EVIDENCE THAT REPLICATION OF HUMAN NEUROTROPIC JC VIRUS-DNA IN GLIAL-CELLS IS REGULATED BY THE SEQUENCE-SPECIFIC SINGLE-STRANDED DNA-BINDING PROTEIN PUR-ALPHA

Initiation of polyomavirus DNA replication in eukaryotic cells require s the participation of the viral early protein T antigen, cellular rep lication factors, and DNA polymerases. The human polyomavirus JC virus (JCV) is the etiologic agent of the fatal demyelinating disease progr essive multifocal leukoencephalopathy in immunocompromised individuals , This virus exhibits a narrow host range and a tissue specificity tha t restricts its replication to glial cells of the central nervous syst em, Restriction of viral DNA replication due to species specificity of the DNA polymerase, coupled with glial cell-specific transcription of the viral early promoter, is thought to account for the brain-specifi c replication of JCV. In this report we demonstrate that overexpressio n of Pur alpha, a protein which binds to single-stranded DNA in a sequ ence-specific manner, suppresses replication of JCV DNA in glial cells . Results from footprinting studies indicate that Pur a and T antigen share a common binding region spanning the single-stranded ori sequenc e of JCV. Further, T antigen was capable of stimulating the associatio n of Pur a with the ori sequence in a band shift assay, Whereas no evi dence for simultaneous binding of Pur ex and T antigen to single-stran ded DNA has been observed, results from coimmuno-precipitation and Wes tern blot (immunoblot) analyses of proteins derived from cells produci ng JCV T antigen indicate a molecular association of JCV T antigen and Pur alpha. The binding of Pur alpha to the single-stranded ori sequen ce and its association with T antigen suggest that Pur a interferes wi th the activity of T antigen and/or other regulatory proteins to exert its negative effect on JCV DNA replication. The importance of these f indings in the reactivation of JCV in the latently infected individual under immunosuppressed conditions is discussed.