SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

Abstract

Our pioneering studies on the interplay between the small ubiquitin-like modifier (SUMO) and influenza A virus identified the nonstructural protein NS1 as the first known SUMO target of influenza virus and one of the most abundantly SUMOylated influenza virus proteins. Here, we further characterize the role of SUMOylation for the A/Puerto Rico/8/1934 (PR8) NS1 protein, demonstrating that NS1 is SUMOylated not only by SUMO1 but also by SUMO2/3 and mapping the main SUMOylation sites in NS1 to residues K219 and K70. Furthermore, by using SUMOylatable and non-SUMOylatable forms of NS1 and an NS1-specific artificial SUMO ligase (ASL) that increases NS1 SUMOylation ϳ4-fold, we demonstrate that SUMOylation does not affect the stability or cellular localization of PR8 NS1. However, NS1's ability to be SUMOylated appears to affect virus multiplication, as indicated by the delayed growth of a virus expressing the non-SUMOylatable form of NS1 in the interferon (IFN)-competent MDCK cell line. Remarkably, while a non-SUMOylatable form of NS1 exhibited a substantially diminished ability to neutralize IFN production, increasing NS1 SUMOylation beyond its normal levels also exerted a negative effect on its IFN-blocking function. This observation indicates the existence of an optimal level of NS1 SUMOylation that allows NS1 to achieve maximal activity and suggests that the limited amount of SUMOylation normally observed for most SUMO targets may correspond to an optimal level that maximizes the contribution of SUMOylation to protein function. Finally, protein cross-linking data suggest that SUMOylation may affect NS1 function by regulating the abundance of NS1 dimers and trimers in the cell.

Sumoylation of Influenza A Virus Nucleoprotein Is Essential for Intracellular Trafficking and Virus Growth

“…We and others have demonstrated that several viral proteins are sumoylated in transfected and infected cells; we previously demonstrated that NS1 is sumoylated at C-terminal residues in infected cells and that sumoylation stabilizes NS1 for efficient viral replication (8). Another group confirmed our finding that the C-terminal lysine is the major sumoylation site and that sumoylation ensures NS1 function (29). Sumoylation of M1 has also been reported and found to be involved in virus assembly (9).…”

Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication

“…The nonstructural NS1 protein is the first discovered and the most frequent SUMO target of IAV, which is SUMOylated to facilitate viral replication73. Our results showed that the SUMOylation of the small GTP-binding protein Rac1 was competitively inhibited by the viral NS1 protein in the host cell, indicating that IAV profoundly relied on SUMOylation to survive in host cells.…”

Proteomic analysis reveals down-regulation of surfactant protein B in murine type II pneumocytes infected with influenza A virus

“…However, PR8 NS1 has been found to inhibit the IFN response by human dendritic cells by a lesser degree than human isolates of IAV (Haye et al, 2009). Furthermore, the multiple functions of NS1 are regulated by post-translational modification, including phosphorylation, SUMOylation, and ISGylation (Hsiang et al, 2012; Santos et al, 2013; Zhao et al, 2010). It is unknown how these modifications of NS1 might affect IFN antagonism by PR8 specifically in ATII cells.…”