A cell-free replication system for human polyomavirus JC DNA

Abstract

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase ␣-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origincontaining plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 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 replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.

Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase α‐primase

“…Proteins SV40 TAg, JCV TAg and the DNA polymerase a-primase complexes (p180-p68-p58-p48) were purified from baculovirus infected insect cells as described previously [22,32,34,35]. Human and murine DNA polymerase a-primase were immunopurified using the monoclonal antibodies SJK237-71 and SJK287-38, respectively [36].…”

“…The extracts were centrifuged at 4°C and 11 000 g. The supernatant was then adjusted to 100 mM NaCl and clarified by a second centrifugation at 100 000 g (S100 extract). Depletion of DNA polymerase a-primase from S100 extracts was performed essentially as previously described [22,27,32].…”

“…These assays were adapted from Ishimi et al [11]. The monopolymerase assay (40 lL) was assembled on ice and contained 0.5 lg pUC-HS (carrying the SV40 replication origin) or 0.5 lg pJC433 (carrying the JCV replication origin [32] . Recombinant DNA polymerase a-primase was added as indicated.…”

“…SV40 DNA replication in vitro was recently shown to require a functional interaction between the SV40 TAg and the C-terminus of the p180 subunit of human DNA polymerase a-primase [26].The genome of JCV is 69% homologous to that of SV40 and expresses an analogous set of proteins [31]. The core origins of replication of the two viruses are also conserved to such an extent that SV40 TAg, which is 72% identical to JCV TAg, can efficiently support JCV DNA replication in vivo and in vitro [32,33]. The high level of conservation between these two primate specific viruses coupled with the fact that JCV DNA replication is inhibited in a nonpermissive host in vivo would imply that the restricted host range of JCV is due to a requirement for human DNA polymerase a-primase, as is the case with SV40 [8].…”

Inhibition of Human BK Polyomavirus Replication by Small Noncoding RNAs

“…Purification of proteins and the inhibitory activities (IAs). Human recombinant topoisomerase I (47), Pol-primase (43), RPA (35,40), BKV TAg, and SV40 TAg (4,28,36) were expressed and purified, and their concentrations and activities were determined as previously described (21).…”