Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic infection of oligodendrocytes by JC Virus (JCV). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. JCVCPN was isolated from the brain of a JCVE patient. JCVCPN contains a unique 143 base pair deletion in the agnogene and has an archetype-like regulatory region (RR), of the type typically found in the kidneys. In this dissertation, we studied the JCVCPN virus to better understand the role of the agnogene and the RR in JCV replication. We used kidney, glial and neuronal cell lines to compare the replication of JCVCPN to the prototype virus JCVMad-1. JCVCPN was able to replicate viral DNA in all cell lines tested, but was unable to establish the high level of infection seen with JCVMad-1. Levels of VP1 capsid protein were undetectable in JCVCPN transfected cells, and few infectious virions were produced. JCVCPN did not have a replication advantage in the neuronal cell line tested. To determine if the agnogene deletion or the archetype-like RR was responsible for the observed phenotype of JCVCPN, we generated a series of chimeric viruses between JCVCPN and JCVMad-1. We found that the phenotype of JCVCPN was due predominantly to the deletion in the agnogene, in particular the loss of the DNA and not the lack of a full length agnoprotein. To further study the role of the agnogene DNA in JCV replication, we introduced a series of small agnogene deletions into a virus with a start codon mutation which prevents agnoprotein expression. We characterized the replication of these additional mutants and found that nucleotides 376-396 are crucial for the expression of VP1 capsid protein. Previous studies have provided evidence for the binding of host cell proteins to the agnogene DNA. We used DNA-Immunoprecipitations with the agnogene to identify candidate binding proteins, but were unable to confirm any candidate proteins as binding specifically to the JCV agnogene. Studying this naturally occurring pathogenic variant of JCV provided a valuable tool for understanding the functions of the agnogene and RR form in JCV replication.