Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum

Abstract

Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RID␣ via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RID␣. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RID␣ did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RID␣-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich "lipid rafts." Collectively, these data show that RID␣ utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes.IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune-mediated pathology have been a particularly rich source of information regarding intracellular protein trafficking. Our studies with the early region 3-encoded RID␣ protein also provided fundamental new information regarding mechanisms of nonvesicular lipid transport and the flow of molecular information at membrane contacts between different organelles. We describe a new pathway that delivers cholesterol from endosomes to the endoplasmic reticulum, where it is esterified and stored in lipid droplets. Although lipid droplets are attracting renewed interest from the standpoint of normal physiology and human diseases, including those resulting from viral infections, experimental model systems for evaluating how and why they accumulate are still limited. Our studies also revealed an intriguing relationship between lipid droplets and innate immunity that may represent a new paradigm for viruses utilizing these organelles.KEYWORDS adenoviruses, cholesterol, endocytic pathway, endoplasmic reticulum, lipid droplet

Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

“…Although ORP1L and Rab7 are the main drivers in this process, NPC1 activity is required for ORP1L function [78]. These findings are based on studies in MelJuSo [27], HeLa [78,90,91] and A549 [92] cells, yet NPC1 mutant CHO cell lines examined in the current study do not express significant amounts of ORP1L. Hence other proteins and mechanisms that allow for MCS formation and cholesterol transfer, including StARD3, need to be considered.…”

“…With regard to cholesterol homeostasis, unsolved mechanisms include the kinetics and directionality of cholesterol transfer. For example, in HeLa cells the StARD3/VAP-A complex mediates cholesterol transport from the ER to late endosomes independently of ORP1L [75,76]; however, using the same protein machinery, transport of cholesterol from late endosomes to the ER was also demonstrated [92]. Based on our findings, one can envisage that late endosome-cholesterol accumulation caused by loss of NPC1 would trigger an elevation of AnxA6 levels, possibly due to inhibition of CMA [83], and a concomitant increased recruitment of AnxA6 to late endosomes [43][44][45].…”

Lipids, inflammasomes, metabolism, and disease

“…Indeed, other crystalline substances also activate NLRP3 inflammasome, 29 [101][102][103] Moreover, perturbations in lipid metabolism underlie diverse human pathologies including cardiovascular disease and Alzheimer's. 83 Finally, genetic defects in lipid and cholesterol trafficking lead to Niemann-Pick and Tangier disease.…”

Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System

“…We tested an alternate model in which ORP1L mediates cholesterol egress at LEL-ER contacts and a defect in this process causes cholesterol accumulation in LELs. ORP1L has not been directly implicated in cholesterol transport from LELs, but it participates in an NPC1independent cholesterol efflux pathway for lipid droplet biogenesis involving receptor internalization and degradation a (RIDa), an adenoviral Rab7 mimetic (Cianciola et al, 2017). ORP1L is also involved in ABCA1-mediated efflux of endosomal cholesterol to apoA-I acceptors (Vihervaara et al, 2011).…”