For this post we are going to take a slight detour and talk about something related to Niemann-Pick C, filovirus infection. I am personally interested in the topic because of my love of viruses – plug for the highly informative TWiV (This Week in Virology) podcast, if you don’t already listen. My first research experience was in Hepatitis C Virus biology. Years later, in graduate school, I engineered Adeno-associated Virus (AAV) vectors for targeted gene delivery, and along the way studied AAV assembly. So of course, when there is an opportunity to combine my interest in virology with my interest in rare genetic disease, I will jump on it.
Since filoviruses enter many different types of cells across species, it has been difficult to uncover the exact mechanism of viral entry. Many cellular proteins have been indicated in playing a role in filovirus entry such as T-cell immunoglobulin and mucin containing protein 1 (Tim1), which might account for the high viral replication in the lungs. Folate receptors have also been indicated as well as a few receptor tyrosine kinases. But the exact mechanisms of entry for each filovirus, across all cell types, have not been elucidated.
Suchita Bhattacharyya and Thomas Hope. Cellular factors implicated in Filovirus Entry.
However, it has been shown that upon attachment, filoviruses enter the cell via the endocytic pathway using macropinocytosis and potentially other entry mechanisms. Once inside the endosome, pH specific proteases cleave GP1 (glycoprotein on the virus surface required for cell attachment), and allow for membrane fusion of the virus to the endocytic membrane. Then once out of the endosome, the virus can enter the cytoplasm and replicate. But what happens in the endosome? How does the cleaved GP interact with the membrane to facilitate fusion? Well that’s where NPC1 transmembrane protein comes in.
It has been shown by a few research groups that NPC1 plays a role in virus nucleocapsid entry into the cytoplasm for viral replication. A 2012 study published by Miller et al., demonstrated that a single luminal domain of NPC1 binds the cleaved GP.
First they expressed human NPC1 in reptilian cells, which are non-permissive, making them highly susceptible to virus infection. Then they expressed truncations of the NPC1 protein in null Chinese Hamster Ovary (CHO) cells, and found that the luminal C domain, not associated with cholesterol transport, is required for viral entry.
This is shown in the image below where NPC1-/- CHO cells expressing various forms of NPC1 (right side of panel) were infected with EBOV and MARV. Only cells with either full-length protein or the CI or AC domains lead to viral entry into the cytoplasm. If the C domain is deleted, as seen in the fourth row of images, virus cannot infect the cells.
The most recent study published by Herbet et al., added additional information regarding EBOV pathogenesis. He and his colleagues challenged Npc1-/-, Npc1 +/-and wildtype (wt) littermates with virus. Wildtype mice lose a significant amount of weight post infection and have detectable virus titers 3 days post infection as does the Npc1 +/- group (A). The Npc1 -/- group does not display significant weight loss post infection (A) or detectable virus titer on day 3 or 7 (C and D). By day 7 the viral titer in Npc1 +/- drops significantly, however the levels in wt remain high (D). The Npc1-/- and Npc1+/- are still alive 20 days post infection while wt mice are not (B).
Now, based on our previous NPC posts you know that individuals with Niemann-Pick type C disease accumulate cholesterol in the endosome/lysosome compartments. Researchers in this study checked to see if it was the accumulation of cholesterol that affected the lack of viral infectivity. They treated NPC2-/-, NPC2 +/- and wt littermates with EBOV. NPC2-/- mice accumulate cholesterol in the lysosome, while still containing functional NPC1. These mice are not resistant to infection even though there is excess accumulation in the lysosome. Meaning that NPC2 is not playing a role in virus infectivity and neither is the cholesterol.
They also followed up with an assay using a cholesterol-clearing compound, hydroxypropyl-beta-cyclodextrin (HP-β CD), which is currently in clinical trial. They treated NPC-/-, NPC +/- and wt littermate mice with HP-β CD, which clears the cholesterol accumulates. And only the wt mice succumb to viral infection. More evidence that cholesterol accumulation is not playing a role in filovirus infection.
Additionally, known NPC1 inhibitors, U18666A and imipramine, inhibit filovirus infection in cell culture. Interestingly, in vivo, these compounds do not protect against weight loss or morbidity post infection (A and B). They do reduce viral replication when checked 5 days post infection (D).
Therefore, there is strong evidence that if therapeutics were developed to inhibit NPC1 from binding to the virus one could protect people from filovirus infection. Just enough protection from the virus could allow for the immune system to kick in and clear the infection. Additionally it is interesting that while carriers do not present the Niemann-Pick type C disease phenotype, they are presumably more resistant to filovirus infection.
There are other examples of genetic mutations leading to severe disease pathology, being beneficial for resistance to viral infection. For example, people carrying the mutation for sickle cell anemia are resistant to malaria infection. Additionally, in cells from people with hypogammaglobulinemia, an N-glycosylation disorder, there is significantly reduced HIV replication. More work is required to uncover the mechanism behind this observation. While on the hunt for therapies and treatments for these devastating rare genetic disorders, researchers will also be uncovering mechanistic information that may help in developing novel antivirals.
