NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes

Abstract

bThe hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase III␣ (PI4KIII␣) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIII␣ complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIII␣-targeting inhibitor. In addition, both the NS5A and PI4KIII␣ classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIII␣. Because of the similarities between the effects induced by treatment with PI4KIII␣ or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIII␣ complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIII␣ complex.T he recent advent of direct-acting antivirals (DAAs) against hepatitis C virus (HCV) is radically transforming the treatment scenario for patients with chronic hepatitis C infection. These new drugs offer the promise of well-tolerated interferonfree oral regimens that are able to cure the majority of infected patients (1).Initially, the effort to identify DAAs focused primarily on inhibitors of two virally encoded enzymes: the nonstructural 3/4A (NS3/4A) protease and the NS5B polymerase. More recently, however, the clinical validation of NS5A inhibitors (2) has generated increasing interest in this target class. The first NS5A inhibitors were discovered by a phenotypic screen based on the genotype 1b replicon system (3, 4). The initial lead compounds had moderate potency and a narrow spectrum of anti-HCV activity, mainly on genotype 1b. Subsequent medicinal chemistry efforts (4) resulted in the design of picomolar inhibitors characterized by a peculiar and highly symmetrical dimeric structure (reviewed in reference 5). The most-studied agent of this "palindromic" NS5A inhibitor class is daclatasvir (DCV, formerly BMS-790052) (6), a highly optimized biphenyl derivative inhibitor for which regulatory approval is currently being sought.Different chemical isotypes were initially claimed to ...

Phosphatidylinositol 4-kinases: Function, structure, and inhibition

“…The compound inhibits replication of HCV with EC 50 ¼290 nM (genotype 1b) and EC 50 ¼ 750 nM (genotype 2 a) [149]. Inhibitors of NS5A seem to interfere with the function of the NS5A-PI4K IIIα complex but not with catalytic function of PI4K IIIα [151]. Another compound with the quinazoline scaffold was identified by focused screening in GlaxoSmithKline.…”

Lipid Tales of Viral Replication and Transmission

“…Frequently cholesterol is also found enriched along with PI4P or PE at replication organelles [7,48-50]. Cholesterol is well known to regulate protein and lipid packing and recent investigations have revealed that it is required for the formation of large (i.e., more than a few nanometers) and stable phosphoinositide or PE enriched lipid domains [51].…”

“…Since PI4P is produced in large quantities continually throughout infection [22], its levels at the replication sites would likely not be significantly depleted. Further support for feedback between PI4P and cholesterol levels during infection comes from inhibition of PI4KIIIα recruitment in HCV infected cells, which leads to a decrease in both PI4P and cholesterol pools at the replication organelles [48]. …”

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication

“…NS5A is a multitasking protein required for the HCV life cycle and thus a good antiviral target (5). It is a phosphoprotein that appears as two bands at 56 and 58 kDa on immunoblots, respectively, referred to as hypophosphorylated (p56) and hyperphosphorylated (p58) NS5A (6).…”