NS5A Promotes Constitutive Degradation of IP3R3 to Counteract Apoptosis Induced by Hepatitis C Virus

Abstract

SUMMARY FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3’s degron in the absence of inositol 1,4,5-trisphosphate (IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85β, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppression of HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.

GGTase3 is a newly identified geranylgeranyltransferase targeting a ubiquitin ligase

“…PTAR1 has long been neglected until it was identified as a gene product required for optimal fitness of cultured cells in a genome-scale screen aimed at defining essential human genes 46 . FBXL2 is a pro-survival gene, whose product, in response to mitogens, inhibits apoptosis by promoting the activation of the PI3K-AKT pathway and by inhibiting mitochondrial calcium overloading 27–29 . Herein, we have shown physical and functional connections between PTAR1 and FBXL2, thereby delineating a post-translational modification cascade critical for cell survival.…”

“…Immunoprecipitation of “Click-IT” lysates were performed under denaturing conditions. Geranylgeranylated FBXL2 was detected with streptavidin covalently conjugated to horseradish peroxidase (STREP-HRP) (Pierce#21126) followed by immunoblotting, as previously described 27,29 .…”

“…FBXL2 has been shown to be geranylgeranylated 26 and, based on the sequence of its CaaX motif, is predicted to be a GGTase1 substrate. We have previously shown that the integrity of the CaaX motif is necessary for FBXL2 to assemble into an active SCF ubiquitin ligase complex and interact with two substrates localized at cellular membranes, p85β, a regulatory subunit of the PI3-kinase (PI3K), and IP3 (inositol 1,4,5-trisphosphate) receptor type 3 (IP3R3) 27–29 . Other independent studies have also reported membrane-localized substrates for both FBXL2 and FBXL20 23,30–32 .…”

“…Other independent studies have also reported membrane-localized substrates for both FBXL2 and FBXL20 23,30–32 . By promoting the degradation of p85β and IP3R3 and, consequently, regulating PI3K signaling and calcium flux from the ER to the mitochondria, FBXL2 plays a critical pro-survival role 27–29 .…”

Control of mTOR signaling by ubiquitin

“…Via these mechanisms, Fbxl2 ensures sufficient activation of the PI3K signaling cascade and promotes cell survival. Intriguingly, viruses (such as hepatitis C) hijack these regulatory mechanisms to inhibit virus-induced apoptosis and establish chronic infection by binding and unmasking IP3R3 degron 13 . In another report, p85 is recruited to HSP70/CHIP-E3 ligase via p42 for ubiquitination and degradation 14 .…”