Exposure to JC Virus (JCV) usually usually occurs during childhood and generally results in a subclinical infection. The virus may persist for life in the kidneys, however in immunocompromised individuals JCV may reach the brain and cause the fatal demyelinating disease PML. In addition to its pathogenic potential in people, JCV has proven to a highly oncogenic in animals; inoculation of JCV into rodents and primates results in a variety of tumors, some of which are among the more frequent types in humans. In contrast to its efficient activity in vivo, JCV exhibits restricted lytic and transforming activities in vitro. Our recent studies indicate that while both regulatory and T antigen coding sequences contribute to this restricted behavior, the latter sequences play a greater role. Our long term objective is to identify those sequences within the early coding region that contribute to the unique biology of JCV. To achieve this goal, our specific aims will revolve around three experimental approaches: 1) Analysis of chimeric viruses that utilize homologous and heterologous regulatory signals to express hybrid T proteins. JCV, BKV, and SV40 differ significantly in their lytic and transforming abilities; can domains of the JCV T antigen be identified that are responsible for JCV's restricted phenotype in vitro? Does the creation of transgenic mice harboring hybrid vs parental viral genomes allow us to identify sequences involved in tumorigenic and pathologic potential? 2) Wild type JCv T protein - overexpression in adenovirus vectors, purification by affinity chromatography, and characterization via biochemical/functional assays. Polyomavirus T antigens are multifunctional proteins that play various roles in DNA replication, transcription"""""""" and oncogenicity. Various regions of the SV40 and Polyoma T proteins are associated with specific functions; are differences in functional domains of JCV T responsible for biological differences between these related viruses? The availability of hybrid and mutant T proteins for similar analyses will enhance these studies. 3) Mutational analysis of the JCV T protein. Mutations will be introduced into the JCV early coding sequences at sites considered to be interesting based on our sequence analysis of wild type and mutant SV40 T proteins. Random and specific mutagenesis be used JCV does conversion of a JCV T to an SV40-like T enhance the lytic and transforming capabilities of JCV in vitro?
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