Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

Abstract

Although classic swine fever virus (CSFV) infection has been reported to induce autophagy, the specific induced mechanism remains unrevealed. Nonstructural protein 5A (NS5A) of CSFV is a multiphosphorylated protein with multiple functions to regulate viral replication and the host cell immune responses. Herein, we demonstrated that CSFV NS5A could induce cellular autophagy and promote viral replication. In the current study, we showed that NS5A expression significantly increased the levels of autophagy-related genes (ATGs), including light chain 3 (LC3), ATG5, and Beclin 1; conversely, degradation of P62/sequestosome 1 (SQSTM1) was observed by Western blotting. The number of autophagy-like vesicles was also obviously increased in NS5A-expressing cells, as analyzed by transmission electron microscopy (TEM). Furthermore, we observed the co-localization of the NS5A and LC3 proteins by confocal immunofluorescence analysis. Direct binding of NS5A to the autophagy-related LC3 protein was confirmed by coimmunoprecipitation in vivo and by a GST pulldown assay in vitro. Through segmentation and point mutation research on the NS5A protein, we found that the N-terminal region and the phosphorylation of amino acids 81 and 92 of the NS5A protein were essential for inducing autophagy. Finally, we demonstrated that the LC3 protein had a positive effect on CSFV replication. These findings emphasize a previously unascertained interaction relationship between NS5A and LC3 in the autophagy process. Furthermore, our research revealed a new role of CSFV NS5A, particularly its N-terminal amino acids serine 81 and serine 92, as a critical regulator of CSFV-induced autophagy and have significance for extending our understanding of the CSFV-autophagy interplay.

Pestiviruses infection: Interferon-virus mutual regulation

“…Jefferson et al demonstrated that pestivirus N pro curbed endogenous mitochondrial apoptosis by inhibiting the activation of the IRF3-dependent pro-apoptotic protein Bcl2-Associated X (BAX), indicating that mitochondria and peroxisomes may be new sites for N pro to regulate IRF3 (Jefferson et al, 2014). Additionally, pestivirus infection induced autophagy to promote virus replication (Pei et al, 2014;Oguejiofor et al, 2019;Zhang et al, 2021). Explored the mechanism and found that CSFV-mediated autophagy restrained the expression of IFN-I.…”

The Multi-Faceted Role of Autophagy During Animal Virus Infection

“…For example, by binding to LC3, the NS5A protein increases the expression of ATGs and activates complete autophagy, thus promoting virus replication. Notably, the phosphorylation of NS5A at 81 and 92 aa within its Nterminal region is essential for activating autophagy (Zhang et al, 2021a). Further, NS3 directly binds to lactate dehydrogenase B (LDHB), a key glycolysis metabolic enzyme that catalyzes the transformation of pyruvate and lactic acid in the anaerobic glycolysis pathway and inhibits LDHB, activating mitochondrial autophagy (Fan et al, 2021).…”

HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

“…For instance, a few studies indicate that NS5A protein interacts with eukaryotic translation initiation factor 3 subunit E (eIF3E), ras-related protein 18 (Rab-18), glucose-regulated protein 78 (GRP78), heat shock protein 70 (Hsp70) and so on to facilitate viral replication (10)(11)(12)(13). Recent studies revealed that NS5A induces autophagy to enhance replication of CSFV (14,15). It has also been shown that CSFV NS5A protein could inhibit the secretion of inflammatory cytokines by suppressing the NF-kB pathway (16).…”