JC VIRUS SMALL T ANTIGEN BINDS RB FAMILY PROTEINS AND THE PHOSPHATASE PP2A AND IS REQUIRED FOR EFFICIENT VIRAL DNA REPLICATION ACTIVITY
Open Access
- Author:
- Hofstetter, Catherine A.
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 25, 2009
- Committee Members:
- Dick Frisque, Dissertation Advisor/Co-Advisor
Richard John Frisque, Committee Chair/Co-Chair
Jean Elnora Brenchley, Committee Member
David Scott Gilmour, Committee Member
Biao He, Committee Member
Kouacou Konan, Committee Member - Keywords:
- PP2A
small T antigen
JC Virus
viral DNA replication - Abstract:
- JC Virus (JCV) is a human polyomavirus which infects approximately 80% of the human population. Five tumor antigen or T proteins are produced from the early coding region of the viral genome; one precursor early mRNA is spliced to generate each of the five mature T protein mRNAs, which encode large Tumor Antigen (TAg), small tumor antigen (tAg), and the three T' proteins, T′135, T′136 and T′165. TAg, the best studied of these, is the key viral protein in facilitating DNA replication and cellular manipulation leading to oncogenic transformation. TAg is capable of binding to many diverse cellular factors, including members of the Retinoblastoma family of tumor suppressor proteins and Hsc70 through its LXCXE and J domains, respectively, to influence cellular proliferation, as well as binding and inactivating p53 to circumvent apoptosis. The 3 T′ proteins play roles in DNA replication and transformation; despite a high degree of sequence similarity to each other, they interact differentially with cellular proteins to modulate cellular proliferation and viral DNA replication. tAg is suspected to be important in host cell proliferation based on studies performed on the related SV40 tAg. In this study, I have generated a collection of DNA constructs which express different combinations of the JCV early proteins. These DNAs were used to analyze the effects of each combination of viral proteins on viral DNA replication and modulation of cellular signaling pathways. In particular, tAg’s specific interactions with cellular proteins and its potential role in pathways affecting cellular proliferation were studied. To examine whether specific conserved amino acid residues contributed to tAg’s functions, I constructed a series of DNAs encoding mutant tAg proteins. To create the first mutant tAg, a conserved proline residue at nucleotide 99 was altered to alanine (P99A); in the second mutant, a conserved cysteine 157, part of tAg’s second LxCxE domain, was changed to alanine (C157A); in the third tAg mutant, histidine 42 in the J-domain was altered to glutamine (H42Q). tAg was found to contribute to viral DNA replication. In contrast to studies which indicate that SV40 tAg is not required for replication of SV40 DNA, I observed no replication of JCV DNA in cells transfected with constructs that did not express tAg. Viral DNA constructs encoding mutant versions P99A or C157A of tAg exhibited decreased replication activity in permissive primary human fetal glial (PHFG) cells relative to a construct expressing wild type tAg. The defective phenotype was more pronounced in the P99A mutant. These data suggest that residues 99 and 157 of tAg facilitate tAg’s contributions to viral DNA replication. I have extended a recently published finding that a tAg-GST fusion protein interacts in vitro with protein phosphatase 2A (PP2A), a ubiquitous cellular protein responsible for dephosphorylation and regulation of many substrates. To determine whether this interaction takes place in vivo, I tested the ability of the P99A and C157A tAgs described above to interact with PP2A. Neither of these point mutations in tAg led to a detectable decrease in PP2A binding. Preliminary data suggests that expression of tAg increases the amount of phosphorylated Akt—a kinase that plays a role in the PI3K-PP2A transduction pathway that influences cellular proliferation—in 3T3 cells; this is predicted to occur through tAg-mediated alteration of PP2A activity. A 3T3 cell line which expresses tAg only, as well as cells which express T+/t+/T′+, T+/P99At+/T′+, or T+/C157At+/T′+, were used to examine interactions between tAg and the Rb family of proteins. These studies revealed that tAg interacts with both p130 and p107. The P99A and C157A mutations did not detectably alter the binding of tAg to either cellular protein. Phosphorylation of specific ser/thr sites regulates multiple functions of many proteins. tAg appears as multiple forms in protein extracts of dividing 3T3 cells subjected to immunoprecipitation and Western blots; here I have determined that these bands represent tAg in both phosphorylated and unphosphorylated forms. The experiments performed here indicate that tAg interacts with PP2A, an important regulator of cell signal transduction pathways, and unlike SV40 tAg, binds Rb proteins and contributes substantially to viral DNA replication.