTRS– double mutant was capable of releasing virus-like particles (VLPs) independently of Env, so it is potentially a tool to study FV assembly independent of the leader peptide-Gag interaction (45).

In the current study, we have further characterized the M+ CTRS– mutant. We confirm that extracellular Gag is detectable, but particles are not infectious. We used transmission electron microscopy (TEM) analysis to examine the intracellular structures of this and additional mutants. Contrary to the previous interpretation, we find that three different viral M signals are incapable of redirecting particle assembly away from the pericentriolar site. In addition, we show that the posttranslational modifications triggered by each M signal, as well as the amino acid changes themselves, cause gross morphological defects in capsid formation. Together these data indicate that normal FV assembly cannot be redirected from the native pericentriolar site and cannot tolerate structural changes at the N terminus of Gag.


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MATERIALS AND METHODS
 
Mutagenesis and cloning. All Gag N-terminal point mutants were generated in the full-length proviral expression construct pCHFV, which is identical to the infectious clone pcHFV13 (18) except that it contains a modified version of the cytomegalovirus immediate-early promoter in place of the U5 region of the 5' long terminal repeat (LTR). N-terminal mutants (Table 1) were created through site-directed PCR mutagenesis using the outer primer pair 5'-TTCCAAAATCTCGTAACAAC-3' and 5'-TTGAGGTTGGTAAGTACGGGGTC-3' and Pfx proofreading polymerase (Invitrogen, Carlsbad CA); specific mutagenic primer sequences available on request. The sequence of each N-terminal mutant was confirmed using ABI Prism Big Dye Terminator v3.1 and 3730XL automated DNA analysis device (Applied Biosystems, Foster City CA). The two deletion mutants were initially created in the cG3 subclone, which contains a 2,885-bp fragment of pCHFV from EagI to SwaI. The {Delta}N2 mutant was created by digestion of cG3 with the restriction enzymes EcoRV and BstEII followed by blunting the 3' end with the T4 DNA polymerase enzyme and religation. The {Delta}N1 mutant was created by inverse PCR using DNA primer oligonucleotides originating at either side of the deletion and the cG3 subclone as a template. The PCR products were subsequently treated with the restriction endonucleases DpnI and ApaI, ligated, and transformed into XL-1 Blue Escherichia coli. Both deletion mutants were then cloned back into the full-length proviral context using the EagI and SwaI sites.


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  		TABLE 1. Infectious particle production correlates with normal targeting and morphology

Cell culture and transfection. 293T cells (6) and FAB cells (43) were maintained in Dulbecco's modified Eagle's minimal essential medium supplemented with 9% fetal bovine serum and antibiotics. In addition to the new mutants described here, the following previously described proviral plasmids were used in transfection: the wild-type (WT) infectious molecular clone pcHFV, the Env deletion mutant {Delta}Env, the CTRS– R50A mutant, the v-src M mutant containing the exogenous myristylation signal, the double mutant v-src M R50W (9), and the Env expression vector pCiES (30, 39). Transfections were performed with either PolyFect reagent (Qiagen, Valencia CA) or 1 mg/ml polyethylimine (Polysciences, Inc., Warrington PA) as previously described (8). Infectious titers from at least three transiently transfected cell supernatants were obtained in duplicate from filtered but unconcentrated supernatants via the FAB assay as previously described (43). Cell lysates and viral supernatants were collected at between 40 and 44 h posttransfection. Pelletable viral material was concentrated from supernatants by filtering through a 0.45-µm-pore-size-filter and pelleting through a 20% sucrose cushion by ultracentrifugation (90,000 x g in a Beckman SW-28 rotor at 4°C). Inhibition of myristylation was performed by replacing the cell culture medium at 12 h posttransfection with medium containing either dimethyl sulfoxide alone or 2-hydroxymyristic acid in dimethyl sulfoxide, and cells were harvested as described above.

Western blotting. Cell lysates and viral supernatants were prepared for immunoblotting as previously described (36). Western blot analyses were performed using anti-Gag polyclonal rabbit serum (1) at 1:5,000 dilution and anti-Env SU monoclonal mouse antibody at a 1:1,000 dilution (44). The commercially available anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) monoclonal mouse antibody (no. 9484; Abcam, Cambridge, MA) was used at a 1:5,000 dilution as a loading control. Blots were visualized using the Li-Cor Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE) after blocking with the Odyssey blocking solution and secondary goat anti-rabbit antibody (no. A21076; Molecular Probes Invitrogen, Carlsbad, CA) and goat anti-mouse antibody (no. 610-132-121; Rockland, Inc., Gilbertsville PA), both at a 1:5,000 dilution.

Electron microscopy. 293T cells were transfected with proviral DNA and incubated at 37° for 40 to 44 h. The growth medium was removed and replaced with 1 ml half-strength Karnovsky's fixative. Cells were then scraped, transferred to microcentrifuge tubes, and held at 4°C for a minimum of 4 h. The fixed cells were processed by the Shared Resources Electron Microscopy Laboratory at the Fred Hutchinson Cancer Research Center for TEM. Thin sections were examined using either a JEOL 1010 or a JEOL 1230 transmission electron microscope and a Gatan digital camera. Only intact nonapoptotic cells were examined for bisection of the centrioles and presence of viral structures.

Iodixanol density gradient centrifugation and fractionation. Equilibrium centrifugation was carried out on a 20 to 55% stepwise gradient in phosphate-buffered saline. Particles pelleted as described above were loaded onto the gradient and centrifuged in a Ti 55 rotor at 32,000 rpm at 4°C for 18 h. Aliquots of 320 µl each were collected from the top, and a total of 14 fractions were prepared to be analyzed for density. Proteins in each fraction were precipitated with 10% trichloroacetic acid and resuspended in 1x sodium dodecyl sulfate (SDS) sample buffer for Western blot analysis.

RPA. At between 45 and 48 h posttransfection, cells were scrapped off from the plates with antibody buffer (20 mM Tris-HCl [pH 7.4], 50 mM NaCl, 0.5% deoxycholic acid, 0.5% SDS, 0.5% aprotinin, 1 mM EDTA [pH 8.0]). The pelletable supernatants were prepared through a 20% sucrose cushion as described above, and pellets were resuspended in TNE buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.4], 1 mM EDTA). The amounts of cellular or viral RNAs were quantitated using a FV LTR-specific riboprobe as described previously (36). Briefly,32P-labeled probe RNA was hybridized to cellular and viral RNAs. RNase protection assays (RPAs) were performed by the method specified for the Direct Protect kit (Ambion, Inc., Applied Biosystems, Foster City, CA). Reaction mixtures were run on 5% polyacrylamide gels, and the dried gels were directly scanned with a Molecular Dynamics PhosphorImager. Radioactively labeled protected bands were quantitated with ImageQuant software (Molecular Dynamics, Inc., GE Healthcare, Piscataway, NJ).


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RESULTS
 
The v-src M signal does not redirect assembly but disrupts normal virion morphology. Figure 1A and B show the features of the FV Gag protein. Full-length Gag (p71) is cleaved at only one site during assembly, near the C terminus, to yield a p68 protein and a 3-kDa peptide (12). This appears to occur in ~50% of the Gag molecules incorporated in virions. Instead of the Cys-His boxes found in orthoretroviral Gag proteins, FV has three C-terminal glycine-arginine-rich domains termed GR boxes, which are thought to have a role in nuclear localization and nucleic acid binding (36, 42). The WT N terminus lacks an M signal but contains a CTRS centered on residue 50.


Figure 1
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  		FIG. 1. Schematic and sequences of the proviral constructs used. (A) Proviral expression is driven by the immediate-early cytomegalovirus (CMV) promoter, which is in place of the native LTR U3 region. The gag open reading frame is shown relative to that of pol, and the sites of the CTRS and GR boxes are indicated. (B) Relationship of the p71 and p68 Gag proteins. (C to E) WT and mutants are listed along with their amino acid sequences in the first 11 residues and at the center of the CTRS at residue 50. Mutated residues are in boldface. (C) Previously published set of mutants (9). (D) New mutants with alternative myristylation signals and respective myristylation-incompetent forms of all three. (E) Additional mutants with amino acid replacements in the WT.

Figure 1C shows the previously published mutants that we have reinvestigated (9), and Fig. 1D and E show the mutants created in this study. Previously it was shown that mutation of the FV CTRS (R50A) (Fig. 1A) abrogated VLP release (9), demonstrating that without the targeting function of the FV Gag CTRS, extracellular particles could not form. Thus, the FV CTRS had a targeting function analogous to that of the CTRS first described for MPMV Gag. VLP release of the FV Gag CTRS– mutant could be restored by addition of an exogenous myristylation signal from the Rous sarcoma virus v-src gene (v-src M R50W [Fig. 1C]) (9). In order to confirm these results and to visualize the intracellular morphology of the mutant particles, we transiently transfected these mutants into 293T cells and examined intracellular and extracellular viral protein using Western blots (Fig. 2). All transfected cells showed approximately equivalent levels of intracellular Gag, although the ratio of uncleaved to cleaved Gag varied (Fig. 2A). The supernatant of WT-transfected cells contained both Gag (Fig. 2B, lane 2) and the Env precursor protein gp130 (PrEnv) and its cleaved SU subunit gp85 (Fig. 2C, lane 2). Deletion of the env gene from the FV genome ({Delta}Env) abrogates Gag release into culture supernatant (1, 23), so the {Delta}Env construct was used as a control for nonspecific viral protein release. No extracellular Gag was detected from this control, as expected (Fig. 2B, lane 3). When the Gag CTRS was disrupted by the single amino acid change R50A, very little extracellular Gag was detectable (Fig. 2B, lane 4). Addition of an exogenous myristylation signal, v-src M, restored Gag release (Fig. 2B, lane 6). The mutant that contained only the v-src M signal also released extracellular Gag at nearly WT levels (Fig. 2B, lane 5). Both v-src M and v-src M R50W showed lower levels of Gag cleavage than the WT (Fig. 2B, lanes 5 and 6). Supernatant from all three mutant transfections contained detectable levels of only the uncleaved Env (Fig. 2C, lanes 4 to 6) and contained no infectious virus (Table 1).


Figure 2
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  		FIG. 2. Western blot analysis of transiently transfected 293T cells expressing proviral mutants. Lane 1, mock-transfected cells; lane 2, WT; lane 3, {Delta}Env; lane 4, CTRS mutant R50A; lane 5, v-src M myristylation signal mutant; lane 6, double mutant v-src M R50W. (A) One percent of each cell lysate probed for Gag expression and cleavage. (B and C) Gag (B) and Env (C) present in pelletable material from the entire volume of transfected cell supernatant.

The lack of Env cleavage in the three mutants was unexpected. One possibility is that the extracellular Env is not associated with Gag particles but represents SVPs comprised solely of Env. These have previously been shown to bud from cells transfected with FV Env alone, but they were reported to contain cleaved SU as well as PrEnv (33). To determine whether the uncleaved extracellular Env seen in Fig. 2C was consistent with SVPs in our transfection system, we examined the viral proteins produced by an Env expression vector (pCiES) (Fig. 3A to C). The control WT and {Delta}Env proviruses expressed Gag intracellularly (Fig. 3A, lanes 2, 3, and 6), whereas pCiES produced only Env (Fig. 3A and B, lanes 4 and 5). The pCiES cell lysate contained much higher levels of intracellular Env than that of WT virus. Even when a 16-fold-higher volume of WT cell lysate was compared to pCiES cell lysate, pCiES synthesized higher detectable levels of PrEnv and SU (Fig. 3B, compare lanes 2 and 4). In the supernatant, Env produced by WT-transfected cells was predominately the cleaved SU subunit (Fig. 3C, lane 2), while the pCiES supernatant contained much more PrEnv than SU (Fig. 3C, lane 3). In the previous study, Shaw et al. (33) found both cleaved and uncleaved Env proteins, but they used both a different antibody and a different Env expression vector than were used in the current work, so the differences in Env cleavage pattern could be attributable to differences in either expression levels or detection. We conclude that in our cell culture system, Env SVPs are composed primarily of uncleaved PrEnv and that the gp130 PrEnv signal detectable in the supernatant of noninfectious mutants (Fig. 2C, lanes 4 to 6) is most likely a result of SVP shedding independent of Gag.


Figure 3
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  		FIG. 3. Western blot analysis of transiently transfected 293T cells expressing proviral mutants. (A and B) Cell lysates probed for Gag (A) and Env (B) expression and cleavage. Lane 1, mock-transfected cells; lane 2, 4% of WT cell lysate; lane 3, 1% of WT cell lysate; lane 4, 0.25% of Env-only expression vector pCiES cell lysate; lane 5, 1% of pCiES lysate; lane 6, {Delta}Env. (C) Env detection in the pelletable material from the entire volume of transfected cell supernatant. Lane 1, mock-transfected cells; lane 2, WT; lane 3, pCiES; lane 4, {Delta}Env.

As previously reported (9), none of the three mutants shown in Fig. 1C had any detectable infectious titer (Table 1). To determine whether the assembly process had been disrupted, we analyzed the intracellular phenotype of the mutants via TEM (Fig. 4). As previously reported (44), FV capsids were found near the MTOC (Fig. 4A). The WT capsids were ~40 nm in diameter and were characterized by a single electron-dense ring around an electron-lucent core (Fig. 4B and C). Viral structures could not be detected in cells transfected with the R50A mutant, despite extensive examination (data not shown), nor were there detectable intracellular structures in cells transfected by the v-src M R50W double mutant, even at the plasma membrane (data not shown). Since this mutant produced extracellular Gag, it was expected that redirected assembly would be evident by virions forming at the plasma membrane. This negative finding does not substantiate the previous conclusion that the v-src M signal can redirect CTRS– Gag assembly to the plasma membrane (9). However, we cannot rule out small numbers of particles assembling at the plasma membrane.


Figure 4
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  		FIG. 4. Analysis of intracellular localization and morphology of the WT and the v-src M mutant via TEM of transiently transfected 293T cells. (A, B, and C) WT particles. (D, E, F, and G) v-src M mutant particles. An arrow denotes the centriole in panels A and D. Panels C, F, and G are enlarged micrographs to compare shape, not size, so scale bars are not included.

Examination of the v-src M mutant, however, showed that viral structures were easily detectable at the same pericentriolar site as for the WT (Fig. 4D). Morphological abnormalities were apparent in the mutant particles (Fig. 4E) (most easily seen at higher magnifications) (Fig. 4F and G; compare to WT in Fig. 4C). The mutant Gag formed a wide range of heterogeneous shapes. Additionally, instead of a single layer of electron density, the v-src M particles contained several distinct layers of electron density. These results suggest that even in the presence of an exogenous myristylation signal, the intact CTRS is dominant and targets Gag assembly to the MTOC. So while the v-src M signal does not redirect assembly, it does disrupt normal particle morphology.

We next asked whether v-src M Gag would behave in a dominant-negative fashion and prevent WT Gag from assembling normally. We found that the v-src M mutant did not prevent the formation of infectious virus when cotransfected at ratios of 1:10, 1:1, and 10:1 to WT (data not shown). This indicates that either the two Gag proteins do not coassemble or incorporation of v-src M Gag into particles is not sufficient to disrupt the normal particle morphology.

Since the data in Fig. 3 suggested that the extracellularly detectable v-src M Gag (Fig. 2B, lane 5) is not infectious because it is not associated with cleaved SU, we asked whether this mutant Gag was otherwise similar to the WT in particle density and genome packaging efficiency. When concentrated extracellular WT particles were centrifuged to equilibrium within an iodixanol density gradient, the majority of particles were found at 1.12 mg/ml (Fig. 5A, fraction 4). The extracellular material from the v-src M mutant, however, had a lower density of 1.08 mg/ml (Fig. 5B, fraction 3). Consistent with earlier observations, only the bona fide WT particles in the gradient had detectable SU; the v-src M mutant lighter-density VLPs were not associated with detectable SU (data not shown). The uncleaved PrEnv was not detectable in the gradient fractions of the v-src M mutant. The v-src M mutant consistently exported more uncleaved than cleaved Gag into the supernatant (Fig. 2B, lane 5), and little or no cleaved Gag was detectable in this density gradient. Particle size, as determined by velocity sedimentation in sucrose, was found to be more heterogeneous for the v-src M mutant than for the WT (data not shown). This fits with the observation using TEM that this mutant forms particles of heterogeneous size (Fig. 4D to G). RNA packaging efficiency was assayed by an RPA after normalization for Gag expression and release (36). All proviruses tested had detectable levels of viral RNA within transfected cells (Fig. 6B, lanes 4 to 6), but only WT particles contained high levels of genomic RNA (Fig. 6B, lane 9). The v-src M mutant had significantly lower packaging efficiency, i.e., only 3% of WT levels when normalized to particle number (Fig. 6B, lane 10). The lighter density and poor packaging efficiency of the v-src M mutant suggests that the extracellularly detectable Gag is not in the form of bona fide viral particles.


Figure 5
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  		FIG. 5. Density analysis of extracellular particles via Western blot analysis of equilibrium gradient fractions. Viral particles from the WT (A) or v-src M (B) were concentrated through a 20% sucrose cushion from the supernatant of transiently transfected 293T cells and then layered onto a 20 to 55% step gradient. Lane 1, 10% of input viral concentrate; lane 2, fraction 2 (fraction 1 was omitted from the SDS-polyacrylamide gel for space); lanes 3 to 7, fractions 3-7. The average density of each fraction is shown below the blots.


Figure 6
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  		FIG. 6. Analysis of viral genomic packaging efficiency via RPA. (A) Schematic of how the probe hybridizes to and protects viral genomic DNA or RNA. nt, nucleotides. (B) Either cell lysate or concentrated viral supernatant from transiently transfected cells was first assayed for equivalent levels of viral protein expression and then hybridized with the radiolabeled probe and subjected to RNase treatment. The size of radiolabeled fragments in the gel is indicated on the left. Lane 1, free probe; lane 2, free probe plus RNase; lane 3, mock-transfected cell lysate; lane 4, {Delta}Env; lane 5, WT; lane 6, v-src M; lane 7, mock-transfected cell supernatant; lane 8, {Delta}Env; lane 9, WT; lane 10, v-src M. The gel image was cropped and rearranged to show only the pertinent transfections, and lines between lanes 9 and 10 indicate gel distortion.

We also examined two Gag deletion mutants that each lacked a functional CTRS and their myristylated counterparts for their ability to escape the cell in an Env-independent manner (Fig. 7A). Mutant {Delta}N1 is missing amino acids 2 to 84, so that it does not contain a functional CTRS. The deletion in mutant {Delta}N2, (amino acids 86 to 202) does not remove the CTRS, so an additional R50A mutation was created. Both mutants were created in an Env-minus background. All mutants express similar levels of intracellular Gag (Fig. 7B, lanes 3 to 6). For each deletion mutant, the myristylated version demonstrated either a shift in mobility, a change in cleavage efficiency, or both. In each case the nonmyristylated deletion mutant could not produce extracellular pelletable Gag in the absence of Env (Fig. 7C, lanes 4 and 6). In contrast, addition of the v-src M myristylation signal allowed significant levels of Gag to be exported from the cells in a pelletable form in the absence of Env expression (Fig. 7C, lanes 3 and 5). Neither of these pelletable deletion mutant VLPs was associated with detectable levels of Pol (data not shown). Since these mutants lack the CTRS (either because it was deleted or because it was mutated by the R50A change), they are unlikely to be assembled and exported in the same manner as the WT.


Figure 7
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  		FIG. 7. (A) Schematic of the FV Gag protein and the relative positions, including the amino acids deleted, of the mutants {Delta}N1 and {Delta}N2. (B and C) Control and mutant proviruses were transiently transfected in 293T cells and analyzed by Western blotting for Gag expression in cell lysate (B) and cell supernatant (C). Lane 1, WT; lane 2, {Delta}Env; lane 3, v-src M {Delta}N1; lane 4, {Delta}N1; lane 5, v-src M {Delta}N2; lane 6, {Delta}N2.

Both myristylation and amino acid changes contribute to aberrant morphology. The v-src M Gag has 8 amino acids changes compared to that of WT Gag (Fig. 1C), so the morphological defect of this mutant could be caused by either myristylation, the mutant amino acids, or both. In order to examine each possibility separately, we created the additional mutants described in Fig. 1D. To address the role of myristylation, two additional M signals were introduced into the N terminus of FV Gag. We chose the M signal from MPMV Gag since it is used as a default targeting mechanism when the CTRS is mutated (28) and the M signal from HIV type 1 Gag since it is required for normal plasma membrane assembly (13). In order to separate the relative contribution of myristylation from that of amino acid changes, the acceptor glycine of each M signal (including the v-src M) was mutated to alanine (G2A mutants, Fig. 1D), since without the acceptor glycine a myristylation signal is incapable of being acylated (24, 25). To address the contribution of specific amino acid changes that might also contribute to a morphology defect, small mutations were introduced into WT FV Gag as indicated in Fig. 1E. Amino acids with long side chains or a rigid shape might interfere in the tertiary structure of the N terminus of FV Gag, and each of the three exogenous M signals contains residues that fit these criteria. In addition, we investigated whether the loss of either the positive charge contributed by two glutamic acid residues at positions 8 and 9 or the potential phosphorylation site at tyrosine at position 10 might contribute to morphological defects, by changing the WT residues to alanine. The results from biochemical analyses and TEM analyses, as well as infectious titers, for all of these mutants are summarized in Table 1.

Both the MPMV M and HIV M signal mutants expressed intracellular Gag and displayed a cleavage pattern more like that of v-src M than like that of the WT in that there is less cleaved than uncleaved Gag (Fig. 8A, lanes 6 and 8). Although both of these mutants produced detectable extracellular Gag, it was primarily uncleaved (Fig. 8B, lanes 6 and 8), and neither mutant was infectious (Table 1). This correlates with the fact that neither mutant had detectable levels of extracellular SU (Fig. 8C, lanes 6 and 8). When examined by TEM, both the MPMV M and HIV M mutants were found in the normal pericentriolar site in transfected cells, but they displayed aberrant capsid morphology (Fig. 9A and B, respectively). Thus, both additional M signal mutants have a phenotype that was similar to that of the v-src M mutant.


Figure 8
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  		FIG. 8. Western blot analysis of transiently transfected 293T cells expressing proviral mutants. The image was cropped and rearranged to match the order of the mutants in Fig. 1 and the text. Lane 1, mock-transfected cells; lane 2, WT; lane 3, {Delta}Env; lane 4, v-src M; lane 5, v-src M G2A; lane 6, MPMV M; lane 7, MPMV M G2A; lane 8, HIV M; lane 9, HIV M G2A; lane 10, 3K; lane 11, v-src M 4A. (A) One percent of cell lysate probed for Gag expression and cleavage. (B and C) Gag (B) and Env (C) present in pelletable material from the entire volume of transfected cell supernatant.


Figure 9
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  		FIG. 9. Analysis of intracellular localization and morphology of selected mutants via TEM of transiently transfected 293T cells. (A) MPMV M; (B) HIV M; (C and D) v-src M G2A; (E and F) 3K. An arrow denotes the centriole in panels B, C, and E. Panels D and F are enlarged micrographs to compare shape, not size, so scale bars are not included.

Next, each M signal mutant in which myristylation is blocked by a G2A mutation was examined, and all three exhibited significant changes in phenotype. The v-src M G2A mutant regained some infectivity compared to its myristylated counterpart, but the level was only about 0.16% of WT (Table 1). This correlated with more complete Gag cleavage (Fig. 8B, lane 5) and the appearance of extracellular SU (Fig. 8C, lane 5). Similar results were obtained when v-src M-transfected cells were treated with the chemical myristylation inhibitor 2-hydroxymyristic acid (5, 20, 21) (data not shown). When examined by TEM, the v-src M G2A mutant was found to still assemble at a site near the MTOC, but the particle morphology was noticeably different from that of the v-src M mutant (Fig. 9C and D; compare to Fig. 4G and F). These aberrant viral structures no longer showed the multiple layers of electron density evident in the myristylated v-src M mutant (Fig. 4D to G). The MPMV M G2A and HIV M G2A mutants regained 45 to 70% of WT infectivity (Table 1) and the supernatant of both these mutants contained detectable levels of SU (Fig. 8C, lanes 7 and 9). TEM analysis of both of these mutants showed them to be indistinguishable from the WT (Table 1). Taken together, these data show that myristylation itself leads to multiple layers of electron density at the particle surface. Further, they imply that the mutated resides in the v-src M signal, but not those in either the MPMV M or HIV M signal, also contribute to aberrant capsid morphology. In other words, the v-src M Gag has disruption caused by both myristylation and mutant amino acids, while the other two M signal mutants are affected only by the addition of the fatty acid.

In order to explore how the rest of the v-src M signal might contribute to the morphological defect, we next tested whether the three lysine residues or the proline from the v-src M signal contributed to the defect by introducing them alone (3K and E8P mutants, Fig. 1E) or in combination (3K/P, Fig. 1E) into the WT Gag protein. The lysines were investigated because they substitute long, positive side chains for small nonpolar or negatively charged amino acids in the WT, and these changes could interfere with the tertiary structure of Gag. The proline was considered because it might interfere with the flexibility of the Gag N terminus. The 3K mutant had 0.3% of the infectivity of the WT (Table 1). 3K also produced detectable extracellular SU (Fig. 8C, lane 10), though at a lower level than the v-src M G2A mutant. TEM analysis of the 3K mutant revealed that it targeted correctly but formed aberrant structures (Fig. 9E). In contrast, the E8P mutant had nearly WT levels of infectious titer and was indistinguishable from the WT in both biochemical and TEM analyses (Table 1), indicating that the proline does not contribute to the morphology defect. When we examined the 3K/P mutant, it showed a low infectious titer that was not significantly different from that of the 3K mutant (Table 1). There was no observable difference in TEM analysis between the morphologies of the 3K and the 3K/P mutants (Table 1). Taken together, these data indicate that the three mutant lysines and myristylation both contribute to the morphological defect seen in the v-src M mutant (Fig. 4D to G). We investigated whether myristylation and insertion of three lysine residues were the only cause of the defective particles in the v-src M mutant, by creating the v-src M 4A mutant (Fig. 1E), which cannot be myristylated and does not have the three disruptive lysine residues. Although this mutant incorporated SU (Fig. 8C, lane 11), it only showed a low level of infectivity (Table 1). This indicates that myristylation and the three lysines are not completely responsible for the aberrant morphology of the v-src M mutant.

The results from the MPMV M G2A and HIV M G2A mutants indicate that none of their mutated amino acids are disruptive to particle morphology, unlike the v-src M signal. To confirm this, we tested whether the two long polar side chains of the MPMV M signal (2Q [Fig. 1E]) or the one long hydrophobic leucine from the HIV M signal (E8L [Fig. 1E]) would be disruptive if introduced into the WT background. Both of these mutants had near-WT titers and were indistinguishable from the WT in biochemical and TEM analyses (Table 1). Finally, when we examined the loss of WT residues with the AAYA and Y10A mutants (Fig. 1E), we found that neither of these mutants had any significant difference from the WT in infectious-titer, biochemical, or TEM analyses (Table 1). This indicates that the loss of three glutamic acid residues or the single tyrosine did not contribute to the defects seen in the v-src M mutant.


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DISCUSSION
 
FV capsid assembly has been difficult to investigate because effects on assembly and release cannot easily be separated, as there is an absolute dependence on Env for particle release. When our previous data suggested that this Env dependence could be relieved by redirecting FV assembly away from its native site, it offered an attractive system to uncouple assembly from budding (9). We therefore further investigated whether myristylated mutants could bud from the plasma membrane in the absence of Env. We present evidence that FV assembly cannot be artificially redirected away from the native pericentriolar assembly site. Without a CTRS targeting motif, FV Gag does not form particles within the cell. If an exogenous myristylation signal is added to the CTRS– FV Gag (as in mutant v-src M R50W), detectable particles still do not form, even though extracellular release of Gag is restored. Release of noninfectious mutant Gag is apparent even when the CTRS is intact, as in the v-src M mutant. The v-src M mutant structures that escape the cell are not infectious, and we show that they do not contain cleaved SU or normal levels of viral genome, nor are they the size or density of WT particles. It is intriguing that genome packaging, which is a function of the C terminus of FV Gag (36) is dramatically perturbed by the N-terminal v-src M mutation. An unanswered question is why Gag cleavage is apparent in mutants that are apparently incapable of assembly. It is possible that some Gag can be cleaved by Pol intracellularly in the absence of normal assembly. The results showing heterogeneous size are consistent with the aberrant particles visualized in TEM analyses. It is clear that the v-src M mutant Gag is capable of escaping the cell as some sort of complex, so we make the distinction that mutant VLPs are not representative of bona fide capsids. In addition, we show that two different exogenous myristylation signals also lead to production of intracellular VLPs that are heterogeneous in size and shape and of extracellular Gag that lacks infectivity. Therefore, while targeting via the CTRS is necessary and sufficient for commencement of particle assembly, it is not sufficient for completion of capsids with normal morphology, as gross morphological defects were apparent in each of the three myristylation signal mutants despite their formation near the MTOC. This suggests that FV Gag contains a signal(s) that is required for both targeting capsid assembly to the pericentriolar site and targeting of completed capsids to the egress pathway of Env interaction at the trans-Golgi network. This is reminiscent of the biphasic targeting pathway of MPMV assembly, except that for FV no default assembly site exists in the absence of a CTRS signal. While we cannot formally rule out some percentage of mutant Gag assembling at the plasma membrane, the significant presence of mutant structures forming at the WT pericentriolar region argues against redirection of assembly.

We also found that artificial myristylation of FV Gag does overcome the dependence on FV Env for particle release. The three M signal mutants all produce extracellular VLPs that are not infectious. Since it is clear that these structures form at the native pericentriolar site, they must escape the cell via a mechanism that is distinct from normal FV egress, most likely via plasma membrane targeting through the exogenous M signal. Mutants with large deletions in the Gag N terminus can also escape the cell as pelletable VLPs if they are myristylated. We cannot distinguish between two possibilities for the egress pathway of the mutant VLPs. The mutant structures could be directly targeted to the plasma membrane, or the plasma membrane targeting could occur after the VLPs traffic as WT to the trans-Golgi network for Env interaction. Both possibilities suggest that the exogenous M signals are either dominant over or interfere with the WT egress targeting mechanism. Both possibilities also indicate that once targeted to a membrane, FV Gag is capable of budding on its own and that the underlying reason for the unique Env dependence in FV is due to the egress targeting mechanism and not an inherent inability of FV Gag to bud. This is supported by a recent study that used a redirected FV Gag assembly system to try to determine if ubiquitination of Gag is involved in late-domain-mediated ubiquitin-dependent budding mechanisms (45). That study used a double prenylation single appended onto the N terminus of a lysine-free FV Gag to relieve Env dependence for Gag release, which was used as a surrogate measure of Gag assembly. However, there were no data presented to show whether or not the extracellularly detectable Gag VLPs were of a size or density similar to that found in WT virions, and no TEM analysis was shown. In light of the data presented here, it is possible that the extracellularly detectable Gag in the aforementioned study is not assembled into bona fide capsids. However, we have not examined any Gag mutants in which a myristylation signal is appended to the N terminus; all of the mutants reported here are N-terminal replacement mutants. We also did not test other prenylation signals, but our evidence that myristylation alone was responsible for massive morphological defects suggests that any long fatty acid on the N terminus of FV Gag would be deleterious to particle formation. However, the work by Zhadina et al. (45) did show that Env-independent VLP release was dependent on an intact late domain. Our myristylated deletion mutants, each of which contains an intact late domain, also produce extracellular VLPs that are not bona fide capsids. The nature of the extracellular mutant structures is unclear, as we have not been able to visualize them by TEM.

The structures formed by the M signal mutant Gag proteins have multiple layers of electron-dense material and are aberrantly shaped. We were able to show that this phenotype had two separable causes. Blocking myristylation of each of the three tested M signals restored the single layer of electron density characteristic of WT capsids but did not restore the uniform spheroid shapes of the WT in the case of the v-src M signal. The aberrant shape component of the v-src M mutant phenotype is attributable, at least in large part, to the three mutant lysine residues. Although there is little evidence that mutant lysine residues are directly disruptive to protein structure, it is interesting to note that a glutamic acid-to-lysine (E200K) mutation in the human prion protein PrPsc is the single most common mutation found in human sporadic Creutzfeld-Jacob disease, where the PrPsc has an abnormal, pathogenic conformation (3, 38). It is also of note that the WT FV Gag is unique among retroviral Gag proteins in that it contains only one lysine residue. The additional lysines might lead to aberrant modifications such as ubiquitination, which could in turn disrupt morphology. While the exact contribution of the mutant lysines to aberrant shapes is not clear, it is clear that FV Gag cannot tolerate the structural changes induced by myristylation at its N terminus. Since the N-terminal of FV Gag is believed to harbor the Env interaction domain (11, 16), it is possible that the three mutant lysines have partially disrupted this domain, either at the primary amino acid level or by spatially interfering with a conformational domain on the face of the fully folded Gag N terminus. The two mutants v-src M G2A and 3K have a phenotype of disrupted morphology, but they retain low levels of infectivity, suggesting that they preserve the ability to interact with Env to some extent. We cannot determine whether Env interaction constitutes an egress targeting mechanism or whether the two functions are distinct. If egress targeting and Env interaction were the same mechanistically, then we would expect that the small number of 3K or v-src M G2A particles that resemble WT capsids at the assembly site would be fully capable of Env incorporation and egress, and the aberrant VLPs at the assembly site would not. However, if egress targeting was intact in the two mutants but Env interaction was disrupted, all the mutant structures would escape the cell and only the small percentage capable of both would contain Env and be infectious. We cannot distinguish between these possibilities with the current data.

In summary, our results extend what is known about FV capsid assembly in that the CTRS and targeting to the MTOC are not sufficient for normal particle assembly. Additional signals, which can be perturbed by changes at the amino terminus of Gag, and possibly including the late domain, are required for normal Gag assembly and particle egress.


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ACKNOWLEDGMENTS
 
We thank Alison Yu for critically reviewing the manuscript. We thank the personnel at the Fred Hutchinson Cancer Research Center electron microscopy facility for all of their assistance.

This work was supported by grant CA 18282 from the National Cancer Institute to M.L.L.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109-1024. Phone: (206) 667-4442. Fax: (206) 667-5939. E-mail: mlinial{at}fhcrc.org Back

{triangledown} Published ahead of print on 23 April 2008. Back


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Journal of Virology, July 2008, p. 6109-6119, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00503-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lee, E.-G., Linial, M. L. (2008). The C Terminus of Foamy Retrovirus Gag Contains Determinants for Encapsidation of Pol Protein into Virions. J. Virol. 82: 10803-10810 [Abstract] [Full Text]  

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