Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway

Area of Science:

• Virology
• Oncology
• Molecular Biology

Background:

• Polyomaviruses are dsDNA viruses implicated in cancer, with alternative splicing producing tumor antigens like large (LT) and small (ST) tumor antigens.
• Some polyomaviruses express middle tumor antigens (MTs) or alternate LT open reading frames (ALTOs), which have distinct gene structures and roles.
• Merkel cell polyomavirus (MCPyV) encodes an ALTO, but its function in viral lifecycle and cancer development remained unknown.

Purpose of the Study:

• To investigate the role of MCPyV ALTO in the viral lifecycle and tumorigenesis.
• To determine the mechanism by which MCPyV ALTO influences cancer cell growth and signaling pathways.
• To explore the evolutionary conservation and function of ALTOs in other polyomaviruses.

Main Methods:

• Functional assays in Merkel cell carcinoma (MCC) cells to assess ALTO's impact on cell growth and signaling.
• Biochemical analyses to identify ALTO's interaction partners, such as SQSTM1 and TRAF2&3.
• Electrophoretic mobility shift assays (EMSAs) and reporter assays to study NF-κB activation and binding to the viral noncoding control region (NCCR).
• Comparative analysis of ALTO and MT sequences across different polyomaviruses.

Main Results:

• MCPyV ALTO functions as a tumor suppressor and is silenced in MCC.
• Restoring MCPyV ALTO expression in MCC cells induced growth arrest and activated NF-κB signaling.
• ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via N-Terminal Activating Regions (NTAR1+2), similar to EBV LMP1.
• Activated NF-κB downregulates viral early transcription by binding the MCPyV NCCR.
• Conserved NTAR motifs in other polyomavirus ALTOs mediate NF-κB activation and viral transcription downregulation, a feature absent in MTs.

Conclusions:

• MCPyV ALTO acts as a tumor suppressor, and its silencing is a critical step in MCC development.
• ALTOs evolved to suppress viral replication and promote latency, utilizing NF-κB activation via NTAR motifs.
• Understanding ALTO function provides insights into polyomavirus-associated cancers and potential therapeutic strategies.