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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 during MVM requires interaction with PCNA and the CRL4Cdt2 ligase complex.A) Illustration of the p21 PIP/degron region (amino acid 136 to 151) of wild-type murine p21 (p21WT), p21Δdegron, and p21ΔPIP. Mutations are shown in red. B) p21Δdegron does not interact with the CRL4Cdt2 complex. 293T were cells transfected with constructs expressing FLAG-tagged p21WT (lane 2), p21Δdegron (lane 3) or control plasmid (lane 1). Cells were harvested at 48 hr. Lysates were immunoprecipitated using with FLAG antibody and blotted against the indicated proteins. C) MVM degradation of p21 requires its interaction with the CRL4Cdt2 ligase complex. Murine A9 cell lines stably expressing p21WT and p21Δdegron were generated as described. Cells were parasynchronized, released into complete media and mock-infected or infected with MVM at an MOI of 10. At 20 hr pi cells were treated with doxycycline to induce p21 expression. Cells were harvested 6 hrs later and processed for western blotting using the indicated antibodies. D) p21ΔPCNA does not interact with PCNA. Experiment performed as for Figure 4B. E) MVM degradation of p21 requires its interaction with PCNA. Experiment performed as for 4C.
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ppat-1004055-g004: p21 degradation during MVM requires interaction with PCNA and the CRL4Cdt2 ligase complex.A) Illustration of the p21 PIP/degron region (amino acid 136 to 151) of wild-type murine p21 (p21WT), p21Δdegron, and p21ΔPIP. Mutations are shown in red. B) p21Δdegron does not interact with the CRL4Cdt2 complex. 293T were cells transfected with constructs expressing FLAG-tagged p21WT (lane 2), p21Δdegron (lane 3) or control plasmid (lane 1). Cells were harvested at 48 hr. Lysates were immunoprecipitated using with FLAG antibody and blotted against the indicated proteins. C) MVM degradation of p21 requires its interaction with the CRL4Cdt2 ligase complex. Murine A9 cell lines stably expressing p21WT and p21Δdegron were generated as described. Cells were parasynchronized, released into complete media and mock-infected or infected with MVM at an MOI of 10. At 20 hr pi cells were treated with doxycycline to induce p21 expression. Cells were harvested 6 hrs later and processed for western blotting using the indicated antibodies. D) p21ΔPCNA does not interact with PCNA. Experiment performed as for Figure 4B. E) MVM degradation of p21 requires its interaction with PCNA. Experiment performed as for 4C.

Mentions: To investigate the importance of these interactions for the MVM-dependent targeting of p21 by the CRL4Cdt2 ligase we generated stable murine cell lines via lentivirus transduction that conditionally expressed FLAG-tagged wild-type or mutant p21 (p21WT, p21ΔDegron, p21ΔPCNA, mutations shown in Figure 4A) in a doxycycline-responsive manner. As expected, MVM infection of a p21WT expressing cell line resulted in degradation of the tagged p21 (Figure 4C and 4E, compare lanes 2 to 4), which could be prevented by treating cells with the proteasome inhibitor MG132 (Figure S4, panel A), or via siRNA knockdown of CRL4Cdt2 components (Figure S4, panels B and C). These results suggested that the depletion of p21 in these cell lines occurred via a similar mechanism to that of the endogenous p21. The lysine at the p21 +4 position, 3′ to its PIP box, has been reported to be required for interaction of p21 with the CRL4Cdt2 ligase complex via Cdt2. Mutation of the +3 to +5 amino acids KRR to AAA (p21Δdegron) abolished p21 interaction with the CRL4Cdt2 complex following its transient transfection as reflected by loss of interaction with DDB1 (Figure 4B, compare lanes 2 to 3). Murine cell lines that expressed the p21Δdegron mutant were generated, and when infected with MVM, in contrast to cell lines expressing wild-type p21 (p21WT), the p21Δdegron protein was resistant to degradation (Figure 4C, compare lanes 6 and 8 to lanes 2 and 4). This suggested that MVM-induced p21 degradation required interaction of p21 with the CRL4Cdt2 ubiquitin ligase complex.


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 during MVM requires interaction with PCNA and the CRL4Cdt2 ligase complex.A) Illustration of the p21 PIP/degron region (amino acid 136 to 151) of wild-type murine p21 (p21WT), p21Δdegron, and p21ΔPIP. Mutations are shown in red. B) p21Δdegron does not interact with the CRL4Cdt2 complex. 293T were cells transfected with constructs expressing FLAG-tagged p21WT (lane 2), p21Δdegron (lane 3) or control plasmid (lane 1). Cells were harvested at 48 hr. Lysates were immunoprecipitated using with FLAG antibody and blotted against the indicated proteins. C) MVM degradation of p21 requires its interaction with the CRL4Cdt2 ligase complex. Murine A9 cell lines stably expressing p21WT and p21Δdegron were generated as described. Cells were parasynchronized, released into complete media and mock-infected or infected with MVM at an MOI of 10. At 20 hr pi cells were treated with doxycycline to induce p21 expression. Cells were harvested 6 hrs later and processed for western blotting using the indicated antibodies. D) p21ΔPCNA does not interact with PCNA. Experiment performed as for Figure 4B. E) MVM degradation of p21 requires its interaction with PCNA. Experiment performed as for 4C.
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ppat-1004055-g004: p21 degradation during MVM requires interaction with PCNA and the CRL4Cdt2 ligase complex.A) Illustration of the p21 PIP/degron region (amino acid 136 to 151) of wild-type murine p21 (p21WT), p21Δdegron, and p21ΔPIP. Mutations are shown in red. B) p21Δdegron does not interact with the CRL4Cdt2 complex. 293T were cells transfected with constructs expressing FLAG-tagged p21WT (lane 2), p21Δdegron (lane 3) or control plasmid (lane 1). Cells were harvested at 48 hr. Lysates were immunoprecipitated using with FLAG antibody and blotted against the indicated proteins. C) MVM degradation of p21 requires its interaction with the CRL4Cdt2 ligase complex. Murine A9 cell lines stably expressing p21WT and p21Δdegron were generated as described. Cells were parasynchronized, released into complete media and mock-infected or infected with MVM at an MOI of 10. At 20 hr pi cells were treated with doxycycline to induce p21 expression. Cells were harvested 6 hrs later and processed for western blotting using the indicated antibodies. D) p21ΔPCNA does not interact with PCNA. Experiment performed as for Figure 4B. E) MVM degradation of p21 requires its interaction with PCNA. Experiment performed as for 4C.
Mentions: To investigate the importance of these interactions for the MVM-dependent targeting of p21 by the CRL4Cdt2 ligase we generated stable murine cell lines via lentivirus transduction that conditionally expressed FLAG-tagged wild-type or mutant p21 (p21WT, p21ΔDegron, p21ΔPCNA, mutations shown in Figure 4A) in a doxycycline-responsive manner. As expected, MVM infection of a p21WT expressing cell line resulted in degradation of the tagged p21 (Figure 4C and 4E, compare lanes 2 to 4), which could be prevented by treating cells with the proteasome inhibitor MG132 (Figure S4, panel A), or via siRNA knockdown of CRL4Cdt2 components (Figure S4, panels B and C). These results suggested that the depletion of p21 in these cell lines occurred via a similar mechanism to that of the endogenous p21. The lysine at the p21 +4 position, 3′ to its PIP box, has been reported to be required for interaction of p21 with the CRL4Cdt2 ligase complex via Cdt2. Mutation of the +3 to +5 amino acids KRR to AAA (p21Δdegron) abolished p21 interaction with the CRL4Cdt2 complex following its transient transfection as reflected by loss of interaction with DDB1 (Figure 4B, compare lanes 2 to 3). Murine cell lines that expressed the p21Δdegron mutant were generated, and when infected with MVM, in contrast to cell lines expressing wild-type p21 (p21WT), the p21Δdegron protein was resistant to degradation (Figure 4C, compare lanes 6 and 8 to lanes 2 and 4). This suggested that MVM-induced p21 degradation required interaction of p21 with the CRL4Cdt2 ubiquitin ligase complex.

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