TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles

Abstract

Host-pathogen dynamics are constantly at play during enteroviral infection. Coxsackievirus B (CVB) is a common juvenile enterovirus that infects multiple organs and drives inflammatory diseases including acute pancreatitis and myocarditis. Much like other enteroviruses, CVB is capable of manipulating host machinery to hijack and subvert autophagy for its benefit. We have previously reported that CVB triggers the release of infectious extracellular vesicles (EVs) which originate from autophagosomes. These EVs facilitate efficient dissemination of infectious virus. Here, we report that TBK1 (Tank-binding kinase 1) suppresses release of CVB-induced EVs. TBK1 is a multimeric kinase that directly activates autophagy adaptors for efficient cargo recruitment and induces type-1 interferons during viral-mediated STING recruitment. Positioning itself at the nexus of pathogen elimination, we hypothesized that loss of TBK1 could exacerbate CVB infection due to its specific role in autophagosome trafficking. Here we report that infection with CVB during genetic TBK1 knockdown significantly increases viral load and potentiates the bulk release of viral EVs. Similarly, suppressing TBK1 with small interfering RNA (siRNA) caused a marked increase in intracellular virus and EV release, while treatment in vivo with the TBK1-inhibitor Amlexanox exacerbated viral pancreatitis and EV spread. We further demonstrated that viral EV release is mediated by the autophagy modifier proteins GABARAPL1 and GABARAPL2 which facilitate autophagic flux. We observe that CVB infection stimulates autophagy and increases the release of GABARAPL1/2-positive EVs. We conclude that TBK1 plays additional antiviral roles by inducing autophagic flux during CVB infection independent of interferon signaling, and the loss of TBK1 better allows CVB-laden autophagosomes to circumvent lysosomal degradation, increasing the release of virus-laden EVs. This discovery sheds new light on the mechanisms involved in viral spread and EV propagation during acute enteroviral infection and highlights novel intracellular trafficking protein targets for antiviral therapy.

Porcine Sapovirus Protease Controls the Innate Immune Response and Targets TBK1

“…Here, we show that PSaV infection in TBK1 deficient porcine cells led to a statistically significant 2.6-fold increase in virus titres. This aligns with observations in studies altering TBK1 levels in both human and murine cells, resulting in a comparable effect on virus proliferation [ 43 , 44 ]. Moreover, the absence of other restriction factors similarly leads to a modest rise in virus titres [ 45 ], underscoring the redundancy of pathways in suppressing viral infection, and highlighting the well-orchestrated defence mechanisms against virus proliferation.…”

Mitochondrial extracellular vesicles, autoimmunity and myocarditis

“…The role of fission in the production of mitochondrial vesicles was confirmed by inhibition/blocking mitochondrial fission machinery using mitochondrial division inhibitor-1 (Mdivi-1) or direct inhibition of Drp1 with siRNA which resulted in less viral replication and fewer/no virus containing EVs in the culture supernatant ( 48 ). Dr. Sin’s group additionally showed that Tank binding kinase I (TBK1) increased phosphorylation of GABA type A receptor associated protein-like (GABARAPL) proteins leading to EVs that contain mitochondria being released from the cells ( 59 ). CVB3 infection has also been shown to perturb syntaxin-17 facilitated mitophagosome-lysosomal fusion, which may lead to build up and release of formed mitophagosomes from the cell ( 60 ).…”

“…It turns out that many viruses are known to localize to mitochondria ( 61 – 64 ), utilize mitochondrial machinery for replication ( 48 , 65 ), evade immune responses within EVs ( 66 ) and modify cellular processes ( 59 , 60 , 67 ). Importantly, most of the viruses that are associated with causing myocarditis [e.g., CVB, influenza, HIV, poliovirus, hepatitis C virus, SARS-CoV-22 ( 68 , 69 )] have been found to target mitochondria to gain a replicative advantage ( 61 – 64 ) and are ejected from cells/tissues in EVs ( 60 , 65 , 70 , 71 ) suggesting that these mechanisms may provide an explanation for how viruses could cause autoimmune disease.…”

“…Dr. Sin's group additionally showed that Tank binding kinase I (TBK1) increased phosphorylation of GABA type A receptor associated protein-like (GABARAPL) proteins leading to EVs that contain mitochondria being released from the cells (59). CVB3 infection has also been shown to perturb syntaxin-17 facilitated mitophagosomelysosomal fusion, which may lead to build up and release of formed mitophagosomes from the cell (60).…”