title: Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal Complex.
abstract: The 90-nm-diameter capsid of coliphage T5 is organized with T=13 icosahedral geometry and encloses a double-stranded DNA genome that measures 121kbp. Its assembly follows a path similar to that of phage HK97 but yielding a larger structure that includes 775 subunits of the major head protein, 12 subunits of the portal protein and 120 subunits of the decoration protein. As for phage HK97, T5 encodes the scaffold function as an N-terminal extension (∆-domain) to the major head protein that is cleaved by the maturation protease after assembly of the initial prohead I form and prior to DNA packaging and capsid expansion. Although the major head protein alone is sufficient to assemble capsid-like particles, the yield is poor and includes many deformed structures. Here we explore the role of both the portal and the protease in capsid assembly by generating constructs that include the major head protein and a combination of protease (wild type or an inactive mutant) and portal proteins and overexpressing them in Escherichia coli. Our results show that the inactive protease mutant acts to trigger assembly of the major head protein, probably through binding to the ∆-domain, while the portal protein regulates assembly into the correct T=13 geometry. A cryo-electron microscopy reconstruction of prohead I including inactivated protease reveals density projecting from the prohead interior surface toward its center that is compatible with the ∆-domain, as well as additional internal density that we assign as the inactivated protease. These results reveal complexity in T5 beyond that of the HK97 system.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA; Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Alexis
authors.initials: A
authors.lastname: Huet
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Duda
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Hendrix
authors.affiliation: Department of Virology, Institute for Integrative Biology of the Cell, UMR 9198 CEA, Centre National de la Recherche Scientifique, Université Paris-Sud, 91191 Gif-sur-Yvette Cedex, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Boulanger
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Electronic address: jxc100@pitt.edu.
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
mesh_terms: D020285:Cryoelectron Microscopy; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D017906:Siphoviridae / Q000502:physiology* / Q000648:ultrastructure; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D014764:Viral Proteins
keywords: capsid assembly; maturation protease; portal; scaffolding; tailed bacteriophage