SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection

Abstract

Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.

Analysis of SARS-CoV-2-controlled autophagy reveals spermidine, MK-2206, and niclosamide as putative antiviral therapeutics

“…The ratio of APs to ALs shifted in SARS-CoV-2-infected cells, indicating strong AP accumulation (Mock= 0.707±0.053; SARS-CoV-2= 0.921 ± 0.058) ( Figure 1e ). Taken together, SARS-CoV-2 strongly reduced the autophagic flux in both cell lines, in a fashion similar to MERS-CoV ( 10 ).…”

“…During autophagy, intracellular macromolecules are recycled by incorporation into LC3B-lipidated autophagosomes (AP) and degradation into their monomers, such as fatty and amino acids, after fusion with low pH lysosomes ( 9 ). In the case of highly pathogenic Middle East respiratory syndrome (MERS)-CoV, we recently showed that autophagy is limited by a virus-induced AKT1-dependent activation of the E3-ligase S-phase kinase-associated protein 2 (SKP2), which targets the key autophagy initiating protein Beclin-1 (BECN1) for proteasomal degradation ( 10 ). Congruently, inhibition of SKP2 by different compounds, including clinically approved drugs, stabilized BECN1 and limited MERS-CoV propagation, indicating that autophagy-inducing compounds hold promise for evaluation as antiviral drugs.…”

“…We aimed to characterize the effect of SARS-CoV-2 infection on autophagy by applying previously established assays ( 10 ) and additionally including detailed analyses of upstream autophagy regulators according to expert-curated guidelines for detecting autophagy ( 11 ). First, we explored whether SARS-CoV-2 reduces autophagic flux, a measure of autophagic degradation activity.…”

“…BafA1 is a specific inhibitor of vacuolar H + -ATPase interfering with lysosome acidification and degradation of AP cargo and preventing the fusion of APs with lysosomes to autophagy-active autophagolysosomes (AL) ( 11 ). Based on our previous experience ( 10 ), we chose a low MOI of 0.0005 and time points 8, 24, and 48 hours post infection to monitor autophagy during the exponential growth of SARS-CoV-2 reaching maximum titers of 10 7 genome equivalents per ml (GE/ml; NCI-H1299) and 10 10 GE/ml (VeroFM) at 48 hours post infection ( Figure S1a ). We found that 100 nM of BafA1 was sufficient to induce maximal LC3B lipidation levels in both cell cultures ( Figure S1b-d ).…”

Targeting the Endocytic Pathway and Autophagy Process as a Novel Therapeutic Strategy in COVID-19

“…For instance, a study using SARS-CoV and MERS-CoV in HEK293T, HeLa and MCF-7 cells found that overexpression of membrane-associated papainlike protease PLP2 (PLP2-TM) of SARS-CoV and MERS-CoV led to blockage of autophagosomeslysosomes fusion and suppression of the autophagic flux [31]. Consistently, a more recent report found that MERS-CoV blocks the fusion of autophagosomes and lysosomes and induction of autophagy reduces the replication of MERS-CoV [32]. Thus, it appears that there is certain type of interplay between the autophagy machinery and CoVs, and the exact nature of such interaction remains to be further elucidated.…”