Foreign and chimeric external scaffolding proteins as inhibitors of Microviridae morphogenesis

Abstract

Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions. Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the Microviridae, whose canonical members are phiX174, G4, and alpha3. However, despite 70% homology on the amino acid level, overexpression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD(r)) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The chiD(r) mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis.

FIG. 1

(A) The Microviridae morphogenetic pathway. In a reaction mediated by the internal scaffolding (or B) protein, 9S and 6S pentamers associate, forming the 12S particle. Twenty external scaffolding (or D) proteins add to this intermediate, which is then organized into the procapsid. The DNA binding (or J) protein enters the morphogenetic pathway during the packaging reaction, perhaps mediating the extrusion of the internal scaffold. Maturation is complete upon dissociation of the external scaffolding protein. (B) The four D proteins associated with each asymmetric unit (2). The subunits are designated D1 through D4; the amino and carboxyl termini are indicated where possible. The first α helices of D1 and D4 are depicted.

FIG. 1

(A) The Microviridae morphogenetic pathway. In a reaction mediated by the internal scaffolding (or B) protein, 9S and 6S pentamers associate, forming the 12S particle. Twenty external scaffolding (or D) proteins add to this intermediate, which is then organized into the procapsid. The DNA binding (or J) protein enters the morphogenetic pathway during the packaging reaction, perhaps mediating the extrusion of the internal scaffold. Maturation is complete upon dissociation of the external scaffolding protein. (B) The four D proteins associated with each asymmetric unit (2). The subunits are designated D1 through D4; the amino and carboxyl termini are indicated where possible. The first α helices of D1 and D4 are depicted.

FIG. 2

(A) Sequence alignment of the φX174 and α3 external scaffolding proteins. (B) Generation of the φX/α3 chimeric gene. (C) Verification of the φX/α3 chimeric gene.

FIG. 3

Sedimentation analysis of particles produced in the presence of foreign and chimeric scaffolding proteins. Symbols: ●, wild-type α3 grown in cells overexpressing the α3 D protein (control); ■, wild-type φX174 grown in cells overexpressing the α3 D protein; ▴, wild-type φX174 grown in cells overexpressing the φXα3 chimeric D protein.

FIG. 4

Nucleotide sequence of the upstream D regulatory region in the forD mutant.