title: Epstein-Barr virus nuclear antigen 3A promotes cellular proliferation by repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1.
abstract: Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF) and p16(INK4a) expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF) and p16I(NK4a). By contrast, p16(INK4a) was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF) was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1), a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1) expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.
authors.affiliation: Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, and the Penn State Hershey Cancer Institute, Hershey, Pennsylvania, United States of America.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Tursiella
authors.affiliation: Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, and the Penn State Hershey Cancer Institute, Hershey, Pennsylvania, United States of America.
authors.forename: Emily R
authors.initials: ER
authors.lastname: Bowman
authors.affiliation: Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
authors.forename: Keith C
authors.initials: KC
authors.lastname: Wanzeck
authors.affiliation: Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Throm
authors.affiliation: Department of Public Health Sciences, Pennsylvania State University College of Medicine, and the Penn State Hershey Cancer Institute, Hershey, Pennsylvania, United States of America.
authors.forename: Jason
authors.initials: J
authors.lastname: Liao
authors.affiliation: Department of Public Health Sciences, Pennsylvania State University College of Medicine, and the Penn State Hershey Cancer Institute, Hershey, Pennsylvania, United States of America.
authors.forename: Junjia
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, and the Penn State Hershey Cancer Institute, Hershey, Pennsylvania, United States of America.
authors.forename: Clare E
authors.initials: CE
authors.lastname: Sample
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000378:metabolism*; D002051:Burkitt Lymphoma / Q000821:virology*; D002460:Cell Line; D049109:Cell Proliferation / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism; D006801:Humans; D051379:Mice
chemical_list: D000956:Antigens, Viral; D050759:Cyclin-Dependent Kinase Inhibitor p21; D019309:Epstein-Barr Virus Nuclear Antigens; C411723:lymphocryptovirus nuclear antigen 3A; D018844:Cyclin-Dependent Kinases; C417029:EBV-encoded nuclear antigen 1