Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

Abstract

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase ␣-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).

Stimulation of BK Virus DNA Replication by NFI Family Transcription Factors

“…The NFIC/CTF1 isotype stimulates the initiation of adenovirus DNA replication by recruiting adenovirus DNA polymerase to the origin of replication (9,19), and the proline-rich transactivation domain of the NFIC/CTF1 isotype stimulates SV40 DNA replication when tethered to the SV40 origin (61). These observations prompted us to assess whether NFIC/CTF1 can stimulate BKV DNA replication in the monopolymerase replication system that contains Pol-primase, RPA, and topoisomerase I (46,85) in the absence of other cellular factors.…”

“…DNA replication assays with transfected cells were performed as previously described (46,84), with transfection procedures optimized by luciferase/␤-galactosidase reporter assays. The in vitro monopolymerase assays were performed as previously described (46,85), with the following slight modifications: the assay mixture included 0.5 g of BKV template DNA, 50 ng topoisomerase I, 1 g replication protein A (RPA), and Pol-primase (as indicated in the figure legends) in a solution containing 30 mM HEPES (pH 7.8); 7 mM magnesium acetate; 0.1 mM EGTA; 0.5 mM dithiothreitol (DTT); 200 M each UTP, GTP, and CTP; 4 mM ATP; 100 M each dATP, dGTP, and dTTP; 10 M dCTP; 40 mM creatine phosphate; 1 g creatine kinase; 0.1 mg/ml heat-treated bovine serum albumin (BSA); and 5 Ci [␣- Elmer) in 40 l. The amount of added NFI protein is indicated in the figure legends. Purified BKV Tag (0.4 g) was added to start the reaction, and after incubation for 60 min at 37°C, the reaction products were precipitated with cold 10% (wt/vol) trichloroacetic acid (TCA) containing 2.5% (wt/vol) sodium pyrophosphate, spotted onto glass fiber filters (GF/C; Whatman), washed with 1 M HCl, and analyzed by scintillation counting.…”

“…1). Adjacent to the core-ori are the early flanking (EF) and the late flanking sequences (the "enhancer"), to which histones, cellular transcription factors, and perhaps also small noncoding RNAs bind and which control viral gene transcription and DNA replication (46,52,84,85). The BKV archetype enhancer, comprised of four single-copy sequence blocks, termed P 68 , Q 39 , R 63 , and S 63 , rearranges by duplication, deletion, and insertion in late-stage PVAN or after passage in cell culture, providing a replication advantage and, perhaps, enhanced tropism (10,30,78).…”

Inhibition of Human BK Polyomavirus Replication by Small Noncoding RNAs

“…Early initiation products, the nascent DNA primers, are then extended by DNA polymerase ␦ and proliferating cell nuclear antigen (PCNA) and the loading factor replication factor C on the leading and lagging strands (37,54). Although the DNA replication machineries are conserved between primates and rodents, BKV does not replicate in mouse cells as a result of species-specific interactions between TAg and cellular Pol-primase, analogous to what has been observed with simian virus 40 (SV40) (26,28,31,32,42,48,52). In contrast to what was observed with SV40, however, a dominant negative factor(s) present in mouse cell extracts acts at the BKV origin of replication to inhibit DNA replication, even in an otherwise supportive environment (28,52).…”

“…In vitro DNA replication assays. Replication of DNAs in vitro was assayed as previously described (28,52) with slight modifications. Briefly, the reaction mixture (40 l) contained the following: 20 mM HEPES-KOH, pH 7.8; 7 mM magnesium acetate (MgAc); 1 mM dithiothreitol (DTT); 4 mM ATP; 200 M each CTP, UTP, and GTP; 50 M dCTP; 100 M each dATP, dTTP, and dGTP; 40 mM creatine phosphate (pH 7.8); 40 g/ml creatine kinase; 5 Ci of [␣-32 P]dCTP (3,000 Ci/mmol, Perkin-Elmer); 0.25 g of origin-containing plasmid DNA; and human HeLa cell extract (100 g of total protein).…”