This post is based on a publication from Nicholas Cianciola out of the Carlin Lab at Case Western.

For my previous post, I wrote about the relationship between Niemann-Pick C and viral pathogenesis. I am going to continue that thread by talking about another virus relationship – how adenovirus infection offers insights into cholesterol metabolism, and how that cholesterol metabolism relates to NPC. Learning how a viral protein affects cholesterol trafficking may also aid in the development of novel therapeutics.

Let’s first start by going over cholesterol metabolism. Cholesterol levels are maintained via homeostatic machinery such as sterol regulatory binding protein (SREBP) and ER localized acyl-CoA:cholesterol acyltransferase (ACAT). When cholesterol levels are low, the N-term of SREBP localizes to the nucleus and upregulates genes involved in the sterol biosynthesis pathway. When excess free cholesterol is available, ACAT esterifies it to be stored in droplets (LDs), ultimately delivered to other compartments.  Additionally, if there is an excess of cholesterol, it is synthesized into oxysterols in the mitochondria, which also affects SREBP and turns on the Liver X Receptor (LXR) in the nucleus. Oxysterols are LXR agonists. Once LXR is turned on, it activates the transcription of genes that increase cholesterol efflux, decrease absorption and increase excretion.

A lot is known about these steps, but the in between is still hazy. How does cholesterol make it to the necessary compartments for homeostasis regulation?

It is known that cholesterol enters the cell as LDL (low density lipoprotein) via the LDL receptor. Both NPC proteins, 1 and 2, and LBPA (LE phospholipid lysobisphosphatidic acid) play a role in allowing for the transport of LDL-cholesterol out of the late endosome/lysosome pathway. And recent research suggests NPC2 transports cholesterol to the mitochondria for oxysterol synthesis.

In 2009 Cianciola and Carlin published that RIDα, an adenovirus protein, rescues the cholesterol metabolism defect in NPC1 deficient fibroblasts, but not in cells containing functional NPC1 protein. Interestingly, RIDα interacts with Rab7 interacting lysosomal protein (RILP) and oxysterol binding protein-related protein (ORP1L). ORP1L also detects late endosome cholesterol and alters the location of late endosomes through interactions with the ER membrane.

In their recent publication, Cianciola et al., uncover that RIDα depends on ORP1L to activate lipid droplet formation for lipid storage. As a first step, it was shown that RIDα expression rescues the cholesterol storage defect observed in NPC1 mutant fibroblasts via filipin staining. If you remember from previous posts, filipin binds to unesterified or free cholesterol, so cholesterol that has not made it to the ER for esterification by ACAT. Here LAMP1 is used as a marker for the presence of late endosomes. When RIDα is expressed, there is a decrease in unesterified cholesterol in the lysosomes.


Additionally, as seen the figure below, in WT fibroblasts loaded with LDL, lipid droplets are observed via the BODIPY stain (green). These lipid droplets are not affected by the expression of RIDα (in red). However, RIDα increases LD formation in NPC1 mutant or deficient cells loaded with LDL, but does not in NPC2 deficient cells. This means that the observed LD formation by RIDα requires the presence of NPC2, not NPC1.


So how does RIDα induce clearance of unesterified cholesterol and LD formation? Researchers decided to see if RIDα aids in the shuttling of LDL cholesterol to the ER for esterification by ACAT. They did this by radiolabeling LDL with [H3] cholesteryl palmitate. Cells were dosed with [H3] cholesteryl palmitate in the presence of excess oleate (oleic acid). If [H3] cholesteryl palmitate leaves the endosome/lysosome pathway it will form [H3] cholesteryl oleate. RIDα expression increases [H3] cholesteryl oleate in NPC1 deficient cells as compared to NPC1 deficient cells not expressing RIDα. This means RIDα expression increases cholesterol esterification.

To additionally support this finding, NPC1 deficient cells expressing RIDα can clear the unesterified cholesterol but the presence of an ACAT inhibitor (S58-035) does not allow for the clearance and leads to unesterified cholesterol accumulation as observed in cells not expressing RIDα.


Interestingly, these effects are not induced by SREBP and LXR regulated gene expression. As previously mentioned these proteins affect the transcription of cholesterol homeostasis genes. Based on real-time PCR results, RIDα is unable to return SREBP regulated genes in NPC1 deficient cells to the homeostatic levels in control cells that are not NPC1 deficient. Additionally, LXR regulated genes are unaffected by the presence of RIDα. Therefore, the observed cholesterol clearance by RIDα is not tied to SREBP or LXR cholesterol homeostasis mechanisms. However, as seen in the figure below, cholesterol ester formation does increase in the presence of RIDα in NPC1 deficient cells as shown below.


In addition, researchers ruled out the possibility that RIDα is releasing NPC1 I1061T mutant protein from the ER so it can function in the endosome and transport the free cholesterol. This is the possible mechanism of action of SAHA, an HDAC inhibitor that also clears the cholesterol accumulations in NPC1 mutant cells. But upon RIDα treatment, NPC1 I1061T remains localized in the ER.

So how is RIDα functioning to increase cholesterol esterification? It has been shown that RIDα interacts with ORP1L. To test if this interaction leads to cholesterol esterification and LD formation, NPC1 deficient cells were treated with RIDα   +/- ORP1L siRNA. In NPC1 deficient cells expressing RIDα there is an observed decrease in free cholesterol, detected via filipin staining. However, when ORP1L is knocked down, free cholesterol remains accumulated. Additionally, when RIDα is expressed and ORP1L is knocked down, lipid droplets do not form, but do when ORP1L is present.



The final proposed endosome-to-ER cholesterol trafficking mechanisms are depicted in the diagram below:


In this study, it was shown that the adenovirus protein, RIDα, is unable to rescue the cholesterol storage defect in NPC2 deficient cells, but can in NPC1 deficient cells, so the function is dependent on the presence of NPC2. All models proposed show that RIDα depends on NPC2 and ORP1L to aid in the transport of cholesterol to the ER for esterification either by direct membrane contact (A), through vesicular transport (B) or via protein binding (C), ultimately leading to LD formation.

There are multiple hypotheses as to how adenovirus uses this to its advantage, but uncovering this novel mechanism for cholesterol transport to the ER for esterification may help in novel therapeutic discovery for the treatment of Niemann-Pick C! There are a lot of great experiments in this publication that were not covered in this post, so I recommend you read it to get all of the details and supporting information, but hope you enjoyed the highlights!

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