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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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p21 degradation is mediated by the CRL4Cdt2 ligase complex.A) Schematic illustrating the experimental protocol for siRNA knockdown of ligase components in Figures 1B and 1C. B and C) p21 degradation requires DDB1 (B) and Cdt2 (C). Murine A9 cells were targeted with control siRNA or siRNA to DDB1 (B) or Cdt2 (C) as depicted in Figure 1A. Uninfected control cells were harvested at the time of release (Mock T0). Infections were done at the time of release at an MOI of 10 before harvest at 24 and 40 hr pi. Western blots were performed using antibodies against the indicated proteins.
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ppat-1004055-g001: p21 degradation is mediated by the CRL4Cdt2 ligase complex.A) Schematic illustrating the experimental protocol for siRNA knockdown of ligase components in Figures 1B and 1C. B and C) p21 degradation requires DDB1 (B) and Cdt2 (C). Murine A9 cells were targeted with control siRNA or siRNA to DDB1 (B) or Cdt2 (C) as depicted in Figure 1A. Uninfected control cells were harvested at the time of release (Mock T0). Infections were done at the time of release at an MOI of 10 before harvest at 24 and 40 hr pi. Western blots were performed using antibodies against the indicated proteins.

Mentions: The CRL4Cdt2 E3 ubiquitin ligase has been implicated in targeting p21 for proteasomal degradation upon S-phase entry and after cellular DNA damage [10]–[12]. Was this ubiquitin ligase also enlisted to target p21 at late times during MVM infection when cells were blocked at the G2/M border? To test this possibility, DDB1 and Cdt2, components of this ligase which are not present in other E3 ubiquitin ligases known to modify p21 [13], [14], were targeted via RNAi in the protocol illustrated in Figure 1A.


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)

p21 degradation is mediated by the CRL4Cdt2 ligase complex.A) Schematic illustrating the experimental protocol for siRNA knockdown of ligase components in Figures 1B and 1C. B and C) p21 degradation requires DDB1 (B) and Cdt2 (C). Murine A9 cells were targeted with control siRNA or siRNA to DDB1 (B) or Cdt2 (C) as depicted in Figure 1A. Uninfected control cells were harvested at the time of release (Mock T0). Infections were done at the time of release at an MOI of 10 before harvest at 24 and 40 hr pi. Western blots were performed using antibodies against the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974872&req=5

ppat-1004055-g001: p21 degradation is mediated by the CRL4Cdt2 ligase complex.A) Schematic illustrating the experimental protocol for siRNA knockdown of ligase components in Figures 1B and 1C. B and C) p21 degradation requires DDB1 (B) and Cdt2 (C). Murine A9 cells were targeted with control siRNA or siRNA to DDB1 (B) or Cdt2 (C) as depicted in Figure 1A. Uninfected control cells were harvested at the time of release (Mock T0). Infections were done at the time of release at an MOI of 10 before harvest at 24 and 40 hr pi. Western blots were performed using antibodies against the indicated proteins.
Mentions: The CRL4Cdt2 E3 ubiquitin ligase has been implicated in targeting p21 for proteasomal degradation upon S-phase entry and after cellular DNA damage [10]–[12]. Was this ubiquitin ligase also enlisted to target p21 at late times during MVM infection when cells were blocked at the G2/M border? To test this possibility, DDB1 and Cdt2, components of this ligase which are not present in other E3 ubiquitin ligases known to modify p21 [13], [14], were targeted via RNAi in the protocol illustrated in Figure 1A.

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