Figure 1.
Wild type tAg interacts with cellular phosphatase PP2A in cells expressing JCV early proteins.
JCV early proteins, expressed in Rat 2 (R2) or MEF cells transformed with pSR:T+/t+/T'+ or in G418-selected 3T3 cells transfected with pSR:T+/t+/T'+ (encodes all 5 JCV early proteins) or pSR:T−/t+/T'− (encodes JCV tAg only) were incubated with anti-T monoclonal antibody PAb 962 (α-T; lanes 4–7) or anti-PP2A antibody (α-PP2A; lanes 9–12). The amount of total cell protein subjected to IP in lanes 9–12 was four times that employed in the corresponding samples in lanes 4–7. Immunoprecipitated proteins were separated on a 20% SDS-polyacrylamide gel, and WB analysis was performed using a cocktail of anti-T monoclonal antibodies to detect JCV early proteins either expressed in the different cell lines (lanes 4–7) or expressed and bound to PP2A (lanes 9–12). Untransfected 3T3 and Rat 2 cells were included as negative controls (no JCV T proteins are present; lanes 1, 2), and α-mouse IgG was used in the IP step with the R2:T+/t+/T'+ cell extract to test for non-specific binding (lanes 3, 8). The asterisks denote antibody light and heavy chains. This figure represents proteins electrophoresed on a single gel and transferred to a membrane, which was then cut in half and each half developed for different lengths of time.
Figure 2.
Comparison of JCV and SV40 tAg amino acid sequences.
The JCV tAg sequence containing 172 amino acids is indicated in red letters, and the 174 amino acid SV40 tAg is shown in black letters. The sequences following the vertical line comprise the unique C-terminal region of each protein. Two conserved cysteine (CxCxxC) clusters (Cluster 1, 2) that contribute to SV40 tAg binding to zinc ions, and possibly to PP2A, are noted in both sequences, as are two recently recognized LxCxE motifs in JCV tAg. Three amino acid residues are highlighted with a blue letter and arrow. Histidine 42 (H42) is a conserved residue in the HPDKGG hexapeptide motif of the J domain that spans most of the N-terminal region of tAg as well as the other JCV and SV40 early proteins; H42 is required for functional interaction with Hsc70. Proline 99 is within a conserved CxxxPxC sequence that influences SV40 tAg binding to PP2A. Cysteine 157 is the central residue of the newly recognized second LxCxE motif in JCV tAg.
Figure 3.
Interaction of mutant JCV tAgs with PP2A.
Interactions between PP2A and wild-type tAg and (A) tAg-mutant P99A or (B) C157A were compared. 3T3 cells were stably transfected with DNA constructs expressing JCV early proteins under the control of SV40 promoter-enhancer signals. Lysates of these cells were subjected to IP with the anti-T monoclonal antibody PAb 962 (α-T) or anti-PP2A antibody (α-PP2A). The amount of total cell protein subjected to IP with anti-PP2A antibody (lanes 5–8, Panel A; lanes 6–10, Panel B) was five times that used with the anti-T antibody (lanes 1–4, Panel A; lanes 1–5, Panel B). Samples were electrophoresed on 18% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. WB was performed using a cocktail of anti-T monoclonal antibodies. Results for two independently-derived cells lines (#1, #2) containing DNA constructs that either encode all 5 JCV wild type early proteins (T+/t+/T′+; Panel A, lanes 1,2,5,6, Panel B, lanes 1,2,6,7) or wild type TAg, T′165, T′136 and T′135 plus tAg mutant P99A (T+/P99At+/T′+; Panel A, lanes 3,4,7,8) or C157A (T+/C157At+/T′+; Panel B, lanes 3,4,8,9) are shown. A single 3T3 cell line expressing tAg only (T−/t+/T′−) was isolated and tested for PP2A binding (Panel B, lanes 5, 10). Panels A and B of this figure each represent proteins electrophoresed on a single gel and transferred to a membrane, which was then cut in half and each half developed for different lengths of time.
Figure 4.
Wild type and mutant JCV tAgs bind the Rb protein p107.
JCV early proteins expressed in Rat 2 (R2) or MEF cells transformed with pSR:T+/t+/T'+ (encodes all 5 JCV early proteins) or in G418-selected 3T3 cells stably transfected with pSR:T+/t+/T'+, pSR:T−/t+/T'− (encodes tAg only) or pSR:T+/C157At+/T'+ (encodes tAg mutated at residue 157 and the other 4 JCV early proteins) were incubated with anti-T monoclonal antibody PAb 962 (α-T; lanes 2–6) or anti-p107 antibody (α-p107; lanes 7–11). Negative controls included EBC lysis buffer immunoprecipitated with anti-T monoclonal antibody PAb 962 (Con, lane 1) and Molt 4 extract with anti-p107 antibody (Molt 4, lane 12). The amount of total cell protein subjected to IP in lanes 7–11 was 10 times that employed in the corresponding samples in lanes 2–6. Proteins were separated on an 18% SDS-polyacrylamide gel and WB analysis was performed using a cocktail of anti-T monoclonal antibodies. The asterisks denote antibody light and heavy chains. This figure represents proteins electrophoresed on a single gel and transferred to a membrane, which was then cut in half and each half developed for different lengths of time.
Figure 5.
Replication efficiencies of DNA constructs expressing combinations of JCV early proteins.
PHFG cells in 60 mm plates were transfected in duplicate with 400 ng of wild type (T+/t+/T'+) or mutant (T+/t−/T'−, muT+/t+/T'+, T−/t+/T'−, T+/t−/T'+, or muT+/t−/T'+) JCV genomes. At each time point, viral DNA was extracted by the Hirt procedure [39], purified, digested with DpnI and EcoRI, electrophoresed on a 0.8% agarose gel, transferred to nitrocellulose membranes and hybridized to a [P32]dCTP-labeled JCV DNA probe. Bands were visualized with a Typhoon phosphorimager and quantitated with ImageQuant software. The marker (M) shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp). The duplicate, independent samples representing each DNA construct were analyzed, and the position of Dpn1-resistent replicating genomes on the Southern blots is denoted by an arrow at days 10, 14 and 21 p.t. Dpn1- and EcoRI-sensitive input DNAs are noted at the day 0 time point. Hirt extracts of uninfected PHFG cells (PHFG) were subjected to the same Dpn1 assay.
Figure 6.
Mutation of tAg residues proline 99 and cysteine 157 reduce JCV DNA replication.
PHFG cells in 60 mm plates were transfected in duplicate with 400 ng of wild type (T+/t+/T'+) or 8 different mutant (muT+/t−/T'+, T+/P99At+/T'+, T+/C157At+/T'+, T+/t+/T'−, T+/t−/T'−, muT+/t+/T'+, T−/t+/T'−, T+/t−/T'+) JCV genomes. Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt [39] on days 0, 7, 10 and 14 p.t. and analyzed using the Dpn1 assay as described in the legend to Figure 5. The marker (M) shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp), and the position of Dpn1-resistent replicating genomes is denoted by an arrow at days 7, 10 and 14 p.t.
Figure 7.
JCV tAg fails to complement replication of a tAg-deficient JCV genome in trans.
PHFG cells in 60 mm plates were co-transfected in duplicate with 400 ng of a tAg-deficient (T+/t−/T'+) JCV genome and 400 ng of a JCV DNA construct expressing either wild type tAg (T−/t+/T′−) or a J domain mutant tAg (T−/H42Qt+/T′−) under the control of the JCV promoter-enhancer. Cells were also transfected with 400 ng of either a T′-deficient (T+/t+/T'−) JCV genome or a positive replication control (T+/t+/T'+). Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt [39] on days 0, 7, and 10 p.t. and analyzed using the Dpn1 assay as described in the legend to Figure 5. The marker shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp), and the position of Dpn1-resistent replicating genomes is denoted by an arrow at days 7 and 10 p.t. Dpn1- and EcoRI-sensitive input DNAs are noted at the day 0 time point.
Figure 8.
JCV early genes expressed from a CMV promoter induce replication of a JCV origin plasmid.
PHFG cells in 60 mm plates were co-transfected in duplicate with 400 ng of the JCV replication origin plasmid, pM1o, and 400 ng of one of the following expression vectors that encode JCV early genes under the control of the CMV promoter: pCMV:T+/t+/T′+ (lanes 2, 3; all five tumor proteins expressed), pCMV:T+/t−/T′+ (lanes 4, 5; TAg and 3 T′ proteins expressed) or pCMV:T+/t−/T′− (lanes 6, 7; TAg only expressed). The ability of the proteins produced by the second plasmid to drive replication of the origin plasmid in trans was tested. Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt [39] on days 0, 3, 4 and 5 p.t. and analyzed using the Dpn1 assay as described in the legend to Figure 5. The marker (M) shown in lane 1 of each blot is 1 ng of linear pM1o (∼2400 bp), and the position of Dpn1-resistent replicating genomes is denoted by an arrow at days 3–5 p.t. Dpn1- and EcoRI-sensitive input DNAs are noted at the day 0 time point.