The small double-stranded DNA human polyomavirus JC Virus (JCV) has been identified as the causative agent of progressive multifocal leukoencephalopathy (PML), a severe neurodegenerative disease resulting from destruction of myelin producing oligodendrocytes in the central nervous system (CNS). JCV also induces malignant tumor formation in animals, and the genome has been isolated from some human cancers.
The JCV genome contains three functional regions: the early region, encoding the large and small T antigens; the late region, encoding the structural proteins VP1, VP2, and VP3 and the late auxiliary agnoprotein; and a noncoding regulatory region. Agnoprotein is a 71 amino acid protein of unknown function.
In JCV infected and agnoprotein transfected cells, the 8kDa protein is found predominantly in the cytoplasm with strong perinuclear localization. However, some agnoprotein is apparent in the nucleus, and a subset of cells express largely nuclear agnoprotein. Transfection with agnoprotein deleted JCV DNA results in radically reduced mRNAs for large T-antigen and VP1, suggesting agnoprotein may have a regulatory role in viral gene transcription. Additionally, agnoprotein directly interacts with T-antigen, the cell-cycle regulated cellular transcription factor YB-1, and host p53; decreases cyclin A and cyclin B1 activity; increases transcription of the cellular cyclin kinase inhibtor p21; and binds the DNA-repair protein Ku70, sequestering it in the cytoplasm. The reported activities of agnoprotein would require nuclear localization at various points of the cell cycle to exert its multiple effects.
Precise and well-timed intracellular localization of proteins is critical for their biological function, and these factors are often controlled by posttranslational modifications. JCV agnoprotein contains six potential phosphorylation sites, and it has been suggested that agnoprotein is phosphorylated by 32P radiolabeling of JCV infected cells; however, the mechanism(s) and function(s) have not been determined. Interestingly, cells treated with the 3’, 5’ cAMP dependent kinase (PKA) inhibitor H-89 resulted in predominantly nuclearly localized agnoprotein, suggesting agnoprotein localization may be regulated by cellular factors.
JCV agnoprotein has a putative highly hydrophobic NES and also two putative NLSs. Although the size of agnoprotein is below the diffusion limit of the nuclear pore complex (NPC), nuclear entry might occur by active import. Considering the implications agnoprotein may have on viral replication, understanding its function is imperative for further study of the mechanisms of JCV lytic infection.
The following specific aims will test the hypothesis that JCV agnoprotein undergoes phosphorylation dependent nucleocytoplasmic shuttling for intimate control of viral transcription and DNA replication.
1) Determine the mechanism and site of phosphorylation of agnoprotein
a. Analyze 32P integration via in-vitro phosphorylation assays using purified kinases.
b. Analyze agnoprotein phosphorylation of infected and transfected cells treated with kinase inhibitors.
c. Determine phosphorylation site(s) by generating site-directed agnoprotein mutants.
d. Generate phosphorylation negative and simulated phosphorylation mutants fused with GFP to prevent passive nuclear diffusion and analyze intracellular localization.
2) Determine requirements for agnoprotein nuclear localization and phosphorylation on viral DNA replication and gene transcription
a. Identify and mutate putative agnoprotein-GFP NLS(s) and analyze for intracellular localization. Identify sites that abolish nuclear localization.
b. Identify and mutate putative agnoprotein-GFP NES or generate nuclearly restricted agnoprotein by fusion of SV40 large T antigen NLS. Block CRM1 mediated nuclear export with leptomycin and analyze.
c. Analyze transcriptional regulation activity of these mutants using T-antigen and a luciferase