INTRODUCTION

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Retrovirus assembly can proceed by either of two morphogenic pathways. D-type retroviruses such as Mason-Pfizer monkey virus (M-PMV) preassemble their capsids within the cytoplasm. These capsids then migrate to the plasma membrane, where they are enveloped and released. In contrast, retroviruses that follow C-type morphogenesis, such as human immunodeficiency virus (HIV) and Rous sarcoma virus, assemble their capsids at the underside of the plasma membrane in a process combined with budding (27). Both assembly processes are driven by the Gag polyprotein precursor. During or shortly after virions are released from the cell, capsids are proteolytically matured by the viral proteinase (14). Cleavage of Gag results in the formation of a compact nucleoprotein core that varies in shape among retroviruses (29). For either morphogenic pathway, viral glycoproteins are translocated into the endoplasmic reticulum, transported to the plasma membrane through the secretory pathway, and incorporated into the virus during capsid envelopment. The RNA genome is presumed to be packaged into the virion at the site of capsid assembly.

All retroviral Gag proteins contain three major domainsmatrix (MA), capsid (CA), and nucleocapsid (NC)as inferred from the matured species following proteolytic maturation (18). Processing of M-PMV Gag results in the production of matured Gag proteins MA, pp24/16, p12, CA, NC, and p4 (Fig. 1). As described for other retroviruses (28) MA is assumed to be associated with the membrane within matured virions, and CA is responsible for the formation of the virion core which contains the genomic RNA complexed with NC. In addition to the canonical retroviral Gag domains, M-PMV contains three additional major domains. The potential functions of these additional mature proteins, pp24/16, p12, and p4 in the mature virion are unknown. However, functional elements within the different domains have been defined that operate in the context of the Gag precursor. The known functional elements, beginning with the N terminus of the precursor protein, are as follows. (i) The N-terminal myristic acid moiety is dispensable for assembly of capsids, but is necessary for the transport of completed capsids to the plasma membrane (20). (ii) The putative cytoplasmic targeting-retention signal (CTRS) within MA appears to be responsible for the transport of Gag precursors to an intracellular site for assembly. This sequence in M-PMV is homologous to a similarly located sequence in the B-type retrovirus mouse mammary tumor virus (MMTV) (21). (iii) A short four-amino-acid motif (PPPY) located within the pp24/16 region functions as the "late budding domain" that is responsible for release of enveloped capsids from the plasma membrane (34). (iv) An acidic stretch of amino acids within the p12 region appears to be a putative internal scaffold domain (ISD) required for the efficient intracellular assembly of immature capsids (24). (v) The major homology region is the most conserved stretch of amino acids within the Gag proteins of retroviruses. Mutations within this sequence can have diverse affects, including the abrogation of particle assembly (26). (vi) The Cys-His motifs within NC, although they have not been directly investigated in M-PMV Gag, have been shown to be critical for the packaging of the viral RNA genome in other retroviruses (reviewed in reference 2).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Identified and potential morphological determinants within the M-PMV Gag precursor protein. The structure as inferred from the major proteolytic products of processing by the viral protease is shown. These domains are indicated from the N terminus to the C terminus as follows: p10MA, matrix; pp24/16, phosphoprotein; p12; p27CA, capsid; p14NC, nucleocapsid; and p4. Within these domains are depicted discrete amino acid sequences for which specific morphologic functions have been predicted or defined. The N-terminal myristic acid modification is also shown. The specific amino acid sequences that comprise the two domains of interest in this report are shown below. For the cytoplasmic targeting-retention signal, a previously defined arginine residue critical for function is displayed in outline.

Evidence for the existence of the CTRS and ISD domains comes from mutational studies in which altered or partially deleted gag genes are expressed in cultured cells. For the CTRS domain, a single amino acid substitution in MA (R55W) was found to redirect the morphogenic pathway of M-PMV Gag from D type to C type (21). While the designation of the region containing R55 as a transport determinant appears logical, it is still possible that the effect of the mutation is due instead to a defect in assembly such that the additional interaction with the plasma membrane becomes necessary for Gag multimerization to occur. For the ISD, it was found that deletions within this region result in an inability of Gag to assemble into immature capsids when the precursor is expressed under the control of the M-PMV promoter. However, when these species of Gag are overexpressed by the vaccinia virus/T7 system, all are able to assemble, a result which led to the hypothesis that this region is not required for assembly but instead influences its efficiency (24). Alternatively, the failure of ISD deletion mutants to assemble could be due to a defect in transport. In this case, the ability to overcome this defect by overexpression could be explained by the intracytoplasmic concentration of Gag reaching a level sufficient for multimerization to occur.

We have extended the analysis of the putative CTRS and ISD regions by examination of Gag precursors containing mutations in these domains in an in vitro assembly system. The rationale for this analysis is to examine the assembly of Gag in a system that lacks the potentially confounding effects of intracellular transport in order to determine unambiguously the nature of the two functional domains. We have previously described a system in which wild-type M-PMV Gag precursors are synthesized by in vitro translation and assemble into structures that, by gradient analysis and electron microscopy, are indistinguishable from immature capsids produced in transfected cells in culture (23). Because of the very low levels of expression possible in vitro, we also conducted our analysis of the CTRS and the ISD by utilizing the very-high-level bacterial T7 expression system for comparison. We report here that the R55W mutation, which lies within the putative CTRS, did not affect the efficiency with which Gag precursors assemble into immature capsid-like particles in either bacteria or in vitro. In contrast, deletions within the p12 domain that removed the putative ISD region rendered Gag incapable of assembly in vitro. Consistent with previous results obtained with an overexpression system in cultured cells (24), all Gag variants containing deletions of sequence within the ISD could efficiently assemble in bacteria.

	MATERIALS AND METHODS

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DNA constructs. Plasmid pTFCG.M100A is a derivative of plasmid pTFCG, which contains the encephalomyocarditis virus cap-independent translation enhancer element and M-PMV gag, pro, and pol flanked by a T7 promoter and a T7 terminator (23). Plasmid pTFCG was modified to create pTFCG.M100A by replacing the methionine codon at position 100 with that for alanine. This was accomplished by substitution of the PacI to SacI fragment of pTFCG with the same fragment containing the modification from pGAG78, an infectious molecular clone of M-PMV based upon pSHRM15 (20). The phenotype of virus produced after transfection of pGAG78 into cells in culture was previously found to be indistinguishable from that of the wild type (22).

In vitro analysis of immature capsid assembly. Transcription and translation were performed simultaneously with the pTFCG series plasmids and the TNT Coupled Reticulocyte Lysate System (Promega) in the presence of [³⁵S]methionine. Products of these synthesis reactions were analyzed on sucrose gradients. Reactions were diluted to a total volume of 200 µl with 30% (wt/wt) sucrose in gradient buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100. Diluted samples were then loaded onto 2.2-ml continuous 30 to 55% (wt/wt) sucrose gradients in gradient buffer. Gradients were centrifuged in a TLS-55 rotor (Beckman Instruments) for 2 h at 55,000 rpm. Approximately 200-µl fractions were taken by hand with a Pipetman (Gilson) from the top of the gradient. The pellet was resuspended in 200 µl of 55% (wt/wt) sucrose in gradient buffer. Aliquots (5 µl) of each fraction were dissolved in sodium dodecyl sulfate (SDS) sample buffer and then loaded onto an SDS-10% polyacrylamide gel. After electrophoresis, radioactive bands were visualized by fluorography of sodium salicylate-impregnated gels.

Expression of Gag species in bacteria. Bacterial expression plasmids of the pET series were transfected into Escherichia coli strain BL21(DE3) which had already been transformed with plasmid pBB131 which contains the Saccharomyces cerevisiae gene for protein N-myristoyltransferase (plasmid pBB131 was the kind gift of J. I. Gordon, Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Mo.). Cells were grown in Luria broth containing 500 µM myristic acid. Production of both Gag and myristoyltransferase was induced with 500 µM isopropyl--D-thiogalactopyranoside (IPTG). Cells were harvested after 4 h of induction by centrifugation at 14,000 rpm in an Eppendorf microcentrifuge (Brinkman).

Electron microscopy. For analysis of in vitro synthesis products by electron microscopy, reaction mixtures were diluted to a total of 1 ml in gradient buffer without detergent and centrifuged at 70,000 rpm for 1 h in a TLA-100.3 rotor with microcentrifuge inserts (Beckman Instruments). For analysis of Gag species produced in bacteria, 1-ml culture samples were centrifuged at 14,000 rpm in an Eppendorf microcentrifuge for 10 min. The resulting pellets, derived from either translation reactions or bacterial cultures, were then fixed overnight in 1% glutaraldehyde in phosphate-buffered saline (pH 7.0) at 4°C. After a rinse in phosphate-buffered saline, pellets were postfixed in 1% buffered osmium tetroxide for 1 h. These pellets were rinsed once more and then dehydrated in a graded series of ethanol solutions beginning with 50% and ending with 100%. After dehydration, pellets were rinsed three times with propylene oxide and then embedded in Polybed. Ultrathin sections were acquired by using a Rechert-Jung Ultra Cut E ultramicrotome. After staining with uranyl acetate and lead citrate, sections were examined and photographed by using a Hitachi-7000 transmission electron microscope.

	RESULTS

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Morphogenesis mutant R55W forms particulate structures as efficiently as the wild type in vitro. As discussed above, the matrix domain mutant R55W alters the morphogenic pathway of M-PMV Gag such that immature capsids assemble under the plasma membrane rather than within the cytoplasm. The altered site of assembly for the R55W variant of M-PMV Gag has been hypothesized to be the result of the disruption of a specific signal within the matrix domain which is responsible for the transport of precursors to the intracellular assembly site (21). Indeed, examination of the nuclear magnetic resonance (NMR) structure of M-PMV MA has revealed that residue 55 is located within the base of an external loop not found in any other MA structure solved to date (8). Such an exposed loop might provide an interaction site for the cellular transport machinery responsible for the intracellular targeting of M-PMV Gag. However, it could be argued that the R55W mutation has no effect upon transport, but, instead, imposes a necessary requirement for membrane interaction in order for Gag multimerization to occur. To distinguish between these two possibilitiestransport defect versus assembly defectwe have analyzed the potential assembly behavior of the R55W variant of Gag in an in vitro translation system.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   Comparison of in vitro-synthesized and -assembled mutant matrix domain Gag species by sucrose gradient analysis. Aliquots of gradient fractions were electrophoresed on an SDS-10% polyacrylamide gel. Lane numbers indicate gradient fractions, beginning with the top fraction at no. 1. Lanes L contain an equivalent aliquot of the translation reactions before gradient fractionation. Lanes P contain material remaining as a pellet in the tube after removal of the gradient. Lanes 7 to 9, containing material indicative of immature capsids, are of a density of ~1.19 to 1.21 g/ml. The numbers to the left indicate the positions of prestained molecular size standards in kilodaltons. Pr78gag and Pr95gag-pro indicate the positions of the Gag and Gag-Pro precursor polypeptides of M-PMV, respectively. The upper and lower panels depict the fractionation of translation reactions programmed with the M100A and R55W/M100A gag genes, respectively. The amino acid sequence of the region homologous with one in MMTV and containing residue 55 is also shown. The residue altered to tryptophan in the R55W mutant is depicted by the outlined W.

An amino-terminal sequence within the p12 domain is necessary for assembly in vitro. Previous characterization of p12 domain deletion mutants had revealed an expression-level-dependent requirement for this domain in assembly (24). When expressed in HeLa cells under control of the long terminal repeat (LTR) promoter, several of these mutants failed to assemble particles; however, all Gag species containing deleted regions of p12 were able to assemble particles, when expressed by the vaccinia virus/T7 system. Thus, overexpression compensates for the defect created by the deletion. The interpretation of these results was that p12 functions to promote Gag multimerization in the process of assembly (24). However, as with R55W, the phenotype of these mutants might be explained by a defect in transport. Loss of specific transport to the site of assembly could lead to a loss of particle formation. This defect could then be overcome by overexpression by providing a sufficiently increased concentration of Gag within the cytoplasm for assembly to occur.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Comparison of in vitro-synthesized p12 domain deletion mutant Gag species by sucrose gradient analysis. The experiment was performed and lane designations and molecular size standards are as described for Fig. 2. Lanes 8 to 10 containing material indicative of immature capsids are of a density of ~1.19 to 1.21 g/ml. M100A, 8-58/M100A, and 1-83/M100A indicate analyses of translation reactions programmed with the corresponding gag genes.

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Further comparative analysis of in vitro-synthesized p12 domain deletion mutant Gag species by sucrose gradient fractionation. The experiment was performed and lane designations and molecular size standards are as described for Fig. 2. Lanes 7 to 9 containing material indicative of immature capsids are of a density of ~1.19 to 1.21 g/ml. M100A, 1-25/M100A, 26-53/M100A, and 54-83/M100A indicate analyses of translation reactions programmed with the corresponding gag genes.

R55W and two partial p12 deletion mutants produce immature capsid-like structures in vitro. Although the above gradient analysis strongly suggests that the several Gag species which sediment into gradients are assembled capsids, we sought to confirm this by electron microscopy. Analysis by electron microscopy was performed as previously described (23). Briefly, whole transcription-translation reaction mixtures were diluted into buffer and then centrifuged at high speed. The resulting pellets were then processed for thin-section electron microscopy. Immature capsid-like structures were observed, along or near the base of the pellet in micrographs, for all mutants in which particulate material sediments into sucrose gradients (Fig. 5). The structures assembled by the R55W Gag precursors were indistinguishable from those composed of M100A, providing further confirmation that R55W is not defective in assembly (Fig. 5A and D). Two p12 deletion mutants, 26-53 and 54-83, also produced structures similar to those found in the M100A samples (Fig. 5B and C). In contrast, capsid-like structures were not observed for Gag species that were unable to assemble as indicated by gradient analysis (not shown).

View larger version (193K): [in this window] [in a new window]   FIG. 5.   Analysis of coupled transcription and translation reactions by thin-section electron microscopy. M-PMV gag genes were expressed in vitro, the reaction mixtures were diluted and centrifuged at high speed, and the resulting pellets were fixed and processed for microscopy. (A to D) Pellets derived from lysates expressing M100A (wild type) 26-53, 54-83, and R55W, respectively. Solid arrows indicate apparently completed immature capsid structures. Open arrows indicate vesicle-like structures in these non-detergent-treated samples.

Mutant Gag species assemble in bacteria. Since the in vitro system provides only a low level of expressed proteinless than can be detected by silver stain of gels (<10 ng [data not shown])it was of interest to compare the above results with those obtained by high-level expression. It had already been established that M-PMV Gag can assemble into immature particle-like structures when expressed at a high level in bacteriaamounts easily detected by Coomassie stain of gels (10 µg [data not shown]) (15). Although E. coli possesses transport machinery, it would not be expected that this machinery would specifically interact with M-PMV Gag. Thus, expression in bacteria would serve as a comparison to the in vitro system in the same way that the vaccinia virus/T7 overexpression system had served as a comparison to LTR-driven expression in cultured cells (24).

View larger version (113K): [in this window] [in a new window]   FIG. 6.   Analysis of M-PMV Gag species expressed in bacteria by thin-section electron microscopy. After 4 h of induction with IPTG, BL21(DE3) cells expressing the various M-PMV Gag species were centrifuged at low speed, and the resulting pellets were fixed and further processed for microscopy. (A to F) Pelleted bacteria expressing M100A, 8-53, 1-83, 1-25, 26-53, and 54-83, respectively. (G and H) Pelleted bacteria expressing R55W. Arrows indicate apparently completed immature capsid structures.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

By the utilization of two different expression systems in which retrovirus particle assembly can be observed in the absence of the complicating action of eukaryotic cytoplasmic transport, we have been able to unambiguously define the functions of two critical domains within the M-PMV Gag precursor protein. The region within matrix containing the R55 residue has now been definitively identified as a transport domain, since alteration of its sequence has no discernible effect upon Gag assembly in vitro and in bacteria. Likewise, the acidic stretch of residues within p12 constitutes an auxiliary assembly domain, since its deletion results in an inability of Gag to assemble under low expression conditions, even in the absence of a eukaryotic transport mechanism. Furthermore, these two systems, because of their contrasting levels of protein expression, varying from minute quantities in vitro to gross overexpression in bacterial cells, have allowed us to make direct comparisons with results from similarly varied levels of expression in tissue culture. The ability of the various p12 deletions to assemble in vitro mirrors their ability to assemble in HeLa cells, where expression levels mimic those of an infection (Fig. 7). Similarly, overexpression in bacteria allows for the assembly of all of the deletion mutants, just as overexpression with the vaccinia virus/T7 system compensates for the defect observed in cultured cells.

View larger version (11K): [in this window] [in a new window]   FIG. 7.   Summary of assembly by p12 deletion Gag mutants. The ability of various p12 deletion species to assemble in vitro and in bacteria is compared to previously reported data from the results of expression in tissue culture cell lines (* [see reference 24]). Schematics of the p12 domain of Gag are shown to the left, with regions of the domain present in each species depicted by the wide bar and regions absent depicted by the thin line. A region apparently critical to assembly under conditions of lower expression is depicted in gray. HeLa refers to results of transfected provirus expression in HeLa cells. Vac/T7 refers to overexpression by the vaccinia virus/T7 polymerase system in CV-1 cells. Plus and minus signs indicate the presence and absence, respectively, of assembled immature capsid structures.

Numerous studies have identified the matrix domain of retroviral Gag precursors as containing a signal or signals important for proper intracellular targeting (9, 17, 35). The R55W mutation lies within a region that is highly conserved between M-PMV and MMTV MA proteins. Since both of these viruses assemble within the cytoplasm, it would be expected for both Gag proteins to contain a similar targeting signal for the intracellular assembly site. In the NMR structure of the M-PMV matrix protein, R55 lies at the base of an external, solvent-exposed loop not found in the structures of any other matrix protein solved to date (8). One could envision this loop as functioning as a docking structure for the cellular transport machinery. Further evidence for the transport function of the R55 loop region of M-PMV matrix has been provided by Choi et al. (7), who have engineered this region into the matrix domain of murine leukemia virus (MuLV) Gag resulting in the production of intracytoplasmic particles. Similarly, the homologous region of MMTV can also induce the assembly of intracytoplasmic immature particles when engineered into the MuLV matrix domain (7). These results led the authors of that study to term this region a cytoplasmic transport-retention signal (CTRS).

Just as the CTRS is found in both M-PMV and MMTV, so too does the auxilliary assembly domain of M-PMV have an apparent counterpart in MMTV. In both cases, the sequence is very highly negatively charged, with seven glutamic acid residues within the M-PMV sequence and nine aspartic acid plus four glutamic acid residues in the homologous p3 region of MMTV (24). How might this region function to increase the efficiency of Gag multimerization and assembly? One possibility is that it functions as an internal scaffold that assists the remainder of the Gag precursor to assemble into the immature particle. Once this particle has formed and budded out of the cell, proteolytic maturation would then excise the p12 domain and leave the matured CA and NC proteins free to reassort into a cylindrical core. Precedence for an internal scaffold domain (ISD) within a virus capsid protein comes from studies of bacteriophage HK97, which unlike most phage, does not have a separate scaffold protein. Instead, the amino-terminal delta domain of the major shell protein is postulated to provide this function (13).

If the ISD indeed functions as an internal scaffold for B- and D-type retroviruses, what then provides the scaffold function for C-type viruses? Campbell and Vogt, in studies of in vitro assembly of Rous sarcoma virus Gag proteins, demonstrated a requirement for RNA for the formation of virus-like particles and hypothesized that this RNA serves as a scaffold upon which Gag proteins assemble (5, 6). However, it seems unlikely that RNA alone can serve as a required scaffold for immature particle formation, since HIV Gag or fragments of Gag can assemble into immature-like particles in vitro in the absence of added RNA (12, 30). Furthermore, since B- and D-type viruses must each incorporate a genomic RNA, the ISD region must be required in addition to RNA for efficient assembly of an infectious virus.

Do all retroviruses have a requirement for a scaffold-like function for efficient assembly? In the in vitro systems using purified Gag or Gag fragments referred to above, assembly is achieved at very high protein concentrationsseveral milligrams per milliliter. Similarly, fragments of HIV Gag, including those lacking the MA domain, can assemble intracellularly when expressed at extremely high levels by recombinant baculoviruses (reviewed in reference 3). This concentration effect is, perhaps, the same phenomenon we observed with p12-deleted M-PMV Gag, in which overexpression compensates for the deletion and allows assembly to occur. Thus, C-type and, indeed, ISD-deleted D-type Gag precursors are competent to assemble without the assistance of a putative scaffold, provided they are sufficiently concentrated. We would propose, therefore, that the ISD functions by increasing the effective concentration of M-PMV Gag to facilitate assembly, independent of it binding to RNA.

If, as postulated, the ISD provides the concentrating factor for B- and D-type retroviruses, what provides this function for the C-type retroviruses? The obvious candidate for this role is the plasma membrane. Several studies have demonstrated that even Gag proteins with very large deletionsprecursors that should be severely handicapped in their ability to assemblecan form particulate structures that bud from the cell provided they reach the plasma membrane. A Rous sarcoma virus Gag fragment, p25^M1, consisting of only MA plus the spacer peptides p2A and p2B between MA and p10 can still produce budded particles, although these are of a lower density than those produced by full-length Gag, due to the absence of the interaction (I) domain in NC (33). Similarly, the matrix protein alone of simian immunodeficiency virus is capable of producing membrane-enveloped particles (10). Thus, membranes may serve as the concentrating factor for C-type viruses and could even be considered to function as a scaffold upon which Gag proteins can multimerize. Consistent with this idea, C-type Gag proteins that are not myristylated fail to assemble, presumably because the precursors are not transported to and/or do not interact with membranes (4, 11, 19, 25). In contrast, myristylation-minus D-type Gag is capable of assembly, although the resulting immature capsids fail to be transported to the cell surface (20). Indeed, in the present study, we have tested the hypothesis that an interaction with the plasma membrane might be necessary to overcome a putative assembly defect in the R55W mutant of M-PMV, but have shown that this variant of Gag, which contains the ISD, is competent to assemble in the absence of such an interaction.

The studies presented here have extended the analysis of two critical morphogenic domains within the Gag precursor of M-PMV and have served to more precisely define their functions. Because it provides the ability to examine assembly in the absence of a functioning cellular transport machinery, the technique of in vitro synthesis and assembly has emerged as a powerful tool for the dissection of transport and assembly functions within the retrovirus Gag precursor.