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Published in final edited form as: Virology. 2012 Jul 27;433(1):12–26. doi: 10.1016/j.virol.2012.06.017

Effect of a triple Phe mutant (F31AF35AF39A) on agnoprotein expression and the viral DNA replication in the viral background. (A) Immunocytochemical analysis of a triple Phe mutant (F31AF35AF39A). SVG-A cells were transfected/infected either with JCV Mad-1 WT or the triple Phe mutant and at day 5, cells were fixed with cold acetone and blocked with 5% BSA prepared in PBST for 2h. Then cells were processed for immunocytochemical detection of agnoprotein as described under the legend for figure 4C. (B) A cumulative effect of all three Phe mutations of agnoprotein on viral DNA replication. DpnI assay for a triple Phe mutant [F31A F35AF39A)=(T-Phe Mut.). JCV Mad-1 Agno WT and JCV Mad-1 Agno-(F31A F35A F39A) mutant genomes were transfected/infected into SVG-A cells and low molecular DNA was isolated at the indicated data points and analyzed by Southern blotting as described for Figure 6A. Replication assays were repeated several times and a representative result is shown here. (C) Quantitation analysis of Southern blots by a semi-quantitative densitometry method (using NIH Image J program) and presentation of the results in arbitrary units. The replication efficiency of the mutant virus was presented relative to that of WT. (D) Western blot analysis. In parallel to Southern blot analysis in 7B, whole-cell extracts were prepared at day 5 and 15 posttransfection from SVG-A cells transfected/infected with either JCV Mad-1 WT or JCV Mad-1 Agno-(T-Phe Mut.) as indicated, and analyzed by Western blotting. In this regard, 40 μgs of whole-cell extracts were separated on a 15% SDS-PAGE, transferred onto a nitrocellulose membrane and incubated with a primary (anti-Agno, polyclonal) (Del Valle et al., 2002) and secondary antibodies. The protein of interest was then detected by using ECL reagent as described in Materials and Methods. In lane 1 and 4, whole-cell extracts prepared from untransfected SVGA cells were loaded as negative controls (- Cont.).

Effect of a triple Phe mutant (F31AF35AF39A) on agnoprotein expression and the viral DNA replication in the viral background. (A) Immunocytochemical analysis of a triple Phe mutant (F31AF35AF39A). SVG-A cells were transfected/infected either with JCV Mad-1 WT or the triple Phe mutant and at day 5, cells were fixed with cold acetone and blocked with 5% BSA prepared in PBST for 2h. Then cells were processed for immunocytochemical detection of agnoprotein as described under the legend for figure 4C. (B) A cumulative effect of all three Phe mutations of agnoprotein on viral DNA replication. DpnI assay for a triple Phe mutant [F31A F35AF39A)=(T-Phe Mut.). JCV Mad-1 Agno WT and JCV Mad-1 Agno-(F31A F35A F39A) mutant genomes were transfected/infected into SVG-A cells and low molecular DNA was isolated at the indicated data points and analyzed by Southern blotting as described for Figure 6A. Replication assays were repeated several times and a representative result is shown here. (C) Quantitation analysis of Southern blots by a semi-quantitative densitometry method (using NIH Image J program) and presentation of the results in arbitrary units. The replication efficiency of the mutant virus was presented relative to that of WT. (D) Western blot analysis. In parallel to Southern blot analysis in 7B, whole-cell extracts were prepared at day 5 and 15 posttransfection from SVG-A cells transfected/infected with either JCV Mad-1 WT or JCV Mad-1 Agno-(T-Phe Mut.) as indicated, and analyzed by Western blotting. In this regard, 40 μgs of whole-cell extracts were separated on a 15% SDS-PAGE, transferred onto a nitrocellulose membrane and incubated with a primary (anti-Agno, polyclonal) (Del Valle et al., 2002) and secondary antibodies. The protein of interest was then detected by using ECL reagent as described in Materials and Methods. In lane 1 and 4, whole-cell extracts prepared from untransfected SVGA cells were loaded as negative controls (- Cont.).