Epstein-Barr virus EBNA3C can disrupt multiple cell cycle checkpoints and induce nuclear division divorced from cytokinesis

Expression of EBNA3C is essential for the immortalization of B cells by EBV in vitro and, in co-operation with activated ras, EBNA3C has oncogenic activity in primary rodent fibroblasts. This suggested that this viral oncoprotein might disrupt the cyclin/CDK-pRb-E2F pathway, which regulates cell cycle progression at the restriction point (R-point) in G1 of the proliferation cycle. An assay was established in which transfected EBNA3C-positive cells could be sorted and simultaneously analysed for their distribution in the cell cycle. This revealed that in NIH3T3 fibroblasts compelled to arrest by serum-withdrawal, EBNA3C induces nuclear division that is often divorced from cytokinesis and so produces bi- and multinucleated cells. This was confirmed using the ecdysone-inducible system for expression of EBNA3C in human U2OS cells and by microinjection of expression vectors into NIH3T3 and U2OS. Further analysis revealed that in the inducible system, EBNA3C expression inhibits the accumulation of p27KIP1 but not the dephosphorylation of pRb. Experiments using the microtubule destabilizing drug nocodazole, showed that EBNA3C could abrogate the mitotic spindle checkpoint.