Hepatocyte Nuclear Factor 4α and Downstream Secreted Phospholipase A ₂ GXIIB Regulate Production of Infectious Hepatitis C Virus

Abstract

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. The life cycle of HCV is closely associated with the metabolism of lipids, especially very-low-density lipoprotein (VLDL) in hepatocytes. Hepatocyte nuclear factor 4␣ (HNF4␣), the most abundant transcription factor in the liver, regulates the VLDL secretory pathway. However, the effects of HNF4␣ on the HCV life cycle are unclear. In this study, we investigated the regulatory effects of HNF4␣ on HCV assembly and secretion. HCV in HNF4␣-deficient hepatocytes showed reduced assembly and secretion but unchanged entry and RNA replication. Bezafibrate, a chemical inhibitor of HNF4␣, suppressed HCV assembly and secretion. HNF4␣ downregulation resulted in rearrangement of cytosolic lipid droplets (LDs), as evidenced by the aggregation of large LDs and distorted cytosolic distribution. Phospholipase A 2 GXIIB (PLA 2 GXIIB), an HNF4␣-regulated factor involved in VLDL secretion, was found to be crucial in HCV secretion. PLA 2 GXIIB expression was upregulated in hepatocytes harboring HCV subgenomic replicons or in HCV-infected hepatocytes. This upregulation was transcriptionally controlled in an HNF4␣-dependent manner after HCV infection. Furthermore, PLA 2 GXIIB combined with microsomal triglyceride transfer protein was found to be responsible for the regulation of HNF4␣-induced HCV infectivity. These results suggest that HNF4␣ and its downstream PLA 2 GXIIB are important factors affecting the late stage of the HCV life cycle and may serve as potential drug targets for the treatment of HCV infection. IMPORTANCEThe assembly and secretion of hepatitis C virus (HCV) are closely correlated with the very-low-density lipoprotein (VLDL) secretory pathway. However, the molecular mechanism by which HCV cooperates with VLDL to facilitate assembly and release is unclear. In this study, we report the function of hepatocyte nuclear factor 4␣ (HNF4␣), the most abundant liver-enriched transcription factor, in HCV assembly and release. HNF4␣ is crucial in VLDL-mediated lipid transport. We found that HNF4␣ abundance was increased upon HCV infection and that HNF4␣ downregulation impaired the assembly and secretion of HCV. We also determined that PLA 2 GXIIB, a direct target factor of HNF4␣ involved in VLDL secretion, was upregulated upon HCV infection. These results suggest that PLA 2 GXIIB is required in the late stage of the HCV life cycle and that the VLDL secretory pathway is important for HCV assembly and secretion. This study provides insights into the mechanism by which HCV cooperates with HNF4␣ to be assembled into and released together with VLDL particles.

Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation

“…4B). Next, shRNA-transduced cells were infected with HCV JFH1 at a low MOI of 0.02, ensuring multiple cycles of infection, which could have a comprehensive effect on the viral life cycle (41)(42)(43). At 72 hpi, we found that silencing of vinexin ␤ (2 sh Vin␤ and 3 sh Vin␤) reduced the intracellular HCV RNA level to 44.7 and 32.7% of that in control (sh NT) cells, respectively (Fig.…”

Cell death-inducing DFFA-like effector B contributes to assembly of hepatitis C virus (HCV) particles and interacts with HCV NS5A

“…To further investigate whether CIDEB is involved in the late stage of the HCV life cycle, we calculated the specific infectivity (infectious titer divided by the HCV RNA copy number) and the ratio of the extracellular HCV titer to the intracellular HCV titer, as previously described 18,26 . The specific infectivity was calculated to evaluate the efficiency of HCV assembly 36 , whereas the ratio of the extracellular titer to the intracellular titer was used to evaluate the level of viral particle secretion 18,26 .…”

“…The specific infectivity was calculated to evaluate the efficiency of HCV assembly 36 , whereas the ratio of the extracellular titer to the intracellular titer was used to evaluate the level of viral particle secretion 18,26 . Results showed that similar to siApoE, siCiDeB significantly reduced extracellular HCV specific infectivity whereas siCD81 had no effect (Fig.…”

“…Lentiviral particles were produced as previously described 46 by transfecting HEK293T cells with the pRlenti construct, the packaging psPAX2 construct (Addgene plasmid 12260), and a construct that expressed the glycoprotein of vesicular stomatitis virus pMD2.G (Addgene plasmid 12259). The siRNA sequences specifically targeting HCV, CD81, PI4KIIIα, ApoE and RACK1 were previously reported 18,47 . The target sequences of the CIDEB siRNA were as follows: 5′-AGAGGAGGATGGAACTGCA-3′ (siCIDEB 213) and 5′-AGTACTCAGGGAGCTCC-3′ (siCIDEB 517).…”

“…For instance, CIDEB mediates VLDL lipidation and maturation by interacting with ApoB 24 ; CIDEB is also required for the biogenesis of VLDL transport vesicles 25 and for chylomicron lipidation in the small intestine 22 . CIDEB is transcriptionally regulated by hepatocyte nuclear factor 4α (HNF4α), the most abundant transcription factor in the liver; HNF4α is crucial for VLDL-mediated lipid transport and participates in HCV assembly/release 18 . As a key transcriptional coactivator of HNF4α, peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) also regulates HCV production 26 , and PGC-1α stimulates VLDL assembly in a CIDEB-dependent manner 27 .…”