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Efficient parvovirus replication requires CRL4Cdt2-targeted depletion of p21 to prevent its inhibitory interaction with PCNA.

Adeyemi RO, Fuller MS, Pintel DJ - PLoS Pathog. (2014)

Bottom Line: PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro.Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not.Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, C.S. Bond Life Sciences Center, University of Missouri-Columbia, School of Medicine, Columbia, Missouri, United States of America.

ABSTRACT
Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

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A stabilized p21 that binds to PCNA inhibits MVM replication.A) p21 degradation by MVM requires a ubiquitin-modifiable lysine. Murine A9 cell lines stably expressing HA-tagged wild-type p21 (p21WT) or a mutant in which all seven lysines have been mutated to arginines (p21K7R) were generated. p21 degradation assay was performed as described in 3C. B) p21K7R retains interaction with PCNA. Control plasmid (lane 1), p21WT (lane 2), p21K7R (lane 3), and p21K7R with additional mutations that disrupt PCNA interaction (p21K7RΔPIP, lane 4) were transfected into 293T cells. At 48 hr cells were lysed, immunoprecipitated with HA antibodies and blotted using the indicated antibodies. The p21K7RΔPIP was expressed at lower levels in this experiment however interaction with PCNA was not detected even upon longer exposure. C) p21K7R inhibits MVM replication. p21WT and p21K7R 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. D) p21ΔPCNA does not inhibit MVM replication. p21WT and p21ΔPCNA cells were treated, processed and standardized as in 4C. E) A p21 peptide competitively binds to PCNA. 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. F) A p21-derived peptide inhibits MVM replication. 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.
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ppat-1004055-g005: A stabilized p21 that binds to PCNA inhibits MVM replication.A) p21 degradation by MVM requires a ubiquitin-modifiable lysine. Murine A9 cell lines stably expressing HA-tagged wild-type p21 (p21WT) or a mutant in which all seven lysines have been mutated to arginines (p21K7R) were generated. p21 degradation assay was performed as described in 3C. B) p21K7R retains interaction with PCNA. Control plasmid (lane 1), p21WT (lane 2), p21K7R (lane 3), and p21K7R with additional mutations that disrupt PCNA interaction (p21K7RΔPIP, lane 4) were transfected into 293T cells. At 48 hr cells were lysed, immunoprecipitated with HA antibodies and blotted using the indicated antibodies. The p21K7RΔPIP was expressed at lower levels in this experiment however interaction with PCNA was not detected even upon longer exposure. C) p21K7R inhibits MVM replication. p21WT and p21K7R 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. D) p21ΔPCNA does not inhibit MVM replication. p21WT and p21ΔPCNA cells were treated, processed and standardized as in 4C. E) A p21 peptide competitively binds to PCNA. 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. F) A p21-derived peptide inhibits MVM replication. 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.

Mentions: Unexpectedly, the p21ΔDegron mutant, although stable, interacted poorly with PCNA for reasons not yet clear (data not shown). As a result, we could not use cell lines expressing this mutant to determine whether stabilized, p21 affected MVM replication via PCNA binding. Thus we generated a murine cell line conditionally expressing a mutant p21 in which all seven lysines in p21 were changed to arginine [p21K7R, a similar mutation has been reported by others [23]]. Similar to the p21ΔDegron mutant, the p21K7R protein was resistant to degradation following MVM infection (Figure 5A, compare lanes 6 and 8 to 2 and 4), yet retained substantial interaction with PCNA in transient transfection assays (Figure 5B, compare lane 3 to lanes 2 and 4). Whereas induction of p21WT expression for 8 hrs after infection had little effect on MVM replication (Figure 5C, compare lanes 1 and 2), p21K7R expression reduced replication by up to 3 fold (Figure 5C, compare lanes 3 and 4). Importantly, in contrast, the p21ΔPCNA mutant-expressing cell line, which expressed a stable p21 which no longer could interact with PCNA (Figure 4D and 4E), failed to inhibit MVM replication upon induction (Figure 5D, compare lanes 3 and 4). This was also the case with the p21ΔDegron mutant-expressing cell lines (data not shown). Furthermore, cell lines expressing a mutant of p21K7R in the PIP box-mutated background (p21K7RΔPIP) (see Figure 5B, lane 4) also failed to inhibit MVM replication (Figure S5), demonstrating that absent PCNA binding, the K7R mutation itself had no deleterious effect on MVM replication. All the mutants tested were recruited to APAR bodies during MVM infection (Figure S6).


Efficient parvovirus replication requires CRL4Cdt2-targeted depletion of p21 to prevent its inhibitory interaction with PCNA.

Adeyemi RO, Fuller MS, Pintel DJ - PLoS Pathog. (2014)

A stabilized p21 that binds to PCNA inhibits MVM replication.A) p21 degradation by MVM requires a ubiquitin-modifiable lysine. Murine A9 cell lines stably expressing HA-tagged wild-type p21 (p21WT) or a mutant in which all seven lysines have been mutated to arginines (p21K7R) were generated. p21 degradation assay was performed as described in 3C. B) p21K7R retains interaction with PCNA. Control plasmid (lane 1), p21WT (lane 2), p21K7R (lane 3), and p21K7R with additional mutations that disrupt PCNA interaction (p21K7RΔPIP, lane 4) were transfected into 293T cells. At 48 hr cells were lysed, immunoprecipitated with HA antibodies and blotted using the indicated antibodies. The p21K7RΔPIP was expressed at lower levels in this experiment however interaction with PCNA was not detected even upon longer exposure. C) p21K7R inhibits MVM replication. p21WT and p21K7R 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. D) p21ΔPCNA does not inhibit MVM replication. p21WT and p21ΔPCNA cells were treated, processed and standardized as in 4C. E) A p21 peptide competitively binds to PCNA. 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. F) A p21-derived peptide inhibits MVM replication. 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.
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ppat-1004055-g005: A stabilized p21 that binds to PCNA inhibits MVM replication.A) p21 degradation by MVM requires a ubiquitin-modifiable lysine. Murine A9 cell lines stably expressing HA-tagged wild-type p21 (p21WT) or a mutant in which all seven lysines have been mutated to arginines (p21K7R) were generated. p21 degradation assay was performed as described in 3C. B) p21K7R retains interaction with PCNA. Control plasmid (lane 1), p21WT (lane 2), p21K7R (lane 3), and p21K7R with additional mutations that disrupt PCNA interaction (p21K7RΔPIP, lane 4) were transfected into 293T cells. At 48 hr cells were lysed, immunoprecipitated with HA antibodies and blotted using the indicated antibodies. The p21K7RΔPIP was expressed at lower levels in this experiment however interaction with PCNA was not detected even upon longer exposure. C) p21K7R inhibits MVM replication. p21WT and p21K7R 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. D) p21ΔPCNA does not inhibit MVM replication. p21WT and p21ΔPCNA cells were treated, processed and standardized as in 4C. E) A p21 peptide competitively binds to PCNA. 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. F) A p21-derived peptide inhibits MVM replication. 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.
Mentions: Unexpectedly, the p21ΔDegron mutant, although stable, interacted poorly with PCNA for reasons not yet clear (data not shown). As a result, we could not use cell lines expressing this mutant to determine whether stabilized, p21 affected MVM replication via PCNA binding. Thus we generated a murine cell line conditionally expressing a mutant p21 in which all seven lysines in p21 were changed to arginine [p21K7R, a similar mutation has been reported by others [23]]. Similar to the p21ΔDegron mutant, the p21K7R protein was resistant to degradation following MVM infection (Figure 5A, compare lanes 6 and 8 to 2 and 4), yet retained substantial interaction with PCNA in transient transfection assays (Figure 5B, compare lane 3 to lanes 2 and 4). Whereas induction of p21WT expression for 8 hrs after infection had little effect on MVM replication (Figure 5C, compare lanes 1 and 2), p21K7R expression reduced replication by up to 3 fold (Figure 5C, compare lanes 3 and 4). Importantly, in contrast, the p21ΔPCNA mutant-expressing cell line, which expressed a stable p21 which no longer could interact with PCNA (Figure 4D and 4E), failed to inhibit MVM replication upon induction (Figure 5D, compare lanes 3 and 4). This was also the case with the p21ΔDegron mutant-expressing cell lines (data not shown). Furthermore, cell lines expressing a mutant of p21K7R in the PIP box-mutated background (p21K7RΔPIP) (see Figure 5B, lane 4) also failed to inhibit MVM replication (Figure S5), demonstrating that absent PCNA binding, the K7R mutation itself had no deleterious effect on MVM replication. All the mutants tested were recruited to APAR bodies during MVM infection (Figure S6).

Bottom Line: PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro.Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not.Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, C.S. Bond Life Sciences Center, University of Missouri-Columbia, School of Medicine, Columbia, Missouri, United States of America.

ABSTRACT
Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

Show MeSH
Related in: MedlinePlus