LAUSR

Dear Editor, The outbreak of SARS-CoV-2 leads global epidemic with high morbidity and mortality. However, the pathophysiology of this deadly virus is complex and largely unknown. Autophagy is a highly conserved homeostatic process that allows cells to recycle their components. Several studies provided evidence that human coronavirus infections are closely related to various cellular aspects associated with autophagy. Autophagy may play a crucial role in the SARS-CoV-2 viral lifecycle. In order to investigate whether the autophagy is altered in response to SARS-CoV-2 infection, we infected GFP-LC3 transfected Vero-E6 cells at MOI of 0.05. In comparation to uninfected cells, SARS-CoV-2 infected cells showed a strong increase of GFPLC3 positive autophagosomes (Fig. 1a). This was also observed in SARS-CoV-2 infected Huh7.0 cells (Supplementary Fig. S1a). The enhanced autophagosome formation was also validated by detecting lapidated LC3-II increased by SARS-CoV-2 infection at 12, 24, and 48 h postinfection (hpi) in Vero-E6 cells (Fig. 1b), Huh7.0 cells (Supplementary Fig. S1b), and Caco-2 cells (Supplementary Fig. S1c). Ultrastructural analysis of SARS-CoV-2 infected Vero-E6 cells by transmission electron microscopy further substantiated the formation of autophagy (Fig. 1c). To determine whether individual SARS-CoV-2 protein can induce autophagy, a series of SARS-CoV-2 nonstructural proteins (NSP5, NSP7, NSP8, NSP9, NSP10, NSP12, and NSP13), and main structural proteins (E, M, S, and N) were investigated. The data showed individual nonstructural protein was not able to induce autophagy (Fig S2a). However, M protein could greatly increase the LC3-II formation in Huh7.0 cells (Fig. 1d), and this increase was in a dose-dependent manner (Fig. S2b). Besides, significant increase of GFP-LC3 positive autophagosomes was also found in M-transfected Huh7.0 cells (Supplementary Fig. S2c). Interestingly, when we extended the time after M transfection, we found that M-induced LC3-II peaked at 24 hpt, and then gradually declined, and M seemed to decline along with LC3-II (Fig. S2d). We proposed that the decrease of LC3-II in the late time may be due to the degradation of autophagosome. To validate this, the cells were transfected with M for 24 h and then were treated with CQ for another 12 h to block the autophagosome degradation by lysosome. We found that the LC3-II in CQ-treated cells was strongly increased compared with control (Fig. S2e). It is worth noting that the content of M seems to be closely related to that of LC3-II. Therefore, we assumed that M may be an interaction partner of LC3. The co-immunoprecipitation assay showed that Flag-tagged M can immunoprecipitate GFP-LC3 (Fig. 1e); vice versa, GFP-LC3 can immunoprecipitate Flag-M (Fig. 1e). Endogenous LC3 was also immunoprecipitated by Flag-M in HEK293T cells (Fig. S2f). This collective data show that M is a binding partner of LC3. Usually, binding partners of LC3 family members typically contain an LC3-interacting region (LIR). LIRs form intermolecular β sheets with LC3 family members by virtue of a consensus W/ Fxxl/L motif. We found that a classical WxxL motif exists in M that is most closely corresponds to those found in p62 and ATG13 (Supplementary Fig. S2g). We then made several mutants in LIR motif to determine their interactions with LC3, and found that the interaction of LC3 with M-W31A, M-L34A, M-W31A/L34A, and M with the LIR deletion (M-△LIR) greatly decreased (Supplementary Fig. S2h). However, M-△LIR was still able to promote the autophagy formation, but this effect was attenuated compared with WT-M overexpression (Supplementary Fig. S2i, j). This indicates the LIR motif in M is critical for the interaction with LC3, and the LC3-II formation, but is not necessary for inducing autophagy. Autophagy can remove organelles upon recognition of autophagic receptors. Therefore, we sought to classify which type of selective autophagy SARS-CoV-2 induced. Transiently expressed M leaded to a significantly decrease of TOM20, TIM23, and p62 in a time dependent manner, but not PA28 (proteasome), L7a (ribosome), and Calnexin (endoplasmic reticulum) (Supplementary Fig. S3a). Besides, we find that M did not alter mitochondrial ROS and dissipate the mitochondrial membrane potential (Supplementary Fig. S6a, b), both of which are closed associated with autophagy. The decrease of the two mitochondrial marker proteins TOM20 and TIM23 were validated after SARS-CoV-2 infection (Supplementary Fig. S3b, c), indicating the turnover of mitochondria. Further result showed that RFP-LC3 was colocalized well with GFP-BID, a mitochondrial marker protein, in SARS-CoV-2 infected cells (Fig. 1f), which suggested mitochondria may be targeted by autophagosome. Because M can induce autophagy, we then determined if M could promote mitophagy. We found that in M-overexpressed Huh7.0 cells, RFP-LC3 was colocalize with GFP-BID. Importantly, we found that M partly colocalized with GFP-BID (Fig. 1g). Besides, our results clearly showed the colocalization of M and mitochondria (mitotracker probes and TOM20 antibody to mark mitochondria) (Supplementary Fig. S3d, e), implying that M may translocate to mitochondria. This was further demonstrated by mitochondrial components separation (Fig. 1h). Of note, in M-expression cells, the level of LC3 in mitochondrial components was significantly higher than control cells, while TOM20 and TIM23 decreased. M-△LIR was also enriched in mitochondrial components, but TOM20 and TIM23 were not significantly changed compared with WT-M (Supplementary Fig. S3f). Mitochondria is a critical platform to converge antiviral innate immune signaling. Therefore, we attempted to determine whether M protein can disrupt type I interferon response. We found M overexpression significantly inhibited SeV-mediated IFN-β activation in a dose-dependent manner (Fig. 1i). Further, we determined that M blocked RIG-I and MAVS, but not TBK1, IKKε, and IRF3-5Dtriggered IFN-β promoter activation (Fig. 1j). It indicated that the inhibitory effect of M on IFN-β activation is mainly through MAVS. As expected, M-△LIR, exhibited an slightly weaker inhibitory effect