Virus particle maturation: insights into elegantly programmed nanomachines

Abstract

Similar modes of virus maturation have been observed in dsDNA bacteriophages and the structurally related herpes viruses and some type of maturation occur in most animal viruses. Recently a variety of biophysical studies of maturation intermediates of bacteriophages P22, lambda, and HK97 have suggested an energy landscape that drives the transitions and structure-based mechanisms for its formation. Near-atomic resolution models of subunit tertiary structures in an early intermediate of bacteriophage HK97 maturation revealed a remarkable distortion of the secondary structures when compared to the mature particle. Scaffolding proteins may induce the distortion that is maintained by quaternary structure interactions following scaffold release, making the intermediate particle metastable.

Figure 1. The bacteriophage P22 assembly pathway

P22 assembles a protein precursor particle called a procapsid, which is the receptacle into which its 43.5 kbp DNA chromosome is packaged. P22 procapsid shells are built from two major components: 415 molecules of coat protein (gp5, the product of gene 5) arranged in a T = 7l icosahedral shell; and roughly 250 molecules of scaffolding protein (gp8) within the coat protein shell. In addition, four other proteins are present in the procapsid. A dodecamer of 84 kDa proteins (gp1) is present at a single unique icosahedral vertex. Six to twenty intravirion molecules of the products of genes 7, 16 and 20 are required for successful DNA injection into susceptible cells and are released from the virion during the injection process. As DNA is packaged, the thick procapsid shell expands from a radius of about 55 nm to a thinner, more angular shell 65 nm in diameter. Despite having a genome 41.7 kbp in length, P22 packages DNA until the capacity of the capsid is reached, which is at 43.5 kbp, a strategy referred to as head-full DNA packaging. Termination of packaging by cleavage of the concatemeric DNA is initiated not by sequence, but when the chromosome is at a defined packing density that is sensed by the portal protein. After DNA is packaged, the tail assembly is constructed by the sequential addition of multiple copies of four gene products (gp4, gp10, gp26 and gp9) to the vertex occupied by the portal ring. (taken from Lander et al 2006)

Figure 2. The HK97 virus-like particle assembly and maturation pathway that is followed when only the coat protein and protease are co-expressed in E. coli

At the top are all the intermediates that have been characterized by crystallography and/or cryoEM. Below is shown the processing that occurs to the capsid protein, gp5, and the residues that form the auto-catalytic crosslink. At right is an enlargement of the final mature particle indicating that the cross-linked gp5 proteins mechanically chain-link the particle together. Each ring of the same color corresponds to 5 or 6 subunits chain-linked together by the isopeptide bond formed by the side chains of Asn 356 and Lys169. Assembly and Maturation: The capsid protein (gp5) immediately assembles into skewed hexamers and pentamers (generically called capsomers) shown in blue. The protease (gp4) is shown in red. The capsomers (60 hexamers and 12 pentamers) and approximately 60 copies of the protease co-assemble to form prohead I, a roughly spherical particle, ~51nm in diameter. The hexamers in this particle are not symmetric, but “skewed”, and correspond roughly to two shifted trapezoids (each composed of 3 subunits) related by 2-fold symmetry[23]. Under normal circumstances, prohead I is transient. The protease becomes active upon assembly and cleaves residues 2-103 (the delta domain) from all the gp5 subunits, creating gp5* as shown. The protease auto-digests and all of the fragments diffuse from openings in the capsid. This creates prohead II (~51nm) that is morphologically closely similar to prohead I (including the skewed hexamers and virtually the same diameter), however the mass of this particle is 13Mdalton, compared to 17Mdalton for prohead I. Prohead I can be stabilized for study by not expressing the protease, or co-expressing a mutant, inactive, protease. Either of these particles can be disassembled and reassembled under mild conditions[27] as indicated by the arrow from capsomers to prohead I. Prohead II is a meta-stable particle that can only transition to EI-1. Conditions that disassemble prohead I cause either nothing to happen or the transition to EI-1. There are many conditions that cause the transition, but dropping the pH from 7 to 4.0 was used for most of the in vitro studies of maturation. The transition to EI-1 (~56.0nm), triggered by the pH drop, has a half-life of ~3 minutes and is stochastic without populated intermediates. EI-1 hexamers are 6-fold symmetric and the particle is crosslink competent, with crosslink initiation commencing virtually immediately after this particle is formed. Approximately 60% of the crosslinks form before the morphology of EI-1 changes (EI-2,3 have the essentially the same morphology as EI-1 with increasing numbers of crosslinks) to EI-4. The process resembles a Brownian ratchet (a process by which thermal energy is captured by driving a process in only one direction) in which the loop containing Lys169 fluctuates until the covalent bond with Asn 356 forms, locking it down and incrementally raising the energy of the particle[31]. When a sufficient number form (~60%) the particle crosses the energy barrier and transitions to EI-4, again without populated intermediates, a round (~ 62.5nm), thin shelled particle eventually forming all but 60 crosslinks[38]. This particle is the end point at pH 4.0 and was studied by crystallography and cryoEM. These studies showed that subunits in the pentamers were not crosslinked and that they were dynamic fluctuating by 1.4nm along the 5-fold particle axes. Neutralizing EI-4 completes the crosslinks with pentamer subunits, forming the fully mature, ~65nm, faceted particle[31].

Figure 3. A cartoon depicting the working hypothesis for creating an energy landscape that makes HK97 maturation exothermic

a. Hypothetical, full length, monomeric, gp5 subunits with the Δ-domain depicted as an extension away from the gp5* digestion product. Without Δ-domain interactions the model conjectures that the gp5* portion of the subunit is undistorted. b. Upon expression the Δ-domains of the gp5 subunits immediately interact with each other forming capsomers with skewed hexamers and distorted subunit tertiary structures as observed in the crystal structure of prohead II. The model posits that interactions between the Δ-domains distort the tertiary structures and balance the energy required for the distortion. c. Capsomers assemble to form prohead I with quaternary and tertiary structure very similar to that seen in prohead II and also package the protease, depicted a blue circles in the cartoon. If proteolysis is inhibited, prohead I can be disassembled under mild conditions and reassembled. d. Following proteolysis, the Δ-domains are removed and the quaternary structure traps the tertiary structure distortion caused by Δ-domain interactions. e. The prohead II particle is now metastable with the energy for the distortion of the tertiary structure trapped by the quaternary structure in the absence of Δ-domain interactions. f. When the metastable particle is perturbed from its local energy minima, the distorted subunits reach their “ground state” structure with the release of energy. This occurs during the transition to intermediate EI-1.