HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest

Abstract

Background: MCM10 regulates initiation of eukaryotic genome replication. Results: HIV-1 Vpr enhances proteasomal degradation of MCM10. Conclusion: HIV-1 Vpr-mediated degradation of MCM10 is essential for induction of G 2 /M cell cycle arrest. Significance: Our study reveals a novel protein target for HIV-1 Vpr whose degradation is involved in the induction of G 2 /M cell cycle arrest by the virus.

HIV-1 Vpr Protein Induces Proteasomal Degradation of Chromatin-associated Class I HDACs to Overcome Latent Infection of Macrophages

“…By binding to VprBP, Vpr enhances degradation of the natural substrates recruited by VprBP (10,13). Furthermore, minichromosome maintenance complex component 10 (MCM10) was recently shown to be the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase (14) and our laboratory showed that proteasomal degradation of MCM10, is also enhanced by Vpr (10). It is believed that Vpr induces most of its functions by interacting with VprBP.…”

“…pWPI-Flag-Vpx was constructed by cloning of Vpx from simian immunodeficiency virus (clone pPBj 1.9) of sooty mangabeys into pWPI using a strategy previously described (10). To generate an expression vector for HIV-1 Vpr, the vpr gene of HIV Gag-iGFP was cloned into pcDNA3.1.…”

“…TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program. Doxycycline-inducible HeLa cell lines (HeLa-iFlag-Vpr, HeLa-iFlag-Q65R) and the control cell line (HeLa-iMock) were described previously (10). Expression of Flag-Vpr was induced by adding 1 g/ml doxycycline.…”

“…Vectors and Virus Constructs-The lentiviral vectors pWPI, pWPI-Flag-Vpr, pWPI-Flag-Q65R, pWPI-Flag-R80A, and their packaging plasmids pCMV-VSV-G and psPax2 were described previously (10). pWPI-Flag-Vpx was constructed by cloning of Vpx from simian immunodeficiency virus (clone pPBj 1.9) of sooty mangabeys into pWPI using a strategy previously described (10).…”

“…Immunoprecipitated proteins and cellular fractions (30 g) were resuspended in Laemmli buffer, heat-denatured for 5 min, and separated on 12% SDS-PAGE gels. Western blots were performed as described previously (10).…”