INTRODUCTION

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Particle assembly of retroviruses is conducted by the Gag polyprotein, which is able to direct budding at the plasma membrane in the absence of the pol and env gene products or of the viral RNA genome (40). Gag proteins are synthesized on free ribosomes in a cytosolic compartment and are then transported to the plasma membrane. At this point, roughly 2,000 Gag molecules interact to drive the budding process. During or shortly after budding, the viral protease (PR) cleaves the Gag molecules into their mature protein products (23), the matrix (MA), capsid (CA), and nucleocapsid (NC); however, proteolytic activity is not required for particle production. For Rous sarcoma virus (RSV), the focus of this report, several additional cleavage productsp2a, p2b, p10, spacer peptides (SP), and PR itselfare liberated upon proteolytic cleavage (see Fig. 1) (2, 21, 29, 31). Particles released when Gag is expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size.

The mechanism by which Gag directs budding is unknown, but significant advances have been made in dissecting this complex event. Studies with RSV and human immunodeficiency virus (HIV) Gag have revealed three small assembly domains, M, L, and I (Fig. 1), that are required for particle formation. The M domain provides a membrane-binding function and is located at the amino terminus in both the RSV and HIV Gag proteins (1, 28, 42). The L domain maps to a proline-rich sequence within RSV p2b and the C-terminal p6 sequence in HIV Gag. It is believed to operate very late in budding, at the membrane-bud separation step (16, 18, 27, 39). The interaction (I) domain, which is present in two copies within NC, mediates productive interactions between Gag proteins to produce particles of the proper buoyant density (1, 38, 43). Further evidence for the important interactions mediated by I domains is the observation that previously characterized large-deletion mutant RSV Gag proteins lacking these domains cannot be rescued into particles when coexpressed with wild-type Gag. However, mutant Gag proteins that retain a functional copy of I but lack either the M or L domain can still be rescued (39, 42).

While mutations within the assembly domains block or severely cripple particle formation, regions that lie outside these domains can be removed with no effect on the release of particles of normal density. Moreover, although the primary amino acid sequences and the order of M, L, and I differ between RSV and HIV, these functions are fully exchangeable between the two Gag proteins (1, 27, 28).

While much progress has been made in understanding how Gag functions during budding, there is little evidence to suggest how it defines the size of a particle. There is some indication in the literature that the CA sequence is important. In the case of RSV, it has been found that large deletions that remove half of CA and most of the p10 sequence result in particles that are very heterogeneous in size (38), but those studies are difficult to interpret because of the severity of the deletions. Other studies have suggested that regions surrounding the CA sequence are critical for particle size and morphology. For instance, it has recently been demonstrated that the addition of CA to the NC sequences of RSV and HIV enables the resulting CA-NC protein, in the presence of RNA, to self-assemble in vitro into tubular structures (3). In the case of RSV, addition of further N-terminal sequences, for example, p10, p2b, p2a, and part of MA, results in the assembly of spherical particles with dimensions similar to those of authentic virions (4). Along with these findings, linker insertions and deletions made within the CA domain of HIV Gag have been found to alter the size of the particles (5, 10, 32). Taken together, these results indicate that additional functions within Gag are needed to constrain the size of the emerging particle but they do not define the exact regions involved.

To precisely map the size-determining regions of RSV, we have employed rate-zonal gradients and electron microscopy (EM) to systematically study the effect of deletions throughout the Gag protein. The only region we found to be critical for determining particle size is the CA-SP sequence, which is the initial capsid species released from Gag and which contains all of the CA sequence plus the spacer peptides. Some regions of this capsid intermediate appear to be more important than others. Thus, while the CA-SP sequence of RSV Gag is dispensable for budding, it is critical for producing particles of uniform and proper size.

	MATERIALS AND METHODS

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Previously described Gag constructs. The RSV gag gene was obtained from pATV-8, an infectious molecular clone of the RSV Prague C genome (33). All of the gag alleles (see Fig. 1) were expressed in simian (COS-1) cells by using the simian virus 40-based mammalian expression vector we have described previously (42). Many of these alleles have been previously reported: pSV.Myr0 (wild type) (42), pSV.Myr1 (42), pSV.Myr1.3h (37), pSV.MA1 (42), pSV.H32R.MB (28), pSV.Myr1.MA6 (25), pSV.Myr1.MA7 (25), pSV.Myr1.MA8 (25), pSV.Myr1.MA9 (25), pSV.Myr0.p2a (39), pSV.Myr0.p2b.ip6 (27), pSV.Myr1.R-3K (39), pSV.Myr1.R-3A (39), pSV.Myr1.R-3J (38, 39), pSV.Myr1.DM1 (38, 39), pSV.Myr1.Es-Bg (8), pSV.Myr1.L171I (8), pSV.Myr1.LOC1 (8), pSV.Myr1.LOC2 (8), pSV.Myr0.SP3 (30), pSV.Myr1.SP9 (7, 30), pSV.Myr0.SP12 (30), pSV.Myr1.Bg-Xm (8), pSV.Myr1.LON1 (8), pSV.Myr1.Sm-Bs (39), and pSV.Myr1.NC (43). In some cases, the activity of the retroviral protease was eliminated in these constructs by substituting the aspartic acid in the active site with either isoleucine (D37I) (6, 34, 41) or serine (D37S) (6), changes which have no effect on particle release, density, or size (see below). All plasmids were propagated in Escherichia coli DH-1 cells and selected by using medium containing 100 µg of ampicillin per ml.

Construction of additional Gag mutants. Mutant pSV.Myr1.MA6E was constructed by digesting pSV.Myr1.MA6 with MluI and SpeI and discarding the small fragment. The sticky ends were then treated with Klenow fragment to realign the gag reading frame, and the plasmid was religated. This deletion encodes a protein which contains one foreign residue (Asp) between amino acids 11 and 99 of Gag.

Transfection of cells. COS-1 cells were grown in Dulbecco's modified Eagle medium (Gibco BRL) supplemented with 3% fetal bovine serum and 7% bovine calf serum (Hyclone, Inc.). RSV-infected turkey embryo fibroblasts were propagated in supplemented F10 medium as previously described (20). COS-1 cells in 60-mm-diameter plates were transfected by the DEAE-dextran-chloroquine method as described previously (41). Before transfection, the plasmid DNAs were digested with XbaI and ligated at a concentration of 25 µg/ml. This step removes the bacterial plasmid sequence and joins the 3' end of the gag gene with the simian virus 40 late polyadenylation signal for high-level expression (41). Plasmid pSV.Myr0.LOC10 was prepared for transfection by digestion with BssHII and ClaI, treatment with Klenow fragment, and religation of the plasmid. This step, which has the same outcome as XbaI digestion, was exploited since an extra XbaI site had been created by the LOC10 deletion. Typically, 1 µg of DNA was applied to each monolayer. For cotransfections, the cells received 0.5 µg of DNA each.

Metabolic labeling and sucrose gradient analysis. At 48 h after transfection, COS-1 cells were starved for 0.5 h in methionine-free, serum-free Dulbecco's medium and then labeled in 0.6 ml of labeling medium supplemented with L-[³⁵S]methionine (50 µCi, >1,000 Ci/mmol) for 8 h. After the labeling period, the medium from each plate was collected and transferred to a microcentrifuge tube, and cellular debris was removed by centrifugation at 15,000 × g for 1 min. For the rate-zonal gradients, labeled particles containing a Gag protein of wild-type density and size was mixed with the samples to provide an internal control. In some cases, infectious RSV was included as the internal control. This virus was grown in turkey embryo fibroblasts and labeled with L-[³⁵S]methionine as described above. The mixture was then layered onto 11.5-ml, 10 to 30% sucrose and centrifuged at 83,500 × g (26,000 rpm) at 4°C for 30 min in a Beckman SW41Ti rotor. Fractions (0.6 ml) were collected through the bottom of each tube and mixed with lysis buffer containing protease inhibitors. The Gag proteins in each fraction were immunoprecipitated with a rabbit antiserum against RSV (reactive with MA, CA, NC, and PR), electrophoresed in a sodium dodecyl sulfate-12% polyacrylamide gel (SDS-PAGE), and detected by fluorography as previously described (37, 38, 41). The resulting films were quantitated by laser densitometry. All gradients were repeated at least once to confirm the results.

Virus-like particle isolation and analysis by EM. At 48 h posttransfection, medium containing virus-like particles was collected and prepared for EM as previously described (34). Briefly, cellular debris was removed from the medium by low-speed centrifugation at 11,950 × g for 10 min in a Sorvall SS-34 rotor. The particles contained in the supernatant were then pelleted through a 15% sucrose-STE (100 mM NaCl, 10 mM Tris [pH 7.5], 1 mM EDTA) cushion at 108,760 × g for 90 min in a Beckman 50.2 Ti rotor. The particle-containing pellets were softened in STE for 1 h at 4°C before resuspension by pipetting and storage at 4°C. Particles were negatively stained with 2% uranyl acetate.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

To ascertain whether particle size determinants could be mapped to a particular region of RSV Gag, we studied the effects of deletions throughout this protein (Fig. 1). To this end, we employed a transient mammalian cell expression system in which RSV-like particles are efficiently produced (41, 42). In this system, the wild-type Gag protein (designated Myr0 to indicate the lack of myristate at the N terminus; 41) drives the release of particles that are identical to authentic virions in terms of their rates of budding, core morphology, size, density, and proteolytic processing of the mature cleavage products (1, 2, 7, 37, 41, 42).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Alterations of the RSV Gag protein. The wild-type (WT) RSV Gag protein (Myr0) is shown at the top. The vertical lines represent viral PR cleavage sites, which separate the mature Gag products (MA, p2a, p2b, p10, CA, SP, NC, and PR) as indicated. The horizontal bar above the Gag protein denotes CA-SP (CA plus the spacer peptides). The shaded region within CA marks the MHR. Numbers refer to amino acid residues. Thick, solid bars underneath the Gag protein indicate assembly domains required for budding (M, L, and I). Illustrated below Myr0 are the deletion and substitution mutants utilized in this study. The open box at the amino terminus of some of the constructs denotes the M domain of the Src oncoprotein (Myr1). The shaded region in H32R.MB and p2b.ip6 indicates substitution of the RSV Gag M and L domains for the HIV equivalents, respectively. The vertical line in Myr1.L171I indicates a substitution of leucine for isoleucine. The column to the left lists the names of the mutants. The column to the right summarizes the size distribution of the mutants as follows: U, particles that are uniform in size; Sm, particles that are uniform in size but smaller in diameter; Het, particles that are heterogeneous in size.

All of the mutants in this study, with just two exceptions, produce particles of normal density (1.16 to 1.18 g/ml) (9). One exception is mutant NC (Fig. 1), which lacks both I domains and therefore produces particles that are dramatically lower in density (1.14 to 1.15 g/ml). The other exception is a group of mutants in which the RSV M domain is completely replaced with smaller membrane-binding domains (Fig. 1, MA1, Myr1.MA6E, and H32RMB). The density shift in this case is minor, however, and the particles band at a density (1.15 to 1.16 g/ml) that overlaps the normal range for wild-type retroviral particles.

Control experiments. Particles produced in the transient expression system were analyzed for size by using rate-zonal sedimentation gradients. For this, culture supernatants containing radiolabeled particles were harvested and cellular debris was removed. A radiolabeled Gag protein of wild-type size was always added to the supernatants to provide an internal control, and the particle mixtures were layered onto 11.5-ml, 10 to 30% sucrose gradients and centrifuged for 0.5 h at 83,500 × g. After centrifugation, the gradients were fractionated and Gag proteins were immunoprecipitated and separated by SDS-PAGE. The resulting X-ray films were then subjected to scanning densitometry to determine the position and amount of Gag protein in the gradients. Under these sedimentation conditions, particles migrate according to their relative sizes. When interpreting the rate-zonal gradients, it is important to note that the position of the peak fraction relative to the internal control and the distribution of the particles in the gradient (e.g., heterogeneous versus uniform) are more important than the heights of the peaks. Rate-zonal gradients provide an important advantage over EM analysis, in that they reveal large differences in particle size and provide information on the total population of particles released from the cell. That is, Gag proteins will be detected whether they are present in particles of recognizable morphology or not.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Control experiments. COS-1 cells transfected with the indicated gag derivatives or RSV-infected turkey embryo fibroblasts were labeled with [35S]methionine for 8 h. After the labeling period, the medium from each plate was collected and mixed with labeled control particles. The mixture was then layered onto 10 to 30% sucrose and centrifuged at 83,500 × g at 4°C for 0.5 h. Fractions were collected through the bottom of each tube, immunoprecipitated with a polyclonal rabbit antiserum against RSV, electrophoresed in an SDS-12% polyacrylamide gel, and detected by fluorography. The autoradiogram was then quantitated by laser densitometry. Arrows indicate the direction of sedimentation.

View larger version (106K): [in this window] [in a new window]   FIG. 3.   Thin-section EM of cells and virus-like particles. At 48 h posttransfection, cells were examined by thin-section EM as described in Materials and Methods. (A) Myr1 particles were homogeneous in size with condensed cores. (B) 3h particles had immature morphology with three rings. (C) Es-Bg particles had noncondensed cores. (D) R-3J particles were heterogeneous in size with condensed cores. Most cores were not in the plane of section or were acentric (arrows). (E) R-3J budding particles; initial bud (left), late bud (center), and released mature particle (right). (F) R-3J.D37S particles were heterogeneous in size with immature morphology. The peripheral material often had gaps. Only two rings were visible. (G) R-3J, flattened patches of protein accumulated on the surface of cells. (H) R-3J.D37S particles budding between patches of accumulated protein. Bars, 100 nm.

View larger version (166K): [in this window] [in a new window]   FIG. 4.   Negative-stain EM of virus-like particles. At 48 h posttransfection, virus-like particles were collected by centrifugation and negatively stained with 2% uranyl acetate as described in Materials and Methods. A, Myr1; B, D37I; C, 3h; D, Es-Bg; E, R-3J.D37S; F, DM1. The arrow in C indicates the striated pattern in D37I and 3h. Bars, 100 nm.

Replacement of the first half of MA with smaller M domains. We began our systematic analysis of RSV Gag with mutants that lack sequences within the first half of MA. We have previously reported that small deletions within the first 85 residues of MA, which constitute the M domain, are defective for budding; however, budding is restored when the membrane-binding domain from Src is placed at the amino terminus (1, 42). In mutants MA1 and MA6E, the complete M domain (contained in segments of 84 and 98 residues, respectively) has been replaced with the small Src membrane-binding domain (Fig. 1). When analyzed for particle size, both mutants produced a uniform population of particles that were slightly smaller than the internal control (Fig. 5A and B). The shift of the peak to a position two fractions higher in the gradient was quite reproducible (data not shown). It may be that removal of the bulky 85-amino-acid M domain of RSV allows the membrane to be pulled closer to the core, thereby reducing slightly the diameter of the particle; alternatively, the lower overall mass of the particles might result in the slight shift (see Discussion). This phenotype was not limited to the Src chimeras but was found in all chimeras in which the RSV M domain had been replaced with a smaller M domain, including mutant H32RMB (Fig. 5C), which has the 32-residue-long M domain of HIV Gag in place of the first 99 residues of RSV Gag (28), and FynMB, in which the first 99 amino acids of MA are replaced with the membrane-binding domain of the Fyn oncoprotein (data not shown). The precise explanation of this minor shift to a higher position in the gradient remains to be determined (see Discussion); however, we conclude from these results that the M domain does not contribute greatly to particle size.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Deletions that lead to smaller particles. Particle sizes were analyzed as described in the legend to Fig. 2.

Deletions within the second half of MA, p2, and p10. The next set of four deletions span the second half of MA, which is dispensable for particle assembly and infectivity in avian cells (25). These mutants (MA6, MA7, MA8, and MA9) collectively lack the residues from 87 in MA to 161 within p2a (Fig. 1). For the most part, these deletions had no effect on particle size (Fig. 6A to D). In the case of MA6, the particles were slightly smaller than the internal control (Fig. 6A) but the density was identical to that of the wild type (data not shown). Moving further down the Gag protein, we found that when all of p2a was deleted, particles of uniform size were released as well (Fig. 6E). It was not possible to analyze a p2b deletion, since this cleavage product contains the proline-rich L domain and its removal blocks particle release (27, 39). However, it was possible to test a chimera, p2b.ip6 (Fig. 1), in which the p2b domain of RSV Gag has been replaced with the L domain from p6 of HIV Gag (27). The foreign amino acid sequence had no effect on particle size (Fig. 6F).

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Alterations in the second half of MA, p2a, p2b, and p10. Particle sizes were analyzed as described in the legend to Fig. 2.

Large internal deletions within Gag. To analyze what impact CA deletions have on particle size, we initially made use of three large internal deletion mutants (R-3K, R-3A, and R-3J in Fig. 1) which lack various amounts of p10 and CA. R-3K, R-3A, and R-3J have been previously shown to produce dense particles at the same efficiency as wild-type Gag (39). However, the particles released by these mutants were found to be extremely heterogeneous in size, with material spread throughout the gradient (Fig. 7A to C). Mutant DM1, which combines the R-3J and 3h deletions (38, 39), produced a heterogeneous profile of particles as well (Fig. 7D). Because p10 deletion mutants are not heterogeneous in particle size, we hypothesized that the defects of these large deletion mutants would map to the CA sequence (see below).

View larger version (23K): [in this window] [in a new window]   FIG. 7.   Large deletions spanning the p10-CA junction. Particle sizes were analyzed as described in the legend to Fig. 2.

Small deletions within CA. Having found that large deletions that extend into CA result in heterogeneously sized particles, we decided to see what effect much smaller mutations solely within CA would have. It was possible that certain regions within CA would be critical for determining particle size, with others being dispensable. Mutants LOC3 through LOC8 lack 10- to 11-amino-acid segments between the beginning of CA and the major homology region (MHR; Fig. 1). All of these mutants produced particles that were heterogeneous in size (Fig. 8A to F). Some produced more of a broad peak which overlapped the control particles (LOC3 to 5), while others produced material that was decidedly larger than the control (LOC6 to 8).

View larger version (32K): [in this window] [in a new window]   FIG. 8.   Small deletions within CA. Particle sizes were analyzed as described in the legend to Fig. 2.

Spacer peptide deletions. The CA sequence is initially released from Gag with a small (12-residue) spacer peptide at its C terminus following cleavage between the peptide and the NC sequences (Fig. 1) (7). This form of CA, previously referred to as CA1 (7), is referred to here as CA-SP for clarity. Over the course of several hours after particle release, cleavages within the spacer peptide result in the appearance of two new products that actually run more slowly in SDS-PAGE (2, 7). Recent studies (30) have demonstrated that in mature virus, the CA protein exists as fully mature CA (formerly named CA2) and a form of CA that retains three residues of the spacer, CA-S (formerly referred to as CA3). When precise deletions of these spacer peptides were made (mutants SP3, SP9, and SP12; Fig. 1) and the peptides were separately expressed in avian cells, virions were efficiently assembled, but none of the mutants were infectious (7, 30). Sedimentation analysis revealed that all three mutants were heterogeneous in size (Fig. 9A to C). Thus, it appears that the CA and SP sequences in Gag provide a very critical size determinant.

View larger version (24K): [in this window] [in a new window]   FIG. 9.   Spacer peptide deletions. Particle sizes were analyzed as described in the legend to Fig. 2.

Deletions within NC. Another mutant, Bg-Xm (Fig. 1), contains a deletion which removes that portion of the CA sequence downstream of the MHR and the first third of NC, effectively removing the spacer peptides and the surrounding sequence. This deletion produced heterogeneously sized particles (Fig. 9D), which we attributed to deletion of the C-terminal region of CA-SP. However, it was also possible that the extension of the deletion into NC contributed to the defect in particle size. This was explored by using NC deletion mutants.

View larger version (20K): [in this window] [in a new window]   FIG. 10.   Deletions within NC. Particle sizes were analyzed as described in the legend to Fig. 2.

Complementation rescue mediated by CA-SP. If CA-SP controls the size of RSV, then it most probably does so through self (i.e., CA-SP-CA-SP) interactions. The properties of mutant NC provided an opportunity to test this idea. This is the only mutant we have found that produces heterogeneously sized particles even though it retains the complete CA-SP sequence. We hypothesized that the CA-SP sequence is properly folded in this mutant but the absence of I domains results in local concentrations of Gag that are too low to permit self interactions (i.e., CA-SP interactions themselves are too weak to create high-density particles).

View larger version (28K): [in this window] [in a new window]   FIG. 11.   Complementation rescue mediated by CA-SP. (Top row) To test the ability of NC to participate in Gag interactions, it was coexpressed with Gag molecules that have all the assembly domains (M, L, and I) and either a mutant or a complete CA-SP sequence. The white boxes indicate the CA sequence. COS-1 cells were cotransfected with NC and the rescuing gag allele. At 48 h after transfection, cells were labeled with [35S]methionine for 8 h. After the labeling period, the medium from each plate was collected. The particles were then layered onto 10 to 50% sucrose and centrifuged to equilibrium at 83,500 × g at 4°C for 16 h. Fractions were collected through the bottom of each tube, immunoprecipitated with a polyclonal rabbit antiserum against RSV, electrophoresed in an SDS-12% polyacrylamide gel, and detected by fluorography. The autoradiogram was then quantitated by laser densitometry. Left column, density gradient of cotransfected R-3J and NC, as well as Es-Bg and NC, particles. Right column, density and rate-zonal gradients of cotransfected 3h and NC particles. Particle sizes were analyzed as described in the legend to Fig. 2. Arrows indicate the direction of sedimentation.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study, we have shown that the size determinants within the RSV Gag protein map to just one region, CA-SP, the segment of Gag consisting of CA plus the spacer peptides located between CA and NC. CA-SP is the first capsid protein species to be released from Gag. It previously was referred to as CA1 (7, 30), but we have adopted the new name to better clarify its structure, which is analogous to that of the initial cleavage product of HIV, variously called p25, CA-p2, or CA-SP1. While the sequence comprising CA-SP lies outside of the assembly domains (M, L, and I) and is completely dispensable for budding, this study demonstrates that it plays a fundamental role in constraining the size of the emerging particle. We use the term "particle" in a broad sense, operationally defined as a Gag protein released into the medium in a particulate form with a density similar to that of wild-type virions. However, in those cases where we have looked, objects with the appearance of true virus-like particles have always been seen (i.e., particles are membrane enclosed, have electron-dense cores, etc.).

The major method we employed to analyze particle size, rate-zonal sedimentation in sucrose gradients, has both advantages and limitations that bear on the interpretation of the data presented. The advantages include the display of the entire population of particles and the standardization provided by internal markers. Among the limitations are the relative lack of sensitivity of sedimentation rate to small changes in size. For particles different in sizes but invariant in density, this rate is directly proportional to the mass and inversely proportional to the frictional coefficient. For example, for a sphere of uniform density, a doubling in mass increases the diameter by the 1/3 power and thus the frictional coefficient (which is proportional to the cross section) by the 2/3 power. As a consequence, the sedimentation rate will increase only by a factor of 2^1/3, or about 26%. This value corresponds to only a few fractions of the collected gradient. Seen in this light, even small changes in the peak positions of the deletion mutants imply significant mass differences, with particles near the bottom of the gradients having masses many times larger than that of wild-type virions. For deletion mutants showing the most heterogeneous sedimentation profiles, the budded particles may not be spherical, as suggested by some of the EM results, and thus may have increased frictional coefficients and even larger sizes than expected of spherical particles with this sedimentation rate. On the other hand, a sedimentation profile coinciding with that of wild-type virions does not necessarily imply a completely homogeneous distribution of particles. Recent cryo-EM measurements of murine leukemia virus (44) and baculovirus-expressed HIV virus-like particles (12) show that, unlike icosahedral viruses, these retroviruses are not entirely uniform in size. It remains to be seen to what degree those results can be extrapolated to avian retroviruses. However, as reported here (Table 1), homogeneous RSV Gag virus-like particles do appear to exhibit variability in particle diameter.

It should be noted that analysis of one of our mutants, SP9, using rate-zonal gradients (Fig. 9B) is at odds with our previously published EM data which showed that this mutant re-leases particles identical to those of the wild type (7). While SP9 can produce particles similar in morphology and size to wild-type virions, a certain subset of the population is more heterogeneous and may have been overlooked by EM. The inability to recognize abnormally sized particles by EM is a limitation of that method of measuring particle size.

Model of RSV particle assembly. Our understanding of how particle assembly occurs during budding is illustrated in Fig. 12. The M, L, and I domains of the RSV Gag protein provide the minimal budding machinery (Fig. 12A). The M domain directs the Gag molecules to the plasma membrane; there, neighboring Gag molecules interact through their I domains via RNA (1, 38, 43). These strong interactions lead to tight packing of the Gag molecules which, in turn, allows the CA-SP sequences to establish contact with one other. The adjacent CA-SP interactions give proper curvature to the bud as it emerges from the surface of the cell. L is believed to function late in budding to separate the viral particle from the cell surface. The result of this process is a viral particle of uniform shape, density, and size.

View larger version (36K): [in this window] [in a new window]   FIG. 12.   Model of RSV particle assembly. Assembly domains M (black ovals), L (black triangles), and I (black circles) are indicated. The squiggly line represents the CA-SP sequence. The horizontal black line denotes the cell membrane. (A) Wild-type Gag proteins interact to form a budding particle of uniform size and density. (B) Gag mutants that harbor a CA deletion produce heterogeneous particles of normal density. (C) Gag mutant that lack functional I domains (open circles) but retain a properly folded CA-SP domain produce heterogeneous particles of low density.

Effects on core morphology. While extensive deletions outside of CA-SP had no effects on particle size, every deletion we analyzed within CA-SP yielded particles that were abnormal in size. Some mutations produced very large and heterogeneous particles (R-3J), whereas others were not as varied (Es-Bg). Es-Bg was particularly interesting because it lacks the MHR and some of the flanking sequence. The MHR is the most highly conserved region within Gag proteins and is involved in the maturation of the virion after budding (8, 24, 35). RSV MHR mutants exhibit defects in core stability and display blocks to infectivity upon entry into the host cell (8). Interestingly, EM analysis of Es-Bg revealed no darkly staining central core but, instead, electron-dense material seemed to be evenly distributed throughout the particle, consistent with a defect in core assembly. This is in stark contrast to R-3J, in which the first half of CA is deleted yet it still forms electron-dense cores. These data suggest that the first half of CA is situated on the outside, while the second half (including the MHR) lies toward the center of the core. This interpretation is compatible with models of the HIV capsid structure (13, 14, 17).

Smaller mutants. While the CA-SP sequence provides the major size determinant, replacement of the large membrane-binding domain of RSV Gag with smaller ones invariably resulted in slightly smaller particles (Fig. 5). Because the RSV membrane-binding domain is in tight contact with the viral membrane, substitution of a physically smaller M domain may take up less space along the membrane and therefore result in a smaller particle. It also appears that MA may contribute to a small degree to the density of the particle. The idea of MA-MA interactions is consistent with previous reports that I domain mutants can still make particles, although these are low in density (1, 38, 43).

Size determinants in other retroviruses. Although a systematic search for size determinants has not been done for any other retrovirus, there are reports in the literature that are consistent with our findings. Linker insertion mutations and deletions within HIV CA have been shown to produce particles heterogeneous in size with diameters of 75 to 315 nm (32). These estimates are similar to the values we have reported for the RSV CA deletions in Table 1 (R-3J, R-3J.D37S, and DM1). Moreover, previously published data on the spacer peptide between CA and NC of HIV demonstrated that cells expressing the protein containing a precise deletion of this region released heterogeneous particles that were noninfectious (22). However, there is reason to believe that our findings with RSV may not apply to all retroviruses. In particular, work in our laboratory suggests that the p6 sequence at the end of HIV Gag is very important (15). Clearly, methodical searches for size determinants in other Gag proteins are warranted.