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. 2009 Jun;83(11):5708-17.
doi: 10.1128/JVI.00300-09. Epub 2009 Mar 18.

Restriction of human polyomavirus BK virus DNA replication in murine cells and extracts

Cathal Mahon  1 , Bo Liang, Irina Tikhanovich, Johanna R Abend, Michael J Imperiale, Heinz P Nasheuer, William R Folk
Affiliations

Restriction of human polyomavirus BK virus DNA replication in murine cells and extracts

Cathal Mahon et al. J Virol. 2009 Jun.

Abstract

BK virus (BKV) causes persistent and asymptomatic infections in most humans and is the etiologic agent of polyomavirus-associated nephropathy (PVAN) and other pathologies. Unfortunately, there are no animal models with which to study activation of BKV replication in the human kidney and the accompanying PVAN. Here we report studies of the restriction of BKV replication in murine cells and extracts and the cause(s) of this restriction. Upon infection of murine cells, BKV expressed large T antigen (TAg), but viral DNA replication and progeny were not detected. Transfection of murine cells with BKV TAg expression vectors also caused TAg expression without accompanying DNA replication. Analysis of the replication of DNAs containing chimeric BKV and murine polyomavirus origins revealed the importance of BKV core origin sequences and TAg for DNA replication. A sensitive assay was developed with purified BKV TAg that supported TAg-dependent BKV DNA replication with human but not with murine cell extracts. Addition of human replication proteins, DNA polymerase alpha-primase, replication protein A, or topoisomerase I to the murine extracts with BKV TAg did not rescue viral DNA replication. Notably, addition of murine extracts to human extracts inhibited BKV TAg-dependent DNA replication at a step prior to or during unwinding of the viral origin. These findings and differences in replication specificity between BKV TAg and the TAgs of simian virus 40 (SV40) and JC virus (JCV) and their respective origins implicate features of the BKV TAg and origin distinct from SV40 and JCV in restriction of BKV replication in murine cells.

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Figures

FIG. 1.
FIG. 1.
Lack of BKV DNA replication in murine cells during viral infection. Human RPTE cells or murine 3T3 cells were infected with BKV. (A) Total cell lysates were harvested at 4 and 7 dpi, and proteins (15 μg) were subjected to Western blotting and probed for expression of TAg and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (a loading control). The numbers above the lanes indicate the days after infection at which the cell lysates were harvested. M, mock-infected lysate. (B) Low-molecular-weight DNA was isolated at 0, 4, and 7 dpi and analyzed by real-time PCR. Data are presented as genome copy number per reaction, normalized to the control plasmid pRL-Null (control for purification efficiency). Samples were assayed in triplicate; results are representative of two independent experiments.
FIG. 2.
FIG. 2.
In vivo DNA replication of BKV and mPyV in human and mouse cells. Vectors expressing BKV TAg (lanes 1 to 8) or mPyV TAg (lane 9) were cotransfected into human HEK293 (lanes 1 to 4) or mouse TCMK-1 (lanes 5 to 9) cells together with plasmids containing the complete BKV origin (lanes 1 and 5, B-B-B), the complete mPyV origin (lane 9, P-P-P), and BKV-mPyV chimeric origins (lanes 2 to 4 and 6 to 8). At 48 h after transfection, DNA was isolated and analyzed by Southern blotting. DNA replication products are marked by arrows.
FIG. 3.
FIG. 3.
Cell-free BKV DNA replication by recombinant BKV TAg. (A) DNA synthesis in the presence of recombinant BKV TAg was measured by incorporation of dNMPs into DNA. Increasing amounts (0, 7, 13, and 26 μg) of High Five cell extracts containing recombinant BKV TAg (about 0, 0.35, 0.65, and 1.3 μg, respectively) were added to HeLa hypotonic extracts in the presence of DNA with a BKV origin of replication (250 ng of B-B-B, ori+, bars 1 to 4) or empty vector (ori−, bars 5 to 8). (B) Cell-free DNA replication in the presence of BKV and SV40 TAg. DNA replication in the presence of 200 ng of purified recombinant BKV TAg or SV40 TAg as indicated was measured using 40 μg of HeLa extract and 500 ng of DNA containing the SV40 or BKV origin of replication (ori+) or an empty vector (ori−). Incorporation of dNMPs into DNA was measured by scintillation counting. The DNA synthesis was determined in duplicate and repeated three times. The averages from these experiments and the standard deviations are presented.
FIG. 4.
FIG. 4.
Modulation of polyomavirus DNA replication in murine cell extracts by human replication factors. In vitro DNA replication in the presence of equal amounts of purified recombinant BKV, mPyV, and SV40 TAg (shaded bars) using mouse FM3A cell extracts is shown. Bars 1 to 4 show DNA synthesis in the presence of mouse cell extracts and either mPyV TAg/P-P-P, BKV TAg/B-B-B, or SV40 TAg/pOriSV40. Incorporation of dNMPs into P-P-P in the absence of TAg served as a negative control. DNA synthesis with mouse cell extract and additional human DNA Pol α-primase (2 units of hPol α-primase) (66) is depicted in bars 5 to 7 (mPyV TAg/P-P-P, BKV TAg/B-B-B, and SV40 TAg/pOriSV40, respectively). The DNA synthesis of mouse cell extracts with an additional 0.5 μg of hRPA using mPyV TAg/P-P-P and BKV TAg/B-B-B is presented in bars 8 and 9, respectively, whereas the influence of human topoisomerase I (hTopo I, 120 ng) on the DNA synthesis in the presence of mouse cell extracts with mPyV TAg/P-P-P and BKV TAg/B-B-B is shown in bars 10 and 11, respectively. All assays were carried out in triplicate, and the results presented are the averages from two independent experiments.
FIG. 5.
FIG. 5.
DNA synthesis with purified human proteins in the presence of BKV and SV40 TAg. (A) Incorporation of dNMPs into DNA containing an SV40 (bar 2) or BKV (bar 4) origin of replication in the presence of 200 ng of the respective viral TAg and 100 ng of purified hPol α-primase, 50 ng topoisomerase I, and 1000 ng RPA was measured (monopolymerase DNA replication system). Vectors without a functional viral origin (ori−) served as negative controls (bars 1 and 3). (B) The effect of human and mouse proteins on DNA synthesis by hPol α-primase was determined with a BKV origin of replication. The incorporation of radioactive dNMPs using the BKV origin of replication as a template was measured in the presence of buffer but no additional proteins or with 15 μg human or mouse cell extracts (bars 1, 2, and 3, respectively). DNA synthesis in the presence of DNA lacking an origin of replication served as a negative control (bar 4). (C) The effect of human and mouse proteins on the DNA synthesis by human DNA Pol α-primase was determined with an SV40 origin of replication. The incorporation of radioactive dNMPs was determined in the presence of buffer but no additional proteins or with 15 μg human or mouse cell extracts (bars 1, 2, and 3, respectively). DNA synthesis in the presence of DNA lacking an origin of replication served as a negative control (bar 4). Incorporation of dNMPs into DNA was measured by scintillation counting. DNA synthesis was determined in duplicate and repeated three times. The averages from these experiments and the standard deviations are presented.
FIG. 6.
FIG. 6.
DNA replication with purified human proteins in the presence of BKV TAg and mouse cell extracts. (A) The replication of polyomavirus DNA was biochemically separated into three consecutive reaction steps: the unwinding, initiation, and elongation reactions. In the presence of RPA, topoisomerase I, ATP, and an ATP-regenerating system, viral TAg unwinds viral DNA at 37°C for 30 min (unwinding reaction). To synthesize primers at the unwound origin of DNA replication (initiation reaction), hPol α-primase and the three remaining ribonucleotides were added, and oligoribonucleotide primers are synthesized during the incubation at 37°C for 30 min, whereas no DNA can be synthesized since deoxynucleoside triphosphates are lacking. Finally, deoxynucleoside triphosphates, which include radioactively labeled dCTP to monitor DNA synthesis via scintillation counting, are added and DNA is synthesized at 37°C for 30 min (elongation reaction). Mouse cell extracts capable of supporting mPyV DNA replication were added to the reactions prior to the specified step (as indicated by the arrows). The addition of buffer served as control for the influence of salt and dilutions. (B) Results of the monopolymerase assay using BKV TAg and template containing the BKV origin of replication. (C) Results of the monopolymerase assay using SV40 TAg and template containing the SV40 origin of replication. In panels B and C, bars 1 and 2 represent dNMP incorporation into DNA in the absence and presence of TAg, respectively, but without mouse proteins. For bar 3, mouse cell extracts were added to reaction components before the addition of hPol α-primase and ribonucleotides (prior to unwinding of DNA). For bar 4, mouse cell extracts were added after the unwinding reaction but before the addition of hPol α-primase (prior to initiation of DNA replication). For bar 5, mouse cell extracts were added after initiation of DNA replication but before addition of deoxynucleoside triphosphates (prior to elongation). Incorporation of dNMPs into DNA was measured by scintillation counting. DNA synthesis was determined in duplicate and repeated three times. The averages from these experiments and the standard deviations are presented.
FIG. 7.
FIG. 7.
Comparison of the replication of DNAs with different origins by polyomavirus TAg proteins in vitro. Replication assays were carried out as described in Materials and Methods. (A to C) Incorporation of dNMPs into DNA was measured in the presence of BKV (A), JCV (B), or SV40 (C) TAg protein and human (HeLa) cell extracts. (D) mPyV TAg-dependent DNA replication in mouse (FM3A) cell extracts. DNA synthesis in the presence of BKV, JCV, SV40, and mPyV origin-containing DNA but without the cognate TAg, as well as DNA synthesis of plasmid DNA without a viral origin in the presence of the indicated TAg, served as negative controls in all panels. All assays were carried out in triplicate, and the results presented are the averages and standard deviations from two independent experiments.
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