title: Functional domains of the HK97 capsid maturation protease and the mechanisms of protein encapsidation.
abstract: Tailed double-stranded DNA bacteriophages and herpesviruses build capsids by co-assembling a major capsid protein with an internal scaffolding protein that then exits from the assembled structure either intact or after digestion in situ by a protease. In bacteriophage HK97, the 102-residue N-terminal delta domain of the major capsid protein is also removed by proteolysis after assembly and appears to perform the scaffolding function. We describe the HK97 protease that carries out these maturation cleavages. Insertion mutations at seven sites in the protease gene produced mutant proteins that assemble into proheads, and those in the N-terminal two-thirds were enzymatically inactive. Plasmid-expressed protease was rapidly cleaved in vivo but was stabilized by co-expression with the delta domain. Purified protease was found to be active during the assembly of proheads in vitro. Heterologous fusions to the intact protease or to C-terminal fragments targeted fusion proteins into proheads. We confirm that the catalytic activity resides in the N-terminal two-thirds of the protease polypeptide and suggest that the C-terminal one-fifth of the protein contains a capsid targeting signal. The implications of this arrangement are compared to capsid targeting systems in other phages, herpesviruses, and encapsulins.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Room 318 Langley Hall, 4249 Fifth Avenue, Pittsburgh, PA 15260, USA. duda@pitt.edu
authors.forename: Robert L
authors.initials: RL
authors.lastname: Duda
authors.forename: Bonnie
authors.initials: B
authors.lastname: Oh
authors.forename: Roger W
authors.initials: RW
authors.lastname: Hendrix
mesh_terms: D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D036022:Capsid Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D016254:Mutagenesis, Insertional; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D021382:Protein Sorting Signals; D010447:Peptide Hydrolases
keywords: EDTA; GuHCl; MALDI-TOF; N-α-tosyl-l-lysyl-chloromethyl-ketone; TCA; TLCK; capsid maturation; ethylenediaminetetraacetic acid; guanidine hydrochloride; matrix-assisted laser desorption/ionization time of flight; protein processsing; protein targeting; trichloroacetic acid; virus assembly