Human Adenoviruses, Cholesterol Trafficking, and NF-κB Signaling.

The interplay between viruses and host factors regulating inflammatory or cytotoxic responses directed against infected cells is well documented. Viruses have evolved a wide array of mechanisms that strike a balance between the elimination of virus and immune-mediated tissue injury by antiviral immune responses. The topic of this mini-review is a series of recent studies demonstrating a link between cholesterol trafficking and innate immune responses in cells infected with human adenoviruses that provide the backbone of commonly used vectors in gene medicine. Besides revealing an unexpected role for lipid metabolism in immune evasion, these studies have important implications for understanding the molecular basis of cholesterol trafficking in normal cells and various disease states. They also describe a previously unappreciated host-virus interaction that may be employed by other pathogens to interfere with the host innate immune system.

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

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 RIDα 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 RIDα. 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, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-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 RIDα 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 RIDα 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.

Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

Molecular pathways for intracellular cholesterol accumulation: common pathogenic mechanisms in Niemann-Pick disease Type C and cystic fibrosis.

It has been less than two decades since the underlying genetic defects in Niemann-Pick disease Type C were first identified. These defects impair function of two proteins with a direct role in lipid trafficking, resulting in deposition of free cholesterol within late endosomal compartments and a multitude of effects on cell function and clinical manifestations. The rapid pace of research in this area has vastly improved our overall understanding of intracellular cholesterol homeostasis. Excessive cholesterol buildup has also been implicated in clinical manifestations associated with a number of genetically unrelated diseases including cystic fibrosis. Applying knowledge about anomalous cell signaling behavior in cystic fibrosis opens prospects for identifying similar previously unrecognized disease pathways in Niemann-Pick disease Type C. Recognition that Niemann-Pick disease Type C and cystic fibrosis both impair cholesterol regulatory pathways also provides a rationale for identifying common therapeutic targets.

Autosomal recessive polycystic kidney disease epithelial cell model reveals multiple basolateral epidermal growth factor receptor sorting pathways.

Sorting and maintenance of the EGF receptor on the basolateral surface of renal epithelial cells is perturbed in polycystic kidney disease and apical expression of receptors contributes to severity of disease. The goal of these studies was to understand the molecular basis for EGF receptor missorting using a well-established mouse model for the autosomal recessive form of the disease. We have discovered that multiple basolateral pathways mediate EGF receptor sorting in renal epithelial cells. The polycystic kidney disease allele in this model, Bicc1, interferes with one specific EGF receptor pathway without affecting overall cell polarity. Furthermore one of the pathways is regulated by a latent basolateral sorting signal that restores EGF receptor polarity in cystic renal epithelial cells via passage through a Rab11-positive subapical compartment. These studies give new insights to possible therapies to reconstitute EGF receptor polarity and function in order to curb disease progression. They also indicate for the first time that the Bicc1 gene that is defective in the mouse model used in these studies regulates cargo-specific protein sorting mediated by the epithelial cell specific clathrin adaptor AP-1B.

Adenovirus RID-alpha activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C.

Host-pathogen interactions are important model systems for understanding fundamental cell biological processes. In this study, we describe a cholesterol-trafficking pathway induced by the adenovirus membrane protein RID-alpha that also subverts the cellular autophagy pathway during early stages of an acute infection. A palmitoylation-defective RID-alpha mutant deregulates cholesterol homeostasis and elicits lysosomal storage abnormalities similar to mutations associated with Niemann-Pick type C (NPC) disease. Wild-type RID-alpha rescues lipid-sorting defects in cells from patients with this disease by a mechanism involving a class III phosphatidylinositol-3-kinase. In contrast to NPC disease gene products that are localized to late endosomes/lysosomes, RID-alpha induces the accumulation of autophagy-like vesicles with a unique molecular composition. Ectopic RID-alpha regulates intracellular cholesterol trafficking at two distinct levels: the egress from endosomes and transport to the endoplasmic reticulum necessary for homeostatic gene regulation. However, RID-alpha also induces a novel cellular phenotype, suggesting that it activates an autonomous cholesterol regulatory mechanism distinct from NPC disease gene products.

Adenovirus RIDalpha regulates endosome maturation by mimicking GTP-Rab7.

The small guanosine triphosphatase Rab7 regulates late endocytic trafficking. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein-related protein 1L (ORP1L) are guanosine triphosphate (GTP)-Rab7 effectors that instigate minus end-directed microtubule transport. We demonstrate that RILP and ORP1L both interact with the group C adenovirus protein known as receptor internalization and degradation alpha (RIDalpha), which was previously shown to clear the cell surface of several membrane proteins, including the epidermal growth factor receptor and Fas (Carlin, C.R., A.E. Tollefson, H.A. Brady, B.L. Hoffman, and W.S. Wold. 1989. Cell. 57:135-144; Shisler, J., C. Yang, B. Walter, C.F. Ware, and L.R. Gooding. 1997. J. Virol. 71:8299-8306). RIDalpha localizes to endocytic vesicles but is not homologous to Rab7 and is not catalytically active. We show that RIDalpha compensates for reduced Rab7 or dominant-negative (DN) Rab7(T22N) expression. In vitro, Cu(2+) binding to RIDalpha residues His75 and His76 facilitates the RILP interaction. Site-directed mutagenesis of these His residues results in the loss of RIDalpha-RILP interaction and RIDalpha activity in cells. Additionally, expression of the RILP DN C-terminal region hinders RIDalpha activity during an acute adenovirus infection. We conclude that RIDalpha coordinates recruitment of these GTP-Rab7 effectors to compartments that would ordinarily be perceived as early endosomes, thereby promoting the degradation of selected cargo.

A tyrosine-based signal plays a critical role in the targeting and function of adenovirus RIDalpha protein.

Early region 3 genes of human adenoviruses contribute to the virus life cycle by altering the trafficking of cellular proteins involved in adaptive immunity and inflammatory responses. The ability of early region 3 genes to target specific molecules suggests that they could be used to curtail pathological processes associated with these molecules and treat human disease. However, this approach requires genetic dissection of the multiple functions attributed to early region 3 genes. The purpose of this study was to determine the role of targeting on the ability of the early region 3-encoded protein RIDalpha to downregulate the EGF receptor. A fusion protein between the RIDalpha cytoplasmic tail and glutathione S-transferase was used to isolate clathrin-associated adaptor 1 and adaptor 2 protein complexes from mammalian cells. Deletion and site-directed mutagenesis studies showed that residues 71-AYLRH of RIDalpha are necessary for in vitro binding to both adaptor complexes and that Tyr72 has an important role in these interactions. In addition, RIDalpha containing a Y72A point mutation accumulates in the trans-Golgi network and fails to downregulate the EGF receptor when it is introduced into mammalian cells as a transgene. Altogether, our data suggest a model where RIDalpha is trafficked directly from the trans-Golgi network to an endosomal compartment, where it intercepts EGF receptors undergoing constitutive recycling to the plasma membrane and redirects them to lysosomes.