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An unexpected twist in viral capsid maturation.

ABSTRACT: Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 A resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 A (ref. 2). A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and beta-sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol(-1) of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process probably present in many dsDNA bacteriophage and possibly viruses such as herpesvirus, which share the HK97 subunit fold.

SUBMITTER: Gertsman I

PROVIDER: S-EPMC2765791 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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An unexpected twist in viral capsid maturation.

Gertsman Ilya I Gan Lu L Guttman Miklos M Lee Kelly K Speir Jeffrey A JA Duda Robert L RL Hendrix Roger W RW Komives Elizabeth A EA Johnson John E JE

Nature 20090208 7238

Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from whic ...[more]

PMID: 19204733

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Project description:Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized "suspended bridges" that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism. Importance: Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

Project description:The herpesvirus capsid assembles in the nucleus as an immature procapsid precursor built around viral scaffold proteins. The event that initiates procapsid maturation is unknown, but it is dependent upon activation of the VP24 internal protease. Scaffold cleavage triggers angularization of the shell and its decoration with the VP26 and pUL25 capsid-surface proteins. In both the procapsid and mature angularized capsid, the apical region of the major capsid protein (VP5) is surface exposed. We investigated whether the VP5 apical region contributes to intracellular transport dynamics following entry into primary sensory neurons and also tested the hypothesis that conserved negatively charged amino acids in the apical region contribute to VP26 acquisition. To our surprise, neither hypothesis proved true. Instead, mutation of glutamic acid residues in the apical region delayed viral propagation and induced focal capsid accumulations in nuclei. Examination of capsid morphogenesis based on epitope unmasking, capsid composition, and ultrastructural analysis indicated that these clusters consisted of procapsids. The results demonstrate that, in addition to established events that occur inside the capsid, the exterior capsid shell promotes capsid morphogenesis and maturation.IMPORTANCE Herpesviruses assemble capsids and encapsidate their genomes by a process that is unlike those of other mammalian viruses but is similar to those of some bacteriophage. Many important aspects of herpesvirus morphogenesis remain enigmatic, including how the capsid shell matures into a stable angularized configuration. Capsid maturation is triggered by activation of a protease that cleaves an internal protein scaffold. We report on the fortuitous discovery that a region of the major capsid protein that is exposed on the outer surface of the capsid also contributes to capsid maturation, demonstrating that the morphogenesis of the capsid shell from its procapsid precursor to the mature angularized form is dependent upon internal and external components of the megastructure.

Project description:The maturation process that occurs in most viruses is evolutionarily driven, as it resolves several conflicting virion assembly requirements. During herpesvirus assembly in a host cell nucleus, micron-long double-stranded herpes DNA is packaged into a nanometer-sized procapsid. This leads to strong confinement of the viral genome, resulting in tens of atmospheres of intracapsid DNA pressure. Yet, the procapsid is unstable due to weak reversible interactions between its protein subunits, which ensures free energy minimization and reduces assembly errors. In this work, we show that herpesviruses resolve these contradictory capsid requirements through a mechanical capsid maturation process facilitated by multifunctional auxiliary protein UL25. Through mechanical interrogation of herpes simplex virus 1 (HSV-1) capsid with atomic force microscopy nano-indentation, we show that UL25 binding at capsid vertices post-assembly provides the critical capsid reinforcement required for stable DNA encapsidation; the absence of UL25 binding leads to capsid rupture. Furthermore, we demonstrate that gradual capsid reinforcement is a feasible maturation mechanism facilitated by progressive UL25 capsid binding, which is likely correlated with DNA packaging progression. This work provides insight into elegantly programmed viral assembly machinery, where targeting of capsid assembly mechanics presents a new antiviral strategy that is resilient to the development of drug resistance. IMPORTANCE Most viruses undergo a maturation process from a weakly assembled particle to a stable virion. Herpesvirus capsid undergoes mechanical maturation to withstand tens of atmospheres of DNA pressure. We demonstrate that this mechanical capsid maturation is mainly facilitated through binding of auxiliary protein UL25 in herpes simplex virus 1 (HSV-1) capsid vertices. We show that UL25 binding provides the critical capsid reinforcement required for stable DNA encapsidation. Our data also suggest that gradual capsid reinforcement by progressive UL25 binding is a feasible capsid maturation mechanism, correlated with DNA packaging progression.

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An unexpected twist in viral capsid maturation.

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An unexpected twist in viral capsid maturation.

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