A CELL-FREE REPLICATION SYSTEM FOR HUMAN POLYOMAVIRUS JC DNA

The human polyomavirus JC virus (JCV) establishes persistent infection s in most individuals and is the etiologic agent of progressive multif ocal leukoencephalopathy. In this report, we describe the establishmen t of a soluble cell-free system that is capable of replicating exogeno us plasmid DNA containing the JCV origin of replication. Replication i n this system is completely dependent on the addition of JCV large T a ntigen (TAg). To prepare JCV TAg for replication analysis, a recombina nt baculovirus containing the JCV TAg-coding sequence was generated. T Ag expressed in insect cells was purified by metal chelate chromatogra phy. JCV TAg supported initiation of JCV DNA replication in the presen ce of DNA polymerase cw-primase, replication protein A, and topoisomer ase I in a dose-dependent manner and was also capable of supporting DN A replication in crude human cell extracts. Point mutation of TAg-bind ing site I strongly diminished TAg binding and concomitantly reduced J CV DNA replication in vivo and in vitro by approximately 50% Point mut ation of TAg-binding site II or deletion of the early palindrome compl etely abolished replication of JCV origin-containing plasmid DNA in vi vo and in vitro, marking these sequences as essential components of th e JCV core origin. A comparison of several TAgs showed that simian vir us 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA re plication in vivo. Therefore, it may be a useful tool for future analy sis of interactions between JCV and its host cell.