A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles.
The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from -8 to -10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.
Mukherjee
Santanu
S
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Thorsteinsson
Marc V
MV
Johnston
Lauren Beth
LB
DePhillips
Pete A
PA
Zlotnick
Adam
A
D004220:Disulfides / Q000378:metabolism; D052162:Human papillomavirus 16 / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D046529:Microscopy, Electron, Transmission; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D019065:Virus Assembly
D004220:Disulfides