title: Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein.
abstract: Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPdeltaN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPdeltaN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPdeltaN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.
authors.affiliation: Centre de Recherche en Infectiologie, Univeristé Laval, Québec, Canada.
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Tremblay
authors.forename: Nathalie
authors.initials: N
authors.lastname: Majeau
authors.forename: Marie-Eve Laliberté
authors.initials: ME
authors.lastname: Gagné
authors.forename: Katia
authors.initials: K
authors.lastname: Lecours
authors.forename: Hélène
authors.initials: H
authors.lastname: Morin
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Duvignaud
authors.forename: Marilène
authors.initials: M
authors.lastname: Bolduc
authors.forename: Nicolas
authors.initials: N
authors.lastname: Chouinard
authors.forename: Christine
authors.initials: C
authors.lastname: Paré
authors.forename: Stéphane
authors.initials: S
authors.lastname: Gagné
authors.forename: Denis
authors.initials: D
authors.lastname: Leclerc
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D036022:Capsid Proteins / Q000235:genetics*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D009154:Mutation; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017863:Potexvirus / Q000235:genetics* / Q000502:physiology; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D019065:Virus Assembly / Q000235:genetics* / Q000502:physiology
chemical_list: D036022:Capsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014357:Trypsin