Coxsackievirus infection induces a non-canonical autophagy independent of the ULK and PI3K complexes

Abstract

Coxsackievirus B3 (CVB3) is a single-stranded positive RNA virus that usurps cellular machinery, including the evolutionarily anti-viral autophagy pathway, for productive infections. Despite the emergence of double-membraned autophagosome-like vesicles during CVB3 infection, very little is known about the mechanism of autophagy initiation. In this study, we investigated the role of established autophagy factors in the initiation of CVB3-induced autophagy. Using siRNA-mediated gene-silencing and CRISPR-Cas9-based gene-editing in culture cells, we discovered that CVB3 bypasses the ULK1/2 and PI3K complexes to trigger autophagy. Moreover, we found that CVB3-induced LC3 lipidation occurred independent of WIPI2 and the transmembrane protein ATG9 but required components of the late-stage ubiquitin-like ATG conjugation system including ATG5 and ATG16L1. Remarkably, we showed the canonical autophagy factor ULK1 was cleaved through the catalytic activity of the viral proteinase 3C. Mutagenesis experiments identified the cleavage site of ULK1 after Q524, which separates its N-terminal kinase domain from C-terminal substrate binding domain. Finally, we uncovered PI4KIIIβ (a PI4P kinase), but not PI3P or PI5P kinases as requisites for CVB3-induced LC3 lipidation. Taken together, our studies reveal that CVB3 initiates a non-canonical form of autophagy that bypasses ULK1/2 and PI3K signaling pathways to ultimately converge on PI4KIIIβ- and ATG5–ATG12–ATG16L1 machinery.

The papain-like protease of coronaviruses cleaves ULK1 to disrupt host autophagy

“…To exclude the involvement of 3CL pro , we performed in- vitro cleavage assay. Time-course treatment revealed that ULK1 was not targeted by SARS-CoV-2 3CL pro , whereas coxsackievirus 3C pro , the positive control [ 21 ], demonstrated efficient cleavage of ULK1 ( Fig. 2 D).…”

In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

“…Our experiments revealed that inhibition of RIPK1, as well as inhibition of RIPK3, led to a reduction of viral replication of approximately 50%, indicating that activity of both kinases is required for viral replication. For intestinal epithelial cells, it was already shown that RIPK3 functions as a positive regulator of CVB3 replication, playing a role in regulation of autophagy, a process that is utilized by CVB3 for replication [22,38]. Although RIPK3 may be involved in the facilitation of CVB3 replication, it is nevertheless a key player of the necroptosis pathway, which may be detrimental to CVB3 replication, especially if the cells die too early before replication is successfully completed.…”

SNAP47 Interacts with ATG14 to Promote VP1 Conjugation and CVB3 Propagation

“…Cell fractionation revealed that SNAP47 and VP1 (a wellknown membrane-associated protein) were enriched on the membrane fractions (Figure 4B). Recent evidence supported the idea that CVB3-induced autophagy bypassed the requirement for canonical autophagy factors, such as the unc-51-like kinase (ULK1) and beclin-phosphatidylinositol 3-kinase (PI3K) complexes, which are crucial for basal and starvation-induced autophagy [16,17]. To test whether SNAP47 functioned as a noncanonical factor participating in CVB3-induced autophagy, we infected SNAP47-KO and WT cells with CVB3.…”

“…However, enteroviruses (EVs), such as CVB3, have evolved to subvert this pathway for pro-viral purposes [5]. It was observed that CVB3 can induce autophagy independent of the canonical initiation complex and subsequently utilize the autophagosome membranes as scaffolds for viral replication [16,17]. CVB3 can also prevent the maturation of the autophagosome by limiting autophagosome-lysosome fusion [18,19], thereby generating very large autophagy-related structures, termed megaphagosomes [20,21].…”