Identification of the Merkel Cell Polyomavirus Large Tumor Antigen Ubiquitin Conjugation Residue

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.

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α‐dependent nuclear localization signals

“…Integration of the viral genome usually occurs upstream of the coding sequence for the LTA helicase domain coding sequence, resulting in a defective viral genome, unable to replicate but still able to sustain cellular proliferation by binding to the Rb protein (Houben et al, 2010 ). In a considerable number of such clones, integration occurs between the NLSm and NLSct coding sequences, generating a truncated LTA fragment whereby NLSct (and thus the bipartite NLS) is destroyed, but NLSm is preserved (Ortiz et al, 2021 ). Therefore, while full‐length MCPyV LTA can be imported to the nucleus in high levels and via multiple IMPα isoforms using a bipartite cNLS, truncated LTA fragments can still interact with the IMPα minor binding site through NLSm (Figure 8H , Figure S6 ) and be actively transported into the nucleus (Figure 8E–G ).…”

A comparative analysis of polyomaviruses Large Tumor Antigens reveals evolution of different importin α-dependent nuclear localization signals

“…Integration of the viral genome usually occurs upstream of the coding sequence for the LTA helicase domain, resulting in a defective viral genome unable to replicate, but still able to sustain cellular proliferation by binding to the Rb protein (57). In a considerable number of such clones, integration occurs between the NLSm and NLSct coding sequences, generating a truncated LTA fragment whereby NLSct (and thus the bipartite NLS) is destroyed, but NLSm is preserved (58). Therefore, while full-length MCPyV LTA can be imported to the nucleus in high levels and via multiple IMPa isoforms thanks to a bipartite cNLS, truncated LTA fragments can still interact with the IMPa minor binding site through NLSm (Fig.…”

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

“…If the immunoblotting result suggested ubiquitination on the substrates, the putative ubiquitinated lysine was further mutated and analyzed by immunoblotting to evaluate whether the mutated lysine was ubiquitinated or not [30,31]. Using this kind of strategy, Ortiz et al found that the ubiquitination level of Merkel cell polyomavirus large tumor (LT) antigen was significantly reduced when K585 was substituted with R585, indicating that K585 is the ubiquitination site [32]. The conventional approaches are the most widely used ones for the detection and validation of ubiquitination for a single protein.…”