The procapsid-to-capsid transition in NωV has only been observed with particles produced in a baculovirus expression-assembly system. Procapsids are only observed in preparations isolated at pH 7.6; capsids are observed when particles are isolated at pH 5. It is likely that the procapsid is a transient intermediate in the formation of authentic virus particles if the assembly occurs in a low pH environment within the cell. A working model for assembly, consistent with this hypothesis, requires that virion formation occur in a low-pH cellular vacuole that depends on viral infection. Expression of only the coat protein in the Sf21 cells apparently does not generate a vacuole, thereby eliminating pH regulation from the assembly. Particles are still able to assemble at the neutral pH of the cytoplasm but are nonnative and are inhibited from maturing into the form of particle observed in the authentic virion. The maturation process occurs in vitro when the pH is reduced to 5.0.
Authentic NωV undergoes an autocatalytic maturation cleavage, and this proteolysis occurs in the VLP capsids at pH 5.0. Like the maturation in nodaviruses (21
), results from the present study show that cleavage does not occur if Asn-570 is replaced by threonine, glutamine, or aspartic acid. The arrangement of residues at the cleavage site in nodaviruses and NωV was shown to be strikingly similar by crystallography (15
), suggesting similar mechanisms for the cleavage in the two virus groups. Although not exhaustively tested, we believe that Asn is absolutely required at the cleavage site. The Asn residue is well documented as contributing to nonprogrammed protein “aging” through deamidation or cleavage on the C-terminal side of the Asn residue (e.g., references 3
). An important example is the formation of cataracts; when these reactions occur in the crystalline protein it forms opaque aggregates in the eye lens (23
). Deamidation at asparagine residues in proteins results from the nucleophilic attack of the peptide nitrogen of the adjacent residue, C-terminal to the Asn, on the side chain carbonyl carbon of the Asn residue, which causes the formation of the succinimide intermediate (Fig. ). Hydrolysis of the succinimide intermediate gives rise to iso- (β) Asn and Asn in 3:1 proportions (24
). While the deamidation is the major reaction that occurs at the susceptible Asn residues, cleavage of the peptide bond following the Asn is also known to occur as a reaction that proceeds via the formation of the succinimide intermediate (23
). In the latter reaction, the side chain amide nitrogen of the Asn residue is responsible for the nucleophilic attack on its own main chain carbonyl carbon atom, causing the cyclic imide intermediate to form and releasing the C-terminal peptide. Hydrolysis of the imide ring produces Asn and β-Asn (aspartyl α-amide) (22
). Such a cleavage reaction at Asn residues is also known to occur during protein splicing and is responsible for the C-terminal cleavage (18
). We propose that this reaction is responsible for the cleavage in both noda- and tetraviruses and is kinetically enhanced by the presence of a catalytic acidic residue in the immediate vicinity of the Asn side chain. In FHV it was shown that altering either Asn-363 or Asp-75 reduced the rate of the reaction below measurability (25
), while the present study showed that replacing Asn-570 has the same effect in NωV. The analogous residue to Asp-75 in NωV is Glu-103. Replacement of this residue in NωV affected assembly, and its role in cleavage could not be independently established. The work is ongoing and will be reported in a separate communication.
FIG. 6. The proposed mechanism of the autoproteolytic cleavage of the NωV coat protein. Glu-103 and Thr-246 are believed to form hydrogen bonds with Asn-570, orienting the asparagine in the ideal position for cleavage to occur. Nucleophilic attack by (more ...)
Inhibition of the maturation cleavage allowed an investigation of the reversibility of the pH-dependent, large-scale quaternary structure of the VLPs. The noncleaving VLP used for the investigation was the N570T mutation. This mutation expressed and assembled with larger yields of particles than the wild-type sequence, as well as the other noncleaving mutations. The reason for the efficiency of production of this particular mutant was not obvious. N570T mutants underwent the transition to the capsid form at pH 5.0, and their sedimentation properties and negative-stain electron microscopy images were indistinguishable from those of the wild-type VLPs; however, cleavage was not detectable by SDS-gel electrophoresis in any of the mutations. As shown in previous studies, the transition from procapsid to capsid in the wild-type particles was not reversible after the subunits cleaved (5
). By labeling the RNA packaged in the wild-type particles with tritiated uridine during expression, these particles were used as a convenient marker for capsid formation, as they sedimented at the same position after maturation, regardless of pH. As anticipated from the previous time-resolved study of the quaternary structure transition (5
), the noncleaved capsid particles expanded to what appeared to be procapsid when the pH was raised to 7.6.
Two features of the particles obtained after raising the pH distinguished them from the native procapsid prior to exposure to pH 5.0. First, the majority of the particles sedimented one fraction slower than the original procapsid, and the major peak was always accompanied by a faster-sedimenting shoulder. Second, the negative-stained electron microscopy images revealed the stain-penetrable phenotype as the native procapsids in the slower-sedimenting peak and a heterogenous mixture of stain-penetrable procapsids and stain-impenetrable capsids in the faster-sedimenting shoulder. The fact that the A260/A280 ratio for all the particles in the peak of the reexpanded particles, where it falls from 1.20 to 1.08, would suggest a loss of encapsidated, cellular RNA during reexpansion. This would explain why these particles sediment a fraction slower than original procapsid particles.
The conversion of procapsids to capsids was previously shown to occur between pH 5.3 and 6.5, with a sharp transition in the titration curve (5
). If the pH was lowered rapidly from 7.5 to 5 in a stop-flow apparatus, the transition occurred in less than 100 ms, the dead time of the solution X-ray scattering apparatus used to measure the transition (5
). The reverse reaction, in which the particles reexpand, is slow, with some never making the transition after 4 days of dialysis against pH 7.6 buffer. The capsid-to-procapsid transition is probably slow because there are extensive protein-protein, and possibly protein-RNA, interactions in the capsid that must be broken during the expansion process. Titration curves were not developed for the N570T VLP transitions; however, qualitatively the procapsid-to-capsid transition occurs in the same pH range and occurs rapidly. The reverse reaction, however, is slow, requiring hours of dialysis against pH 7.6 buffer to convert the majority of capsids to the procapsid-like particles. This observation suggests that the transition can be viewed as having two components: the pH-dependent driving force and a “cargo” that is coupled to the driving force. Previous structural studies of the procapsid and capsid of wild-type particles demonstrated that almost all of the NωV subunit behaves as a rigid body during the transition and that the procapsid cryoEM density can be modeled with high fidelity by the X-ray coordinates determined from the capsid structure (6
). The only region of density that showed an obvious quaternary and possibly secondary and tertiary structure change in the two particles occurred on the inside of the subunit in the region occupied by the helical domain of the proteins. It is known that these helices have different configurations in the different subunits observed in the T=4 particle and that they function as the switches that determine the angle of contact between twofold-related subunits in the authentic virus (15
). Residues 608 to 641 are visible as helices in the C and D subunits of the X-ray structure of the capsid but are invisible in the A and B subunits. As previously discussed (6
), the quasi- and icosahedral twofold axes of the procapsid are nearly indistinguishable from each other in the procapsid, while they differentiate into distinct, flat (dihedral angle, 180°) and bent (dihedral angle, 144°) twofold contacts in the capsid. A working hypothesis, currently being tested, proposes a pH-induced helix-coil transition that occurs in the interior, helical region of the subunit. It is likely that these regions exist as helices in the procapsids and that, after protonation at lower pH, they transform into coils initiating the conformational transition to capsids. The different environments established by flat dihedral angles between C and D subunits and bent dihedral angles between A and B subunits alter the pH of the helix-coil transition in those two environments. This becomes apparent in the crystal structure of NωV, where the C terminus is visible as a helix in the C and D subunits but is invisible in the A and B subunits. Protonation of specific residues at low pH initiates the helix-coil transitions of these polypeptides. The transition occurs at a pH near 6, suggesting histidine as a possible candidate for this protonation, based on pKa
, but the influence of tertiary and quaternary structures may create environments that alter the pKa
of various residues. The protonation would be proceeded by electrostatic repulsions causing the helices to unfold into random coils, thus initiating the driving force for the conformational change. This region of the subunit can be viewed as a pH-driven engine, while the remainder of the subunit is the cargo that is pushed or pulled as a rigid body. When cleavage occurs, the cargo is uncoupled from the engine and the particle remains in the capsid state regardless of pH. When the cleavage does not occur, the cargo remains coupled and the whole process is reversible (Fig. ). The great number of protein-protein interactions created when the capsid forms undoubtedly causes the hysteresis and the slow reversible transition back to the procapsid.
FIG. 7. Schematic representation of the relationship of coat protein cleavage and the pH-induced conformational change in NωV. The region of the helical domain of each coat protein subunit believed to be responsible for the helix-coil transition represents (more ...)
The NωV particle displays the properties of a molecular machine as it undergoes the transitions required to achieve the formation of a complex quaternary structure. Unlike comparable transitions in bacteriophages, and fusion glycoproteins in alphaviruses, flaviviruses, and influenza viruses that depend on the generation of meta-stable particles and states that irreversibly mature, the NωV particle maturation is pH dependent and reversible as long as cleavage of the coat protein is inhibited, allowing a detailed analysis of the machinery by mutagenesis and biophysics.