Inhibition of cyclophilins alters lipid trafficking and blocks hepatitis C virus secretion

Abstract

Host cyclophilin (cyp) inhibitors, such as NIM811, efficiently inhibit replication of hepatitis C virus (HCV) and have shown significant promise in recent clinical trials for the treatment of chronic HCV. It is therefore important to fully understand the mechanism of action of these therapeutic agents. Data obtained from comprehensive systems biology approaches have led to the hypothesis that the antiviral activity of cyclophilin inhibitors is mediated through impairing the cellular machinery on which HCV relies to traffic cofactors necessary for formation of the replication complex. Indeed, our results demonstrate when cyclophilins are inhibited by NIM811, lipid and protein trafficking within the VLDL pathway is impaired. Following treatment of replicon or HCV infected cells with NIM811, intracellular lipid droplets (LD) more than double in size and decrease in number. Changes in the LDs in response to cyclophilin inhibition are dependent upon expression of viral proteins. Additionally, in cells treated with NIM811, apoB accumulates in a crescent or ring shaped structure surrounding the enlarged LDs and is no longer secreted. Silencing of cypA or cyp40 using siRNA had a similar effect on LD size and apoB localization as compound treatment, suggesting these cyclophilins may play an important role in lipid and apoB trafficking. Interestingly, the decrease in apoB secretion correlates with a decrease in release of viral particles in HCV infected cells. Altogether, these results add a new level of complexity to the mechanism of action of cyclophilin inhibition, and suggest the role for cyclophilins in the virus life cycle extends beyond replication to virus release.

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

“…In the present study, HNF4␣ or PLA 2 GXIIB downregulation induced abnormal aggregation of large LDs and reduced the number of cytosolic LDs. Such LD rearrangement was accompanied by the reduced expression and distorted cytosolic distribution of HCV proteins, similar to that of nordihydroguaiaretic acid and cyclophilin inhibitors (33,34). These results echo the notion that cytosolic LDs are important organelles for HCV assembly and secretion (11).…”

“…The rearrangement of LDs reportedly impairs HCV infectivity (32)(33)(34). Huh7.5.1 cells were infected with HCVmutJFH1 in the presence of bezafibrate and then observed under a confocal microscope at 72 h postinfection to investigate whether or not HNF4␣ regulates the rearrangement of LDs.…”

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

“…Electron microscopy analyses suggest that NS5A-positive structures correspond, in part, to double membrane vesicles, which are a hallmark of the membranous HCV replication factory, designated membranous web (52). A NS5A "cluster" phenotype similar to that of the PW turn mutants was previously found in cells treated with NS5A or Cyp inhibitors (46,52,53), as well as in PI4KIII␣ knockdown cells. This phenotype was shown to correspond to lipid droplets or aberrant double membrane vesicles, respectively (47).…”

“…9B, right column). This altered phenotype correlated with a strong accumulation of large structures where NS5A is present, reminiscent of the cluster distribution in cells treated with NS5A and Cyp inhibitors (46,52,53). Notably, the proportion of cells with the cluster phenotype varied from 24 to 67% for I315G and P314A mutants, respectively, compared with 5% for WT (Fig.…”

Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

“…CyPA binding to, and presumably isomerization of, NS5A domain II has been proposed to change the local conformation of the CyPA-binding site (51), which in turn can "activate" NS5A to maintain the integrity of the viral replication complex (30). In addition, Anderson et al (2) reported a distinct change of localization and trafficking of lipid molecules upon CPI treatment. Because NS5A localizes to lipid droplets (LDs), possibly to influence viral assembly, CPIs might affect NS5A trafficking and HCV assembly as well (12,26).…”