Deciphering HCV-host interactions by identifying substrates of viral-induced selective autophagy

Autophagy is a catabolic process by which cells break down the intracellular components to maintain cellular homeostasis. Autophagic response has long been known to be a bulk and non-selective proteolysis pathway. However, mounting lines of evidence recently indicate that autophagic process can selectively sequestrate and target the damaged organelles and unwanted proteins to degradation via lysosomal proteases. Several viral infections, including hepatitis C virus (HCV), activate autophagy to benefit viral growth in the infected cells. We previously demonstrated that HCV infection induces the complete autophagy throughout to mature autolysosome to promote viral replication via suppressing antiviral innate immunity (Journal of Clinical Investigation, 2011; Autophagy, 2011). However, whether and how the HCV-activated autophagy selectively eliminates intracellular organelles and/or proteins to regulate the viral life cycle and modulate host cellular responses still remain largely unknown. Our current studies demonstrated that HCV infection led to engulfment of lipid droplets (LDs) and mitochondria within autophagic vacuoles. In addition, HCV infection also promoted the degradation of cargo receptor proteins of selective autophagy, including sequestosome 1 (SQSTM1), neighbor of BRCA1 gene 1 (NBR1), nuclear domain protein 52 KDa (NDP52), and optineurin (OPTN). Interference with the autolysosome maturation by pharmacological inhibitors restored the expressions of these cargo receptors and also coincidently inhibited HCV replication in the infected cells. Moreover, the cargo receptor proteins were shown to be sequestrated within the LC3B-labeled autophagosome in the HCV- infected cells. Most importantly, HCV infection led to accumulations of polyubiquitinated proteins within autophagic vacuoles. Therefore, these results not only indicate that HCV infection activates selective autophagy to specifically degrade intracellular organelles and proteins, but also support a notion that selective autophagy plays a vital role in regulating HCV-host cell interactions. The overall goal of this proposal is aim to identify the substrates targeted to the HCV-induced selective autophagy and to investigate the molecular mechanism underlying how these substrates are selectively degraded thorough autophagic process. Finally, we aim to explore the functional role of selective autophagy in the balance of HCV replication and host cellular responses. To this end, we set up four specific aims to completely explore how HCV activates selective proteolysis to selectively eliminate the degradative substrates and to unveil how selective autophagy regulates HCV-host cell interactions. The specific aim I will investigate whether HCV activates selective autophagy to degrade the known autophagic substrates. As to specific aim II, we will comprehensively identify the unknown substrates targeted to HCV-activated selective autophagy. The specific aim III will delineate the molecular basis of how selective autophagy degrades the intracellular substrates in the HCV-infected cells. In the specific aim IV, we will plan to decipher the physiological significance of selective autophagic proteolysis in the maintenance of HCV-host cell interactions. Accomplishment of these studies will promote our knowledge of how cytoplasmic substrates are polyubiquitinated by specific ubiquitin E3 ligases, recognized by cargo receptors of selective autophagy, and targeted to selective proteolysis via HCV-induced autophagic process. Most importantly, the results obtained in this proposal will first uncover a novel mode of how autophagic process maintains the balanced interactions between HCV and host cells through selective proteolysis. In perspective, these results not only promote our understanding on the pathogenesis of HCV-associated liver diseases, but also shed insight into developing a new treatment strategy against HCV infection.

Project ID:PG10501-0482
External Project ID:NHRI-EX105-10322SC