Published! Role of a Nuclear Localization Signal … in BK Polyomavirus Nuclear Entry

My second research paper from my graduate school lab finally came out, and I would like to try to summarize it for you here, in layman’s terms and with cartoons instead of figures because I’m not sure how copyrights work and I don’t feel like searching for old data. The headings are taken directly from the paper.

Introduction

In my first paper, I showed evidence that BK virus travels from the cellular compartment called the ER into the fluidy part called the cytosol on its trip towards the nucleus within a cell. Another big question in the field is how the virus enters the nucleus. The nucleus of the cell contains our DNA, so obviously it doesn’t let just any protein-sized object into it!

So as a follow up to my previous paper, I set out to determine whether the virus gets into the nucleus from the cytosol using the pathway that other nuclear proteins use, where a specialized import protein recognizes a “signal sequence” on the nuclear protein that signals the import protein to send it into the nucleus. This was the hypothesis on which I based my experiments. It is summarized here in this figure below.

????????????????Figure: After coming out of the ER, the virus (in red) uses a signal on one of its structural proteins to interact with nuclear-import proteins (in green) to enter the nucleus (through the Pore):

Lysine 319 on VP2 and Lysine 200 on VP3 are critical for nuclear localization of the minor capsid proteins.

The first thing that we had to do was to look for the nuclear localization signal on the virus. Based on what the signal looks like on other proteins, we hypothesized that a couple of amino acids on the minor structural protein provided the signal. So we mutated one specific amino acid in that area of the protein and looked at the protein within a cell.

We found that normally the protein goes to the nucleus. However, with the mutation, the protein remains outside of it. (We determined this by microscopy and fluorescently labeling the protein). This suggests that the amino acid we mutated was needed for signaling nuclear localization.

????????????????The VP2/3 NLS is important for entry in RPTE cells

The more important question about this nuclear localization signal is whether it is needed when it is part of the whole virus capsid, and not just a lone protein. So, we made virus containing this mutation in its minor capsid structural protein. Then, we infected cells with this mutant virus to see if it can infect cells as well as the normal (“wild-type”) virus. We found that it could not infect the cells as well as the wild-type virus. We could deduce from this finding that the one amino acid in that one protein was important for… well, something… during the infection (although we hypothesize that it is needed in nuclear entry, this data alone doesn’t give us conclusive evidence… so, next experiment).

????????????Importin β – mediated nuclear import is involved during infection

The other way we can look at this “pathway” is from the other side of the picture – does the import protein that is floating around in the cell, looking for nuclear localization signals, play a role in infection? This protein is call “importin” – because it imports proteins into the nucleus, clever right?

We looked at this question in two ways, the first of which was to take away that specific importin protein in a process that we call a knock-down. We introduce a RNA molecule into the cells that will cause the importin to no longer be made. Then, we infect those cells with the virus, and compare the infection with cells that have not had the protein knocked down. What we found was that without importin, the cells were not as easily infected. Again, this tells us that importin was important for something during the infection.

Model KDThe second way we tested this pathway was to use an inhibitor called ivermectin that targets importin’s function. This chemical gets in the way when importin tries to interact with its target – in this case, the virus. So we treated cells with ivermectin, and then infected with virus (and compared to what? You guessed it, cells that were not treated with ivermectin). What we saw was that the cells treated with ivermectin did not become infected as easily, implying that infection needed something that the ivermectin was blocking.

???????????????Discussion

In this paper, combined with my previous work, I identified components of a kidney cell that BK virus uses to cause infection – specifically, the nuclear import protein importin (and likely its helper proteins).

Are there alternative interpretations to these conclusions? Like a court case, we base a lot of things on the best interpretation of the evidence – are there possibilities that we haven’t considered? It is likely – things that scientists haven’t discovered yet about the cell, for example. It is also possible that our interpretation is missing some details. That is why we present the data, along with our methods and conclusions, so that other members of the scientific community can evaluate it and use it to make other hypotheses to reach our end goal of helping sick people get better.

If each of these methods blocked infection in the cells, can’t we use them now to block infection in people? In general we are able to manipulate the cells easily because they are in a plate. In a person, there are so many different types of cells, and a lot of those cells would probably need the importin protein to keep working to keep that person alive. The same is true for the treatment with ivermectin – it is actually very toxic, and although it prevents the cells in the plate from being infected, after a couple days the cells would die anyway from the treatment.

Progress depends on what has already been done, and ideas that have been presented, to support and invoke new ideas and hypotheses about how nature works. My work on how BK virus gets to a cell’s nucleus has brought us one step closer to treatment of BK virus infection. Since other viruses may use the same pathway, if a drug is discovered that targets BK virus, it may also be useful for those other viruses. It will be so exciting to see what is discovered next!

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