Purified JC virus T and T ' proteins differentially interact with the retinoblastoma family of tumor suppressor proteins

The amino termini of polyomavirus T antigens contain LXCXE and J domains, w hich are necessary for binding and inactivating the retinoblastoma family o f tumor suppressors. Both of these motifs are found in the JC virus (JCV) e arly proteins T-135' T-136' and T-165' leading to the suggestion that these recently discovered proteins complement the cell-cycle-deregulating functi on of the JCV large T antigen (TAg). To investigate this hypothesis, the th ree JCV T' proteins were produced in a baculovirus expression system and pu rified by immunoaffinity chromatography. To facilitate purification, hybrid omas that secrete antibodies recognizing amino-terminal epitopes of JCV ear ly proteins were produced. Potential interactions between the early viral p roteins and the cellular proteins pRB, p107, and p130 were investigated by incubating purified JCV TAS and T' proteins with extracts of MOLT-4 cells, a human T cell line. The four viral proteins preferentially bound hypophosp horylated species of the cellular proteins and exhibited the highest bindin g affinity to p107 and the lowest affinity to pRB. TAS and T-165' bound mor e pRB and less p107 than did T-135' and T-136'; T-165' also bound less p130 than the other three early proteins. Results of these in vitro interaction s were compared to those obtained in vivo using POJ cells, a transformed hu man glial cell line that expresses JCV early proteins, relatively high leve ls of pRB and p107, and low levels of p130. Most of the pRB in POJ cells is hyperphosphorylated, and only a fraction of the hypophosphorylated form(s) of pRB is bound by the viral proteins. In contrast, only hypophosphorylate d p130 is detected in the transformed cells, and most of this protein was f ound in complex with the viral proteins. Finally, nearly all of the p107 in POJ cells is bound by the JCV proteins. (C) 2000 Academic Press.