Citation

  • Authors: Ortiz LE. et al.
  • Year: 2021
  • Journal: Int J Mol Sci 22 7169
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell type: HEK-293
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293, 293

Method

HEK293 cells were cultured in DMEM with 10% premium grade fetal bovine serum (FBS). Transfections with expression vectors were performed using jetOPTIMUS (Polyplus Transfection, New York, NY, USA) according to the manufacturer’s instructions. HEK293 cells were transfected with an LT expression vector (LT.wt) and Rep+ (MCPyV ER transcription reporter) using jetOPTIMUS (Polyplus Transfection) in 12-well plates. Episomal DNA was collected by salt-precipitation at 48 h post-transfection.

Abstract

Merkel cell polyomavirus (MCPyV) large tumor (LT) antigen is a DNA binding protein essential for viral gene transcription and genome replication. MCPyV LT interacts with multiple E3 ligases in a phosphorylation-dependent manner, limiting its own viral replication by enhancing LT protein degradation, which is a unique mechanism for MCPyV latency. Thus, identifying LT ubiquitination sites is an important step toward understanding the biological role of these virus-host interactions that can potentially result in viral oncogenesis. The ubiquitin (Ub) attachment sites in LT were predicted by using Rapid UBIquitination (RUBI), a sequence-based ubiquitination web server. Using an immunoprecipitation approach, the lysine (Lys, K) 585 residue in LT is identified as the ubiquitin conjugation site. Lysine 585 is deleted from tumor-derived truncated LTs (tLTs), resulting in stable expression of tLTs present in cancers. Substitution of lysine 585 to arginine (Arg, R) increased LT protein stability, but impaired MCPyV origin replication, due to a loss of ATP hydrolysis activity. These findings uncover a never-before-identified ubiquitination site of LT and its importance not only in the regulation of protein turnover, but also in MCPyV genome replication.

Pubmed