A stabilized p21 that binds to PCNA inhibits MVM replication.

<p><b>A</b>) <i>p21 degradation by MVM requires a ubiquitin-modifiable lysine</i>. Murine A9 cell lines stably expressing HA-tagged wild-type p21 (p21^WT) or a mutant in which all seven lysines have been mutated to arginines (p21^K7R) were generated. p21 degradation assay was performed as described in 3C. <b>B</b>) <i>p21^K7R retains interaction with PCNA</i>. Control plasmid (lane 1), p21^WT (lane 2), p21^K7R (lane 3), and p21^K7R with additional mutations that disrupt PCNA interaction (p21^K7RΔPIP, lane 4) were transfected into 293T cells. At 48 hr cells were lysed, immunoprecipitated with HA antibodies and blotted using the indicated antibodies. The p21^K7RΔPIP was expressed at lower levels in this experiment however interaction with PCNA was not detected even upon longer exposure. <b>C</b>) <i>p21^K7R inhibits MVM replication</i>. p21^WT and p21^K7R cell lines were parasynchronized, released and infected with MVM at an MOI of 0.5. At 16 hr pi cells were treated with doxycycline to induce p21 expression and harvested 8 hrs later. Cells were processed for Southern blotting (top panel), or for western blotting using the indicated antibodies (bottom panels). A representative experiment is shown; quantifications in the text reflect three independent experiments. <b>D</b>) <i>p21^ΔPCNA does not inhibit MVM replication</i>. p21^WT and p21^ΔPCNA cells were treated, processed and standardized as in 4C. <b>E</b>) <i>A p21 peptide competitively binds to PCNA</i>. 293T cells were transfected with a construct expressing FLAG-tagged p21 (lanes 2 to 4) or control plasmid (PCDNA, lane 1) and processed for co-immunoprecipitation as in 4D. Treatment with wild-type but not scrambled peptide reduced interaction of p21 with PCNA. <b>F</b>) <i>A p21-derived peptide inhibits MVM replication</i>. Murine A9 cells were parasynchronized, released and infected with MVM at an MOI of 1. At 18 hr pi control cells were harvested (lane 1) and remainder were treated with vehicle (lane 2), wild-type peptide (lane 3) or scrambled peptide (lane 4) for 6 hrs. At 24 hr pi cells were harvested and processed for Southern blotting. A representative experiment is shown; the experiment was done three times.