Folding and particle assembly are disrupted by single-point mutations near the autocatalytic cleavage site of Nudaurelia capensis omega virus capsid protein

Abstract

Protein subunits of several RNA viruses are known to undergo post-assembly, autocatalytic cleavage that is required for infectivity. Nudaurelia capensis omega virus (Nomega V) is one of the simplest viruses to undergo an autocatalytic cleavage, making it an excellent model to understand both assembly and the mechanism of autoproteolysis. Heterologous expression of the coat protein gene of Nomega V in a baculovirus system results in the spontaneous assembly of virus-like particles (VLPs) that remain uncleaved when purified at neutral pH. After acidification to pH 5.0, the VLPs autocatalytically cleave at residue 570, providing an in vitro control of the cleavage. The crystal structure of Nomega V displays three residues near the scissile bond that were candidates for participation in the reaction. These were changed by site-directed mutagenesis to conservative and nonconservative residues and the products analyzed. Even conservative changes at the three residues dramatically reduced cleavage when the subunits assembled properly. Unexpectedly, we discovered that these residues are not only critical to the kinetics of Nomega V autoproteolysis, but are also necessary for proper folding of subunits and, ultimately, assembly of Nomega V VLPs.

Figure 1.

The crystal structure of NωV reveals that the cleavage site is located on the interior of the capsid in the helical region, near the β-barrel domain of the coat protein subunit. The helical region is colored dark blue in A and B. (A) A ribbon diagram of the X-ray structure of the NωV A subunit. The NωV capsid is composed of 60 copies each of A, B, C, and D subunits, which are from the same gene and related by quasi-equivalence. (B) Magnified view of the cleavage site of the NωV coat protein with ball-and-stick representations of residues believed to be involved in the autocatalytic cleavage that occurs between Asn-570 and Phe-571. (C) Electron density of intrasubunit residues near the cleavage site of NωV contoured at 0.8 σ. (D) The crystal structure of NωV capsid at the icosahedral fivefold axis of symmetry and (E) the icosahedral twofold axis of symmetry from the inside of the capsid looking out. A subunits are displayed in blue, B subunits in red, C subunits in green, D subunits in yellow, and asparagine-570 of each coat protein subunit as white spheres in D and E. The ordered N termini of neighboring subunits are approximately 10–14 Å from carbonyl carbons of Asn-570. A, B, D, and E were made with Molscript (Kraulis 1991) and Raster3D (Merritt and Murphy 1994). C was made with the program O (Jones et al. 1991).

Figure 2.

Constant UV absorbance (OD₂₅₄) of 10%–40% sucrose gradients used to purify wild-type and mutant NωV VLPs at pH 7.6 and pH 5.0. Arrows in each window indicate where wild-type VLPs sediment under identical conditions.

Figure 3.

Negative stained electron micrographs of purified wild-type and mutant NωV VLPs at pH 7.6 and pH 5.0. E103Q (g,h) and E103S (e, f ) formed aberrant assembly products. E103D (c,d) and T246S (i, j ) formed particles that resembled wild type (a,b). No particles were detected for T246A, K521A, and K521R. Arrows in (e) and ( f ) indicate E103S mutant particles that resemble wild-type particles in size and appearance. Scale bar is 200 nm.

Figure 4.

4% to 12% Bis-Tris gel used to detect cleavage of the NωV coat protein, α(70 kDa), into β(62 kDa) and γ(8 kDa) polypeptides. Cleavage of wild-type coat protein occurs only after acidification to pH 5.0. E103D and T246S mutants cleaved at pH 5.0, but significantly less than wild type under identical conditions. No cleavage was detected in the aberrantly assembled E103Q and E103S mutants, even at pH 5.0.

Figure 5.

Proposed autocatalytic cleavage mechanism of NωV coat protein based on the deamidation mechanism of the protein crystalline. See text for details.