Day 259: Trojan exosome hypothesis debunked?

Let me start by saying that I’m a product of the Hildreth lab, so I am fairly well-versed in the trojan exosome hypothesis. Basically, the hypothesis, as presented by Drs. Hildreth and Gould, is that HIV particles are basically glorified exosomes. They postulate that the HIV budding machinery usurps the normal exosome budding machinery to produce virions. Accordingly, HIV virions are pretty similar to exosomes, except that they carry the viral payload. There are a number of lines of evidence that suggest this, most of them outlined in the PNAS hypothesis paper published about 5 years ago.

 

Now, there are a number of people who don’t believe this is the case—some of them pretty prominent. I’m not writing to either support or refute the exosome hypothesis, but rather to discuss a paper that came out of David Ott’s lab claiming to refute the trojan exosome hypothesis.

 

A number of groups have noticed that CD45 is missing in viral particles. This observation is part of the evidence for HIV budding from lipid rafts—although I don’t really believe lipid rafts exist; I am much more partial to tetraspanin enriched membranes. In any event, one way to get highly purified viral particles is to run supernatant from infected cells through a series of density gradients, take the pellet, and then deplete for CD45. This gets rid of contaminating vesicles that contain CD45, leaving purified viral particles. A number of groups claim that exosomes also do not contain CD45, which would mean that CD45 depletion would leave both exosomes and HIV particles. Lori Coren reports in Retrovirology that depleting supernatant from Jurkat and SupT1 cells of CD45 removes exosomes, suggesting that HIV particles and exosomes differ in CD45 composition and, therefore, likely do not share the same budding process.

 

They really state their case with one figure, although it’s split into six parts. They isolated exosomes from the two cell lines, depleted half for CD45 and then blotted for CD45, actin and did silver stains for total protein. After depleting they observed no CD45, no actin, and I think no protein (I think they mislabeled part of the figure). They did the same thing for infected cell lines, except that they also blotted for p24. They found pretty much the same thing, although actin shows up in the Jurkat/HIV supernatant that was CD45 depleted and you can see protein (mostly viral proteins) in the CD45 depleted samples. They claim that CD45 depletion leaves virus (we know that), but not exosomes. They claim that the reason other groups didn’t find CD45 in exosomes was because they were using anti-CD45 antibodies that don’t recognize the cytoplasmic domain of the protein, while they use a pan-CD45 antibody that will recognize more isoforms. I’m not sure how immunoprecipitating with antibodies that recognize the cytoplasmic domain of a protein will pull out intact vesicles, but with this paper, it’s beside the point.

 

Now, their findings may be true, but this paper has several flaws preventing me from accepting their conclusions. Anyone who has tried to isolate exosomes knows that it is a total pain in the ass. They are hard to define biochemically, which is why in almost every paper you see on exosomes, exosome presence is confirmed by electron microscopy. Based on this paper, we have no idea what this group is actually working with. You can’t show the depletion of exosomes if you don’t show that the exosomes are there in the first place. And there is no control for depletion using markers we know are on exosomes (like CD81) and markers that we know are excluded. I would be much more inclined to believe the results if they showed depletion using some unrelated antibody, like an anti-GFP, didn’t produce the same effect. How do we know that their immunoprecipitation itself didn’t affect the outcome? There’s no protein left after immunoprecipitation and it’s not like Jurkats and SupT1s pump out exosomes; they may just not have enough protein in their sample following depletion to detect by Western blot. And why don’t you blot for exosome markers while you are at it? If you are going to do biochemical analyses, at least do it thoroughly.

 

So, I think if they added some controls, this could be an interesting observation. As it is, it doesn’t clarify anything.

 

M. Linde