Capsid-Glu73Gln difference map. (A) Difference density at each of four quasi-equivalent cleavage sites (residue Asn570 is shown as a space-filling model). The locations of the cleavage sites were determined from the capsid X-ray structure (details in Fig. S2). Significant positive difference density was observed at the B and C active sites, whereas almost no difference density was observed at the comparable A and D positions. The nature of these differences strongly support the hypothesis that cleavage has occurred in A and D where the structure is equivalent to the fully mature particle, and has occurred to only a limited degree in B and C where the differences from the mature particle are great. (B) The X-ray model viewed from inside the particle looking out at the “flat” interface. The switch helices (residues 627–639) of the C and D subunits are colored black. The regions where the mutation was made are indicated by arrows and they correspond to salt links (black stick model) adjacent to the switch helices in the wild-type sequence that were broken by the mutation (also see Fig. S3 for more detail). (C) The same view as shown in B with difference density (mature–mutant) shown in pink and density for the mutant shown in gray. Clearly the switch helix in the mutant is not positioned equivalently as in the fully mature particle since significant positive difference density is observed in the γ region.

NωV maturation pathway. (A) A comparison of difference density maps showing the exterior of the particle of the capsid-Glu73Gln (50% cleaved) mutant density and the capsid-T1 time point density of the wild-type particles. The crystal structure of Ig-like domains in an icosahedral triangle viewed from the exterior, directly down an icosahedral 3-fold axis, as in Fig. 1A, is shown with the difference density. Unexpectedly, density of greater significance (difference maps are contoured at 0.9σ) is observed in capsid-Glu73Gln, where ∼50% of the subunits were cleaved, than in difference density for the capsid-T1 particles (∼15% cleaved). The dissimilarity between the mutant and time-resolved WT difference density shown is due to the failure of the switch helix to be properly positioned at the quasi-2-fold axes (Fig. 2C) in the mutant, which in turn prevents Ig domains at the quasi-3-fold axes from achieving their final positions. In contrast, at the earliest time point in WT maturation, the helix is properly positioned at the quasi-2-fold axes and the Ig domains at the quasi-3-fold axes are closer to their final form than in the mutant. However, the quasi-3-fold interactions continue to change with time in WT, with the difference density being reduced at later time points (Table S1). (B) A schematic representation of the NωV maturation pathway. The structure of the procapsid at pH 7.6 (Left) is dominated by trimeric interactions on the inside of the particle that relate the subunits shown within the triangles. Dimeric interactions between the pairs of subunits shown dominate the outer part of the procapsid particle (6). (1) Lowering the pH from 7.6 to 5.0 results in a LCC that occurs in ∼2 min (16). Subunits adjacent to the 5-fold symmetry axes (A subunits, blue) and the 3-fold symmetry axes (D subunits, yellow) assume a final conformation in this time period with autocatalytic cleavage detectable within 2–3 min after the LCC. (2) The proper positioning of the A and D subunits facilitate the placement of the helical switch polypeptides (residues 627–639 of the C and D subunits) along the line between icosahedral 2-fold (quasi-6-fold) axes. (3–5) Proper positioning of the switch helices, which takes ∼3 min following the LCC, is required for subsequent folding and autocatalysis of the B and C subunits and their movement toward the quasi-3-fold axes (shown by the arrows in 3 and 5). As shown in 3, the folded state and position of the B subunits are almost complete within 30 min after the helical rearrangements, whereas the same changes in the C subunits take place in the time frame of hours (shown in 5). The structural changes near the quasi-3-fold axes result from a hierarchy of relatively slow tertiary and quaternary structural changes in the B and C subunits as described. The proper regulation of subunit contacts induced by the switch helices is essential for the formation of the T = 4 icosahedral architecture.

NωV subunits arrangement and kinetics of autocatalytic cleavage. (A) A color-coded diagram of a T = 4 surface lattice with the four quasi-equivalent subunits shown as blue (A subunit), red (B subunit), green (C subunit), and yellow (D subunit). Icosahedral and quasi-symmetry elements are shown as open and filled symbols, respectively. The insets show how interactions between the same subunit surfaces are differentiated through the ordering or disordering of residues 627–639 (“molecular switch helices”) of the γ peptides. These residues (contributed by the C and D subunits) are ordered at the flat contact. Because of the hinge motion of the bent contact, there is no space for the corresponding region of the A and B subunits and they are invisible in the crystal structure. (B) The kinetics of cleavage at pH 5.0. The fraction of subunits cleaved was previously determined by SDS gel analysis (7). Cleavage of the Glu73Gln mutant stopped at the ∼50% point. The particles for cryo-EM analysis were frozen at indicated time point (T1, T2, and T3) after lowering pH to 5.0.

Time-resolved analysis of NωV maturation. (A) Difference density maps near the cleavage sites of the four quasi-equivalent subunits corresponding to the time points at 3 min (T1), 30 min (T2), and 4 h (T3) after lowering the pH from 7.6 to 5.0. Difference density maps correspond to fully mature capsid-T1, capsid-T2, and capsid-T3. Maps are contoured at 0.9σ. More details are provided in Fig. S4. (B) A hypothetical graphical representation of NωV autoproteolytic kinetics consistent with the rate of cleavage-site formation in the four different subunits shown in A. The cleavage-sites of the A and D subunits form rapidly and are almost complete after the LCC, thus activating cleavage at these sites and accounting for 50% of the autoproteolysis seen at T2. The B subunit folding is slower than that of the A and D, accounting for the majority of the cleavage between time points T2 and T3. The C subunit, which requires hours to complete the maturation process, accounts for most of the cleavage observed after the T3 time point.