Efficient Parvovirus Replication Requires CRL4Cdt2-Targeted Depletion of p21 to Prevent Its Inhibitory Interaction with PCNA

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.

Minute Virus of Mice Inhibits Transcription of the Cyclin B1 Gene during Infection

“…However, both proteasome and protease inhibitors had only a negligible ability to restore cyclin B1 levels during infection (data not shown). Additionally, efficient small interfering RNA (siRNA) knockdown (done as previously reported [24]) of the CDC20 E3 ubiquitin ligase, which targets cyclin B1 for ubiquitination during G 1 , also had no effect on the accumulation of cyclin B1 in MVM-infected cells (data not shown). However, analysis of RNAs from the 18-h and 24-h samples from the experiment shown in Fig.…”

“…Infections were carried out at a multiplicity of infection (MOI) of 10 unless otherwise indicated. Lentivirus constructs designed to express cyclin B1 were generated by cotransfecting equal concentrations of HIV Gag/Pol, vesicular stomatitis virus glycoprotein G (VSV-G), and pINDUCER20 plasmids containing versions of the cyclin B1 gene, as described previously, into HEK293T cells (24,43). Stable doxycycline-inducible A9 cell lines were generated by infection of A9 cells with a pseudotyped pINDUCER20 virus.…”

“…A9 cells were parasynchronized in G 0 by isoleucine deprivation, as previously described (44), and then infected with MVMp at the time of release into complete medium. Entry into S phase occurs approximately 12 h after release into complete medium (24). Virally infected cells harvested at 24 h thus represent an approximately 12-h transit into S phase.…”

“…The FLAG-tagged wild-type cyclin B1 gene was cloned into pDONR221 (Invitrogen) and pINDUCER20 by use of BP and LR Clonase kits (Invitrogen), respectively. pINDUCER reagents were a gift from Guang Hu (NIH/NIEHS) (24,43). pcDNA-dCas9-HA was produced by Charles Gersbach (Addgene plasmid 61355) (45).…”

“…At 15 h postrelease, cells were treated with 200 nM doxorubicin as indicated. At the indicated time points (18,24, and 32 h postinfection), cells were processed as described in Materials and Methods for ChIP using antibodies directed against FoxM1. Samples were analyzed by qPCR as described in Materials and Methods.…”