Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris.
Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.
Acosta-Rivero
Nelson
N
Division of Vaccines, Electron Microscopy Laboratory, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, Cuba. nelson.acosta@cigb.edu.cu
Rodriguez
Armando
A
Musacchio
Alexis
A
Falcón
Viviana
V
Suarez
Viana M
VM
Chavez
Liudmila
L
Morales-Grillo
Juan
J
Duenas-Carrera
Santiago
S
D001665:Binding Sites; D004279:DNA, Viral / Q000737:chemistry* / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D008970:Molecular Weight; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry* / Q000648:ultrastructure*; D016601:RNA-Binding Proteins / Q000737:chemistry; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus