Identification of FBL2 As a Geranylgeranylated Cellular Protein Required for Hepatitis C Virus RNA Replication

Abstract

We recently reported that Hepatitis C virus (HCV) RNA replication requires one or more geranylgeranylated host proteins. Using a combination of [(3)H]mevalonate labeling, coimmunoprecipitation, and bioinformatic search, we identified a geranylgeranylated host protein required for HCV RNA replication. This protein, FBL2, contains an F box domain and a CAAX motif (CVIL). It forms a stable immunoprecipitable complex with the HCV nonstructural protein 5A (NS5A). The association of FBL2 with NS5A requires the CAAX motif of FBL2, but not the F box. Deletion of the F box created a dominant-negative protein that inhibited replication of HCV RNA when overexpressed in Huh7-K2040 cells; this inhibition was overcome by coexpression of NS5A. siRNA-mediated knockdown of FBL2 mRNA by 70% in Huh7-HP cells reduced HCV RNA by 65%; this reduction was overcome by expression of a cDNA encoding a wobble mutant of FBL2. The current data indicate that geranylgeranylated FBL2 binds to NS5A in a reaction crucial for HCV RNA replication.

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

“…Host Cell Factors Involved in HCV RNA Replication-Apart from the viral proteins and CREs, several host cell factors play an important role for HCV RNA replication based on the criteria that RNA interference-mediated knockdown of each of these cellular genes and/or overexpression of dominant negative mutants decreases viral replication (55)(56)(57). The first one is the human vesicle-associated membrane protein-associated protein A (VAP-A) that was initially identified as an interaction partner of NS5A and NS5B (58).…”

“…The third host cell factor involved in HCV RNA replication is the geranylgeranylated protein FBL-2 that also interacts with NS5A (55). Inhibition of geranylgeranylation in cells by disrupting the function of 3-hydroxy-3-methylglutaryl-CoA reductase or by impairing the enzymatic activity of geranylgeranyl transferase I led to the disassembly of replicase complexes and abolished HCV RNA replication (61).…”

Dysregulation of distal cholesterol biosynthesis in association with relapse and advanced disease in CHC genotype 2 and 3 treated with sofosbuvir and ribavirin

“…non-structural protein 5A [18]. However, the antiviral efficacy of statin monotherapy in HCV patients has proved to be insignificant [41][42].…”

“…Post-translational modification of geranylgeranyl facilitates the membrane association between a key protein, F-box and leucine-rich repeat protein 2 (FBL2) with HCV NS5A [18]. However, the role of FBL2 in viral replication has not been fully established across all HCV genotypes.…”

Function follows form: The structure of the N-terminal domain of HCV NS5A

“…3 In addition, numerous other functions and a plethora of interaction partners have been postulated for NS5A. 4,5 However, only few of these potential interactions have been validated in a meaningful experimental context involving active HCV RNA replication, 6,7 and surprisingly little effort has been devoted to the basic biochemical characterization of this protein.…”