Abstract
JC virus (JCV), a human polyomavirus, is an important pathogen of human central nervous system (CNS) demyelinating disease. The purpose of this thesis is to investigate the molecular characteristics of the major structure protein, VP1, of JC virus and assembly mechanism of the virus particles. First of all, we amplified a new JCV genome (Taiwan-3, TW-3) from an autoimmune diseases patient by PCR and then cloned into a prokaryotic replicative plasmid, pGEM-7Zf(-). This clone is an important material to study the biological functions of TW-3. The TW-3 genome was sequenced and found to comprise of 5,111 base pairs. The major capsid protein VP1 gene of JCV was amplified from the TW-3 JCV genome and cloned into a prokaryotic expression plasmid. The VP1 protein expressed in E. coli was self-assembled into capsid-like particles and caused hemagglutination of human O-type red blood cells. Cesium chloride density gradient centrifugation analysis found that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The pseudovirion packaged DNA and RNA molecules but the pseudocapsid did not contain any nucleic acid. The DNA binding activity of VP1 was also demonstrated by the Southwestern probing method in vitro. The pseudocapsid was further demonstrated to be able to deliver exogenous DNA into human fetal kidney epithelial cells. In order to study the mechanism of DNA packaging, we deleted the first 12 amino acids of JCV VP1. The expressed truncated VP1 (△N12VP1) failed to encapsidate the host DNA although the integrity of the capsid-like structure was maintained. In addition, capsid-like particles of △N12VP1 did not package exogenous DNA in vitro. The results indicate that the N-terminal region of the human polyomavirus major capsid protein VP1 may be involved in viral genome encapsidation during progeny maturation. The calcium ions play an important role in the assembly process of JCV progeny. In this thesis, we tried to find out the calcium binding sites in the VP1 molecule for capsid assembly. Site-directed mutagenesis was performed to replace Glu149 and Glu152 within EF loop, Asp239 and Glu240 within GH loop and Asp338 within C-arm by alanine respectively. Sucrose cushion and CsCl density centrifugation demonstrated that Asp239 and Asp338 mutated VP1 proteins could not form a capsid-like particle. These results indicate that Asp239 and Asp338 may be involved in calcium binding and then cause associations of inter-pentameric molecules for capsid assembly.