Study On The Mechanism Of AP1 Complex In HCV Infection

Hepatitis C virus (HCV) chronically infects almost 150 million people worldwide and is a leading cause of liver cirrhosis, end-stage liver disease and hepatocellular carcinoma. Virus life cycle depends on the interactions between viral elements and host factors. Essential host factors for HCV entry and replication were characterized extensively, such as virus receptors and replication dependency factors. However, the mechanisms of host regulation on HCV protein trafficking, assembly and release at the late stage of HCV life cycle remain incompletely understood.In this study, using a human 50k shRNA library, we systematically screened the host critical factors related to HCV infection, and 206 potential factors were identified. Among the 15 selected candidates, AP1S3 was identified as the most significant for its association with intracellular protein trafficking and was selected for functional validation.Adaptor protein (AP) complexes are trafficking heterotetramers that sort transmembrane proteins by assembling small transport vesicles in the cytosol. AP1 contains four subunits, two large (y and β1), one medium (μ1), and one small (σ1), which exist several isoforms encoded by different genes, including twoγy (AP1G1 and AP1G2), two μ1 (AP1M1 and AP1M2), and three σ1 (AP1S1, AP1S2, and AP1S3). AP1 mediates transport between the trans-Golgi network (TGN) and endosomes by recognizing PI4KIIa and-b, meanwhile, AP2 facilitates endocytic events at the sites of envelopment. Many viruses hijack the intracellular trafficking functions of API for assembly and budding, including human immunodeficiency virus (HIV) and African swine fever virus (ASFV).To analyze the role of AP1S3 throughout the HCV life cycle, different virus models were applied, including HCV pseudo-particles (HCVpp) platform, HCV replicon system (2-3+) and HCV assemble and release experiment. According to the results, silencing AP1S3 significantly decreased the production and infectivity of HCV progeny particles. And the other subunits of AP1 complex (AP1B, AP1M1 and AP1S1) could lead to similar effects of anti-virus.To determine the mechanism of API in HCV infection, Immunoprecipitation analysis was performed and revealed that API interacted with the HCV core and E2 proteins by distinct subunits via two conserved motifs, YXXO and (D/E)XXXL(L/I). With these interactions, AP1 could protect HCV core and E2 from proteasomal degradation. Knocking-down AP1 enhanced the poly-ubiquitination level of both HCV core and E2. Using in vivo and in vitro ubiquitylation assay, we identified that the E3 ubiquitin ligase E6 associated protein (E6AP) was not noly associated with HCV core, but also HCV E2, and mediated their ubiquitylation.AP1 is also involved in the regulation of different viral envelope proteins during the viral life cycle. Four different viral envelope proteins were selected for degradation experiments, including two flaviviridae virus Japanese encephalitis virus (JEV) and West Nile virus (WNV) and two non-flaviviridae virus Ebola virus (EBOV) and Influenza. According to the results, silencing AP1 decreased the expression of several virus envelope proteins, particularly those that are highly glycosylated, such as influenza hemagglutinin and Ebola virus glycoprotein. In addition, since the YXXΦ and [D/E]XXXL[L/I] motifs are essential for AP1-core/E2 binding, we synthetized a peptide by ligating a TAT domain with a core YXXΦ motif and a E2 [D/E]XXXL[L/I] motif to test its inhibition of HCV. Treatment with synthetic peptides dramatically inhibited HCV infection in Huh7.5.1 cells.Together, AP1 represents a novel host network that is required by viruses during infection provides a potential target for developing broad-spectrum anti-virus strategies.