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Architecture of a dsDNA viral capsid in complex with its maturation protease.

ABSTRACT: Most double-stranded DNA (dsDNA) viruses, including bacteriophages and herpesviruses, rely on a staged assembly process of capsid formation. A viral protease is required for many of them to disconnect scaffolding domains/proteins from the capsid shell, therefore priming the maturation process. We used the bacteriophage HK97 as a model system to decipher the molecular mechanisms underlying the recruitment of the maturation protease by the assembling procapsid and the influence exerted onto the latter. Comparisons of the procapsid with and without protease using single-particle cryoelectron microscopy reconstructions, hydrogen/deuterium exchange coupled to mass spectrometry, and native mass spectrometry demonstrated that the protease interacts with the scaffolding domains within the procapsid interior and stabilizes them as well as the whole particle. The results suggest that the thermodynamic consequences of protease packaging are to shift the equilibrium between isolated coat subunit capsomers and procapsid in favor of the latter by stabilizing the assembled particle before making the process irreversible through proteolysis of the scaffolding domains.

SUBMITTER: Veesler D

PROVIDER: S-EPMC3939775 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Architecture of a dsDNA viral capsid in complex with its maturation protease.

Veesler David D Khayat Reza R Krishnamurthy Srinath S Snijder Joost J Huang Rick K RK Heck Albert J R AJ Anand Ganesh S GS Johnson John E JE

Structure (London, England : 1993) 20131219 2

Most double-stranded DNA (dsDNA) viruses, including bacteriophages and herpesviruses, rely on a staged assembly process of capsid formation. A viral protease is required for many of them to disconnect scaffolding domains/proteins from the capsid shell, therefore priming the maturation process. We used the bacteriophage HK97 as a model system to decipher the molecular mechanisms underlying the recruitment of the maturation protease by the assembling procapsid and the influence exerted onto the la ...[more]

PMID: 24361271

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Project description: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.

Project description:Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the ?-genome maturation complex. The ?-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient ?-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

Project description:Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating.Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.

Dataset Information

Architecture of a dsDNA viral capsid in complex with its maturation protease.

Publications

Architecture of a dsDNA viral capsid in complex with its maturation protease.

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