A novel inducible expression system to study transdominant mutants of HIV-1 Vpr.
In this study, an episomal system for ecdysone-inducible gene expression was developed. Human embryonic kidney 293 cells (293VE) expressing a heterodimer of modified ecdysone and retinoid X receptors and the Epstein-Barr nuclear antigen-1 were screened. Plasmids containing the EBV replication origin, oriP, and the ecdysone-response element could replicate and persist in 293VE cells to inducibly express luciferase or Vpr. The induction level, tested with luciferase reporter plasmid, varied among cell lines from 254- to 2056-fold. In one highly inducible cell line, HIV-1 Vpr was expressed well and caused G2 cell cycle arrest in the presence of the inducer, while in the absence of the inducer, no Vpr protein or cell cycle arrest could be detected. Using different selection markers, HIV-1 Vpr was coexpressed with Vpr mutants defective in phosphorylation at Ser79 and G2 cell cycle arrest activity. These Vpr mutants were transdominant to wild-type Vpr for G2 cell cycle arrest activity, but did not alter wild-type Vpr phosphorylation. It is likely that the transdominant mutants and wild-type Vpr compete for a downstream target(s) of G2 cell cycle arrest.
Zhou
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Y
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Ratner
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L
D000595:Amino Acid Sequence; D002460:Cell Line; D019281:Dimerization; D004440:Ecdysone / Q000494:pharmacology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000494:pharmacology; D005434:Flow Cytometry; D016195:G2 Phase; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D016336:Gene Products, vpr / Q000096:biosynthesis / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D004854:Herpesvirus 4, Human; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D017510:Protein Folding; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000494:pharmacology; D014162:Transfection; D054325:vpr Gene Products, Human Immunodeficiency Virus
D019309:Epstein-Barr Virus Nuclear Antigens; D016336:Gene Products, vpr; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D054325:vpr Gene Products, Human Immunodeficiency Virus; D004440:Ecdysone