Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

Abstract

<jats:p>Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV’s effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-localize. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.</jats:p>

Dose–Response Effects of 7-Dehydrocholesterol Reductase Inhibitors on Sterol Profiles and Vesicular Stomatitis Virus Replication

“…Conditions that alter homeostasis ,, and introduce oxidatively reactive sterols into the cellular lipidome have the potential to perturb critical steps in the infection process. Indeed, there is evidence that cellular 7-DHC gives highly reactive electrophilic oxysterols upon peroxidation, see Figure A. − These species adduct to several lipid synthesis and transport proteins, prominent among these proteins being constituents of COPI, the transport complex − involved in traffic between the Golgi and endoplasmic reticulum and ACSL4, an acyl-CoA synthetase.…”

“…64−69 Likewise, many viruses use cholesterol-rich plasma membrane lipid raft microdomains to bud from the infected host cells, 69−71 and cholesterol is important for intracellular trafficking processes for the assembly of replication complexes and for viral genome replication. 72,73 Conditions that alter homeostasis 3,74,75 and introduce oxidatively reactive sterols into the cellular lipidome have the potential to perturb critical steps in the infection process. Indeed, there is evidence that cellular 7-DHC gives highly reactive electrophilic oxysterols upon peroxidation, see Figure 6A.…”

BmCH25H, a vertebrate interferon‐stimulated gene(ISG) homolog, inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori

“…Modulation of the activity of DHCR24 has been observed during infection of mammalian viruses (Tallorin et al. , 2020), confirming the important role of cholesterol metabolism in viral infections.…”

Hepatitis C Viral Replication Complex

“…PI4P could attract lipid transport proteins (oxysterol-binding protein (OSBP), four-phosphate adaptor protein 2 (FAPP2), NPC1, and ceramide transfer protein (CERT) to deliver glycosphingolipids, cholesterol, and ceramide to RO, respectively [ 16 , 59 , 60 ]. Recently, it was reported that HCV NS3/4A protease controls the activity of 24-dehydrocholesterol reductase (DHCR24), catalyzing the conversion of desmosterol to cholesterol and regulating the lipid environment for HCV RNA replication [ 61 ]. In contrast, cholesterol-25-hydroxylase induced by interferon could block MW formation via the production of 25-hydroxycholesterol and thus restrict HCV replication [ 62 ].…”