In Vitro Assembly of the øX174 Procapsid from External Scaffolding Protein Oligomers and Early Pentameric Assembly Intermediates

Abstract

Bacteriophage øX174 morphogenesis requires two scaffolding proteins: an internal species, similar to those employed in other viral systems, and an external species, which is more typically associated with satellite viruses. The current model of øX174 assembly is based on structural and in vivo data. During morphogenesis, 240 copies of the external scaffolding protein mediate the association of 12 pentameric particles into procapsids. The hypothesized pentameric intermediate, the 12S⁎ particle, contains 16 proteins: 5 copies each of the coat, spike and internal scaffolding proteins and 1 copy of the DNA pilot protein. Assembly naïve 12S⁎ particles and external scaffolding oligomers, most likely tetramers, formed procapsid-like particles in vitro, suggesting that the 12S⁎ particle is a bona fide assembly intermediate and validating the current model of procapsid morphogenesis. The in vitro system required a crowding agent, was influenced by the ratio of the reactants and was most likely driven by hydrophobic forces. While the system reported here shared some characteristics with other in vitro internal scaffolding protein-mediated systems, it displayed unique features. These features most likely reflect external scaffolding protein-mediated morphogenesis and the øX174 procapsid structure, in which external scaffolding–scaffolding protein interactions, as opposed to coat–coat protein interactions between pentamers, constitute the primary lattice-forming contacts.

Introduction

Bacteriophage øX174 morphogenesis combines features of seemingly disparate prokaryotic and eukaryotic viral systems. Capsid assembly involves the association of pentameric assembly intermediates,¹ a feature shared with polyoma, papilloma and picornaviruses.2, 3, 4, 5 However, as observed with many bacteriophages and herpesviruses,6, 7, 8, 9 morphogenesis requires scaffolding proteins to lower thermodynamic assembly barriers and ensure morphogenetic fidelity.7, 10 Most scaffolding-dependent systems utilize a single internal scaffolding protein, and the assembly-reactive coat protein is usually a monomer or a dimer, which does not display a defined stoichiometry with the scaffolding protein.11, 12, 13 In contrast, the øX174 internal and external scaffolding proteins, B and D, respectively, exhibit strict stoichiometric relationships with the coat protein in both the procapsid and the early assembly intermediates.

Like the “classical” P22 and herpes simplex virus scaffolding proteins,14, 15 the øX174 B protein prevents coat protein aggregation and facilitates minor structural protein incorporation,16, 17, 18 whereas protein D assembles pentameric intermediates into the procapsid (Fig. 1b). Viable multiple mutants that no longer require the internal scaffolding protein have been isolated.¹⁹ Most of the mutations in this “B-free” strain affect the external scaffolding protein, its interactions with the viral coat protein and its expression level, which is grossly exaggerated. Along with the atomic structure of the procapsid, 20, 21 these data indicate that the external scaffolding protein is more critical for morphogenesis. This may be a general feature of dual scaffolding protein systems. In vitro, the Sid protein, an external scaffolding protein encoded by satellite virus P4, can assemble P2 coat proteins into P4 procapsids,²² circumventing the in vivo requirement of the P2 internal scaffolding protein. Despite this similarity, proteins D and Sid may operate via different mechanisms. The Sid protein alters capsid dimensions by forming a dodecahedral lattice that forces a growing coat protein shell into a smaller procapsid.22, 23, 24 In contrast, the øX174 external scaffolding protein is the major lattice-forming molecule.20, 21, 25 As can be seen in Fig. 1a, removing the external scaffolding protein density from the procapsid cryo-electron microscopy structure reveals underlying coat protein pentamers that make little or no contact with each other.²⁵ Thus, the coat protein pentamers act as a template on which external scaffolding proteins associate, but scaffolding–scaffolding interactions both form and maintain procapsid integrity.

Although in vitro capsid assembly systems have been established for many viruses,2, 3, 12, 13, 22, 26, 27, 28, 29, 30, 31, 32 the complexity of the øX174 procapsid has complicated in vitro reconstitution. Dissociation–reassociation studies were hindered by the heterogeneity of the dissociation products and the internal scaffolding protein's autoproteolytic activity.²⁵ Purifying proteins individually from cells expressing the requisite genes proved equally problematic. Consequently, an alternate strategy was developed. It utilized a recently isolated, putative, assembly intermediate that contains a stable internal scaffolding protein.³³

The current version of the øX174 assembly pathway is illustrated in Fig. 1b. It differs from previous pathways1, 18, 34 by the inclusion of two B protein-containing early intermediates, the 9S⁎ and 12S⁎ particles. These particles accumulate in the absence of the external scaffolding protein and can be easily purified.³³ The 12S⁎ particle is composed of five copies each of the coat, spike and internal scaffolding proteins and one copy of the DNA pilot protein H. In vitro, it was assembled into procapsid-like particles by the external scaffolding protein, presumably in the form of a tetramer, suggesting that the 12S⁎ particle is a bona fide assembly intermediate. While the assembly system reported here shared some characteristics with other in vitro scaffolding protein-mediated systems, it displayed unique features that most likely reflect external scaffolding protein-mediated morphogenesis and lattice formation.