Assembly dynamics of the nucleocapsid shell subunit (P8) of bacteriophage phi6.
Phi6 is an enveloped dsRNA bacteriophage of Pseudomonas syringae. The viral envelope encloses a nucleocapsid, consisting of an RNA-dependent RNA polymerase complex within an icosahedral shell assembled from approximately 800 copies of a 16 kDa subunit (protein P8, encoded by viral gene 8). During infection, the nucleocapsid penetrates the host plasma membrane and enters the cytosol, whereupon the P8 shell disassembles and the polymerase complex is activated. To understand the molecular mechanisms of shell assembly and disassembly-processes that have counterparts in most viral infections-we have investigated the structure, stability, and dynamics of P8 in different assembly states using time-resolved Raman spectroscopy and hydrogen-isotope exchange. In the presence of Ca(2+), which promotes shell assembly, the highly alpha-helical conformation of the P8 subunit is stabilized by rapid assembly into shell-like structures. However, in the absence of Ca(2+), the P8 subunit is thermolabile and unstable, manifested by a slow alpha-helix --> beta-strand conformational change and the accumulation of aberrant aggregates. In both properly assembled shells and aberrant aggregates, the P8 subunit retains an alpha-helical core that is protected against deuterium exchange of amide NH groups. Surprisingly, no additional protection against amide exchange is conferred by the shell lattice. Time-resolved assembly and disassembly experiments in deuterated buffers indicate that the regions of P8 involved in subunit/subunit interactions in the intact shell undergo rapid exchanges, presumably due to local unfolding events that are characterized by low activation barriers. Such localized dynamics of P8 within the shell lattice may mediate the nucleocapsid/host membrane interactions that are required in the cytosol for particle assembly during maturation and disassembly during infection.
Tuma
R
R
Division of Cell Biology & Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
Bamford
J K
JK
Bamford
D H
DH
Thomas
G J
GJ
D017126:Bacteriophage phi 6 / Q000737:chemistry*; D002942:Circular Dichroism; D003903:Deuterium; D006859:Hydrogen; D019251:Nucleocapsid / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012995:Solubility; D013059:Spectrum Analysis, Raman
D006859:Hydrogen; D003903:Deuterium