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. ). To this end, we employed a transient mammalian cell expression system in which RSV-like particles are efficiently produced (41
). 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
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 ΔNC (Fig. ), 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. , ΔMA1, Myr1.ΔMA6E, and H32RΔMB). 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.
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.
To determine whether Gag proteins produced in our transient expression system are of a size similar to authentic, wild-type virus, infectious RSV produced in turkey embryo fibroblasts were run in a gradient along with Gag-only particles produced from COS-1 cells. To distinguish the proteins in the transiently produced particles from those of the authentic virus, a Gag mutant was employed that lacks protease activity (Myr0.D37S) and therefore releases only uncleaved Gag precursors. Following centrifugation, the two types of particles were found in the same fractions (Fig. A), and the distribution of particle sizes was uniform and homogeneous in each case. Thus, proteolytic maturation of the Gag protein does not influence the size of the particles. This was confirmed in an experiment in which the protease-positive and protease-negative Gag-only particles were both produced by transient expression (Fig. B). The addition of a foreign membrane-binding domain to the N terminus of Gag (i.e., the Src membrane-binding domain, which is present in many of our constructs) also did not affect particle size (Fig. C). Moreover, this substitution, combined with nearly complete deletion of the protease (mutant 3h, which lacks the last 117 amino acids of Gag), also had no effect on size (Fig. D), as previously reported (38
). From these control experiments, it appeared that the extremities of Gag do not control particle size.
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 (more ...)
Further evidence of this was obtained by examining the Src chimera (Myr1) and protease deletion mutant (3h) by thin-section EM. Both produced budding structures and released particles typical of C-type retrovirus morphogenesis (Fig. ). Due to the presence of an active protease, Myr1 was processed to produce electron-dense cores, as expected (Fig. A). Mutant 3h particles lack the PR domain and therefore retained the concentric ring structure typical of immature particles (Fig. B). Three concentric rings were observed. The outer ring was associated with the lipid envelope. Ten nanometers further toward the center was another, lighter-staining ring. The innermost, darkly staining ring had a diameter of about 40 nm and was located about 10 nm central to the middle ring. Consistent with the rate-zonal gradient data, the particles produced by both Myr1 and 3h were homogeneous in size and shape.
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 (more ...)
The Gag derivatives also were examined by negative staining. Most of the particles did not allow the stain to penetrate the lipid envelope and therefore only provided information on the overall shape and size of the particles. In some instances, the stain did enter the particle to reveal the internal structure (Fig. ). Central cores produced by proteolytic maturation were evident in Myr1 (Fig. A). Penetration of the stain into the center of the particles which had an inactive PR (D37I in Fig. B) or lacked a PR (3h in Fig. C) suggested that the center of the particle was hollow, as expected, with the protein located at the periphery. Striations similar to those reported for immature HIV (19
) were also clearly visible in D37I and 3h (Fig. B and C, respectively).
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; (more ...)
The diameters of individual negatively stained particles were determined from photographic negatives (Table ). The average diameters of Myr1, D37I, and 3h were essentially the same and identical to previously reported measurements for other retroviruses (9
). However, it is interesting that even these homogeneous particles are not identical in size but display some variability in particle diameter. This has been observed for other retroviruses, too. For instance, it has been reported that authentic HIV particles vary in diameter between 90 and 160 nm (32
) or 95 and 175 nm (10
), which is remarkably consistent with the values obtained for Myr1, D37I, and 3h (Table ). Because all of these Gag derivatives make normal-size particles, they could be used as controls in subsequent experiments to map the genetic determinants of particle size.
TABLE 1 Analysis of size distribution of virus-like particles by negativestaininga 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
). In mutants ΔMA1 and ΔMA6E, the complete M domain (contained in segments of 84 and 98 residues, respectively) has been replaced with the small Src membrane-binding domain (Fig. ). When analyzed for particle size, both mutants produced a uniform population of particles that were slightly smaller than the internal control (Fig. A 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 H32RΔMB (Fig. C), which has the 32-residue-long M domain of HIV Gag in place of the first 99 residues of RSV Gag (28
), and FynΔMB, 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.
Deletions that lead to smaller particles. Particle sizes were analyzed as described in the legend to Fig. .
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 (ΔMA6, ΔMA7, ΔMA8, and ΔMA9) collectively lack the residues from 87 in MA to 161 within p2a (Fig. ). For the most part, these deletions had no effect on particle size (Fig. A to D). In the case of ΔMA6, the particles were slightly smaller than the internal control (Fig. A) 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. E). 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
). However, it was possible to test a chimera, Δp2b.ip6 (Fig. ), 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. F).
Alterations in the second half of MA, p2a, p2b, and p10. Particle sizes were analyzed as described in the legend to Fig. .
Next, we analyzed mutants that lack various amounts of the p10 sequence. ΔQM1 lacks the first third of p10, while Δp10.31 and Δp10.52 have internal deletions (Fig. ). ΔQM1 released particles as efficiently as the wild type, but Δp10.31 and Δp10.52 exhibited reduced levels (data not shown). The lower yield of particles from these two p10 mutations was consistent with previously published deletions within p10 (11
). The reduction in budding for these latter two mutants is probably due to a conformational problem because large mutants with most of p10 and a large amount of CA deleted (such as R-3A and R-3J [Fig. ]) release particles at wild-type levels (39
). Nevertheless, particles produced by all three of our p10 mutants were homogeneous and uniform in size (Fig. G to I). Δp10.31 and Δp10.52 appeared to sediment slightly more slowly than the control particles, similar to the M domain substitutions (Fig. ); however, unlike ΔMA1, ΔMA6, and H32RΔMB, the p10 deletion mutants possessed wild-type density (data not shown). It may be that large deletions within p10 decrease the distance between the membrane-binding domain and the core, resulting in smaller particles (see Discussion), but this does not explain why ΔQM1 is not shifted to the same extent.
Collectively, the results shown so far indicate that deletions within MA, p2, and p10 (i.e., the first third of RSV Gag) have no effect on uniform particle release. Our next step was to determine whether deletions within the CA sequence would alter particle size.
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. ) 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. A to C). Mutant DM1, which combines the R-3J and 3h deletions (38
), produced a heterogeneous profile of particles as well (Fig. D). 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).
Large deletions spanning the p10-CA junction. Particle sizes were analyzed as described in the legend to Fig. .
To corroborate the rate-zonal gradient data and demonstrate that the heterogeneous profile of particles was not a result of aggregation, we analyzed three of these large deletion mutants by thin-section and negative-stain EM. R-3J and R-3J.D37S differ in having or not having an active protease, respectively. Both produced heterogeneously sized particles ranging in size from normal to extremely large, disrupted particles (Fig. D and F, respectively). For the larger particles produced by R-3J, the cores were either not present within the plane of section, aberrant, or off center (Fig. D). It was not clear whether the core was located at a fixed distance from the lipid envelope, which in normal-size particles would place it at the center, or whether the off-center cores represent a random distribution of free-floating cores. When the PR was inactivated (R-3J.D37S), the viral protein remained associated with the lipid envelope in discontinuous patches of electron-dense material and no cores were evident (Fig. F). A horseshoe-shaped patch of dense material was commonly observed, as if the circumference of the particles was not completely enclosed with protein. Similar results have been reported for HIV when small deletions were made in the N-terminal half of CA (10
). Normal budding structures were present for both R-3J and R-3J.D37S (Fig. E), but large accumulations of protein underneath the plasma membrane, with little if any curvature, were the predominant structures observed in the cells (Fig. G and H, respectively).
Negative-stain EM analysis of R-3J.D37S (Fig. E) and DM-1 (Fig. F) confirmed that these particles are heterogeneous in size. Penetration of the stain into the center of the particles with an inactive PR (R-3J.D37S in Fig. E) or lacking the PR sequence (DM1 in Fig. F) suggested that the center of the particle was hollow, as expected, with the protein located at the periphery. When the size distributions of R-3J, R-3J.D37S, and DM-1 were quantitated from negative-stain images, the particles were found to have a very heterogeneous profile, as predicted by the sedimentation analysis (Table ). The EM results may underrepresent the number of larger particles because numerous disrupted particles were observed, but only spherical particles were counted. In particular, the R-3J and R-3J.D37S Gag proteins differed only in PR activity, but R-3J produced smaller particles on average. Presumably, the largest mature particles from R-3J were less stable during purification or negative staining than the same-size immature particles from R-3J.D37S. DM-1 produced an even wider range of particle sizes as measured by EM, but such differences could not be detected in gradients.
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. ). All of these mutants produced particles that were heterogeneous in size (Fig. A 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).
Small deletions within CA. Particle sizes were analyzed as described in the legend to Fig. .
A larger deletion mutant, Es-Bg, which lacks the MHR along with some flanking sequences, produced particles with a broad peak that overlapped control particles (Fig. G). Thin-section EM (Fig. C) and negative-stain EM (Fig. D) of this mutant revealed particles that were heterogeneous in size. However, both EM and rate-zonal gradient data analysis demonstrated that Es-Bg particles are not as heterogeneous in size as some of the other CA deletions. The internal morphology of Es-Bg was interesting. Instead of a central, collapsed core, material seemed to be evenly distributed throughout the volume of Es-Bg particles (Fig. C and D) even though an active protease is present (data not shown; see reference 8
). The thin sectioning and negative staining suggested that Es-Bg has a defect in core assembly.
To look more closely at the MHR, we made use of mutant L171I (8
), in which a conserved Leu residue within the MHR is replaced with Ile. L171I has no effect on particle release, but when this point mutation is built back into the viral genome, the resulting viruses are noninfectious in avian cells. When analyzed for size, homogeneous particles were observed (Fig. H). Thus, although the MHR region may be critical for proper maturation of the viral core (8
), it does not play an important role in defining particle size.
Four additional deletions within the last quarter of the CA sequence also produced heterogeneously sized particles (Fig. I to L). Mutants LOC1 and LOC2 appeared as broad peaks that overlapped the internal control, whereas LOC9 and LOC10 particles were more heterogeneous. While the smaller deletions within CA produced particles with various degrees of heterogeneity, all of the CA mutations analyzed (with the exception of L171I) had some effect on particle size. Thus, it appears that CA provides a very critical determinant of particle size.
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. ) (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
). 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 ΔSP3, ΔSP9, and ΔSP12; Fig. ) and the peptides were separately expressed in avian cells, virions were efficiently assembled, but none of the mutants were infectious (7
). Sedimentation analysis revealed that all three mutants were heterogeneous in size (Fig. A to C). Thus, it appears that the CA and SP sequences in Gag provide a very critical size determinant.
Spacer peptide deletions. Particle sizes were analyzed as described in the legend to Fig. .
Deletions within NC.
Another mutant, Bg-Xm (Fig. ), 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. D), 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.
LON1 and Sm-Bs, which lack sequences within NC and retain one copy of the I domain (Fig. ), produced particles that were uniform and homogeneous in size (Fig. A and B). The slightly smaller size relative to the internal control of these uniformly sized particles might be a consequence of the reduced mass resulting from these large deletions (see Discussion). In contrast, ΔNC, with almost all of NC deleted, produced particles that were heterogeneous in size (Fig. C). The latter result was not surprising, since ΔNC lacks both copies of the I domain and releases particles with low density (43
). If the I domains are not present, proper interactions cannot take place among the Gag proteins and heterogeneous particles with low density are produced. Thus, along with having an intact CA-SP domain, the Gag protein must have at least one copy of the I domain to produce particles that are uniform and homogeneous in size.
Deletions within NC. Particle sizes were analyzed as described in the legend to Fig. .
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 ΔNC 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).
To test the ability of ΔNC 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 (illustrated in Fig. , top row). When CA deletion mutant R-3J or Es-Bg was used, no interactions were observed, as shown by the continued appearance of mutant ΔNC in particles of lower density (Fig. , left column). Similar results were obtained with mutant R-3K (data not shown). In contrast, when ΔNC was coexpressed with mutant 3h (which lacks protease but retains CA-SP), it was found in particles normal in both density and size (Fig. , right column). The simplest interpretation of this result is that the CA-SP sequence of ΔNC is indeed properly folded and provides a means for the mutant Gag protein to be rescued into normal particles.
FIG. 11 Complementation rescue mediated by CA-SP. (Top row) To test the ability of ΔNC 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. (more ...)