Maturation converts an assembled retrovirus particle into an infectious particle. Maturation occurs as a result of concerted cleavages at a small number of sites within the Gag, Pol, and (in some retroviruses) Env protein molecules of the virion, and it is accompanied by a wide variety of changes in the particle. Our goal in these experiments was to gain some insight into the contribution that each of the three cleavages in MLV Gag makes to the physical changes in the particle and in its acquisition of infectivity.
One of the most striking changes associated with retroviral maturation is the conversion from the immature morphology, with a densely staining ring surrounding a relatively electron-lucent interior, to the mature form, in which the ring is absent and the center of the particle is filled with densely staining material. We found that site 1, site 2, and site 3 mutants all lacked the ring and contained densely staining material in their interiors, in contrast to fully immature PR− particles. Thus, no single cleavage event is absolutely required for the loss of the ring or deposition of this material. Perhaps the interior core in mature particles is composed of RNA associated with NC, and cleavage anywhere in Gag is sufficient for the release of RNA, together with RNA-bound proteins, from the membrane-associated MA domain.
Our results with individual mutants can be briefly summarized as follows. First, mutants at site 1 (preventing cleavage between p15MA
and p12) are released at essentially the same level as wild-type MLV. S1KK particles (in which the leucine and tyrosine residues at the C terminus of MA have been replaced with lysines) are slightly smaller than wild type. (Obviously, this is an effect of the change in Gag sequence on the assembly process itself and tells us nothing about the significance of site 1 cleavage.) Particles in which this cleavage was blocked were still infectious (Table ), with a specific infectivity only ~10-fold lower than that of wild-type control particles. Thus, the cleavage event between MA and p12 appears to be merely “fine-tuning,” improving the efficiency of replication by roughly an order of magnitude. Perhaps this is not surprising, since other studies have shown that a substantial portion of p12 is tolerant to alanine substitution (52
), and under some conditions virtually the entire MA domain of HIV-1 can be deleted without loss of infectivity (32
In contrast, site 2 mutants displayed no detectable infectivity: their specific infectivity was at least 1,000-fold lower than that of wild-type particles. Like site 1 particles, they were slightly smaller than wild-type controls. Further, they were frequently different in appearance from either mature or immature particles: unlike wild-type particles, they did not possess a distinct outline or “capsid shell” around the core, but unlike PR−
particles they often contained a compact body within the interior of the virion (Fig. to ). We also observed that if cleavage at site 2 were blocked, then the cleavage between CA and NC was somewhat inefficient (for example, note the accumulation of a larger intermediate, as well as p42, in S2D particles [Fig. ]). This observation suggests that cleavage at site 1 and/or site 3 is partially dependent upon cleavage at site 2. Indeed, in normal MLV maturation, cleavage at site 2 precedes that at sites 1 and 3 (28
At site 3, replacement of the C-terminal leucine in CA with arginine allowed the formation, at low efficiency, of virions roughly similar to mature MLV particles (Fig. ). However, many of these particles were abnormal in appearance, with a small accumulation of electron-dense material acentrically placed within the “core” of the particle. All of the other changes that we made at the C terminus of CA reduced the ability of Gag to assemble into released virions by a factor of approximately 100. The amount of genomic RNA per amount of Gag protein was somewhat lower in the S3R particles than in wild type, and it also appeared to be more susceptible to degradation than that in controls. The deficit in genomic RNA in these particles may be partially compensated for by an increase in rRNA content.
Particles in which cleavage was blocked at site 1 or 2 still contained mature dimeric RNA, with the same thermostability as that extracted from mature MLV particles (Fig. ). The fact that inhibition of cleavage at site 1 or site 2 still permits the stabilization of genomic RNA dimers accompanying viral maturation strongly suggests that the separation of CA and NC is the event leading to stabilization of the viral RNA. (We cannot exclude the alternate possibility that other PR-mediated cleavages, such as those in the Pol region, are responsible for the stabilization.) It would seem reasonable to imagine that when NC is released from CA during maturation, it can coat the genome and then, by virtue of its nucleic acid chaperone activity, catalyze the conformational rearrangement of the dimeric RNA. (In avian retroviral particles, chemical cross-linking of NC to RNA in mature particles is several orders of magnitude more efficient than cross-linking of Gag to RNA in immature particles [41
].) However, studies with recombinant HIV-1 proteins have shown that the Gag polyprotein possesses nucleic acid chaperone activity very similar to that of its cleavage product, NC (7
). Indeed, both HIV-1 NC and the HIV-1 Gag polyprotein can stabilize dimeric linkages in transcripts containing retroviral sequences in vitro (7
). Therefore, the requirement that Gag be cleaved before the genomic RNA within the virion attains its most stable conformation is probably due to a change in the access of the viral proteins to the RNA accompanying maturation, rather than a difference in the chaperone activities of Gag and NC. The present results suggest that it is cleavage between CA and NC that affords the NC domain increased access to the RNA. It is also possible, of course, that Gag and NC differ quantitatively or qualitatively with respect to their chaperone activity in ways that our in vitro assays (7
) failed to detect. Our results here would then imply that the difference in activity depends upon cleavage of NC from CA. A prior study with HIV-1 also concluded that cleavage of Gag at the N terminus of NC is critical for maturation of the genomic RNA dimers (39
It seems likely that the p10NC moiety of the p40 fusion protein is bound to RNA within the site 3 mutant particles; perhaps this is the acentrically located mass visible in electron micrographs of these particles (Fig. ). The fusion protein is apparently less effective than wild-type NC in protecting the genomic RNA against nucleolytic degradation, either within the particle or during extraction. The attachment of this protein to RNA may also explain the fact that a substantial fraction of p40 remains pelletable after disruption of the particle with NP-40 (Fig. ).
We also found that the ratio of genomic RNA to Gag protein in S3R particles was somewhat lower than in wild-type particles (Table ). This observation implies that genomic RNA encapsidation is reduced by the S3R mutation, and it might suggest that the region of Gag responsible for the encapsidation extends a short distance into the CA domain. However, it is also possible that each S3R particle contains the same amount of viral RNA as a wild-type particle but is, on average, somewhat larger and contains two to three times as much Gag protein as a normal particle. We also cannot completely exclude the possibility that some of the RNA detected in viral pellets comes from cellular debris, not from virions.
It is interesting to compare our results with those of prior studies on HIV-1 maturation. In HIV-1, the first cleavage event is not at the N terminus of CA but at the N terminus of NC (30
). CA is directly linked to MA in HIV-1 Gag. Interfering with the cleavage between MA and CA prevents the formation of the normal conical capsid shell, resulting instead in particles with dense, acentric cores (15
). One explanation for this phenotype could be that the retention of CA at the membrane, through its linkage to MA, prevents its condensation into the normal mature core. Alternatively, the phenotype might result from the lack of the normal CA N terminus. Structural studies on the HIV-1 CA protein show that there is a rearrangement of the N-terminal region of CA following the release of the N terminus during maturation (13
) and that this rearrangement (in which the conserved proline at the N terminus forms a buried salt bridge with a conserved aspartate or glutamate residue in CA) leads to new CA-CA interactions (46
). Virions in which the proline residue at the N terminus is replaced by leucine, an alteration which does not interfere with cleavage (10
), have a morphology similar to those in which the cleavage is blocked (15
); this is also true if the aspartate at position 51 in CA, the other partner in the buried salt bridge, is changed to alanine (45
). These observations strongly support the idea that the morphogenesis of the conical capsid shell in normal HIV-1 maturation depends upon formation of the salt bridge between the N-terminal proline and the internal aspartate residues. This salt bridge is not, however, required for the deposition of dense material in the interior of the particle.
The most dramatic effect of the S2D mutation upon the morphology of MLV particles was the elimination of the border that surrounds the core of a normal mature particle. It is important to remember that in MLV there is a protein between MA and CA and that cleavage at site 1 still occurs in site 2 mutant particles (Fig. ). Therefore, the CA-containing fusion protein (p42) is not tethered to the membrane in these particles and should be free to “collapse” into the interior of the particle. The absence of the outer boundary of the core in S2D particles is thus not due to retention of CA at the membrane; rather, these results are consistent with the hypothesis that release of the normal N terminus of CA from Gag is essential for formation of this border, in close analogy with those described above in HIV-1. While the densely staining bodies seen within many S2D particles (Fig. ) may be NC-RNA complexes, cleavage of NC from the p42 fusion protein is somewhat inefficient in these particles (Fig. ); thus, another possibility is that these structures are composed of complexes of the p12-CA-NC fusion protein with RNA.
Our observation that even small alterations at the C terminus of MLV CA can drastically disrupt the normal process of particle assembly (Fig. ) is similar to results with HIV-1; it is clear that this region plays a critical role in determining both the efficiency of assembly and budding and the radius of curvature of the resulting particle (15
). The effects of our mutations on assembly are obviously due to the changes we introduced into the Gag protein, not to the interference with site 3 cleavage. In the case of HIV-1, it has been suggested that a crucial element in this region is an α-helical structure spanning the CA-spacer cleavage site (1
). In MLV, the C-terminal region of MLV CA is important for assembly (47
) and contains an extraordinary run of charged residues. Some deletions in this region produce results very similar to those we observed with point mutations at the extreme C terminus, with a dramatic loss of the ability of Gag to assemble into particles with the normal radius of curvature. The properties of the deletions in this region supported the hypothesis that it contains an α-helix (5
In conclusion, we have found that no individual cleavage event is required for the loss of immature morphology of PR− particles. However, cleavage between p12 and CA is essential for formation of the normal core structure found in mature MLV particles. Cleavage between CA and NC is probably required for the stabilization of genomic RNA dimers associated with MLV maturation. Cleavage between MA and p12 plays no critical role in the conversion of MLV particles to an infectious form.