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The Transplant Patient's Friend
I’m going to tell you about a protein inside the cell that recognizes virus particles that have escaped the immune system, and alerts cellular degradation machinery to its presence. This provides a mechanism of intracellular immunity, and is especially important in people that have compromised or suppressed immune systems, such as transplant patients.
This is a narrative, though a complex and somewhat boring one…until the end! Stay with me, here, there are pictures to guide you.
Before I begin, here is the citation of the paper I will be describing here:
Using antibodies produced by white blood cells, the immune system can “tag” viruses for attack by other parts of the immune system. Some virus particles can escape this fate.
TRIM21 is a cytosolic protein capable of binding to these antibodies, with extremely high affinity. Its activity is conserved across mammals.
Since antibodies and their receptors are extracellular proteins, it seemed unlikely to the authors that TRIM21 would be both highly conserved and highly specific for these anitbodies, and yet not play a role in providing protection against virus particles. Thus, they are going to hypothesize that TRIM21 mediates intracellular protection against viruses that have escaped detection by the immune system.
To investigate this, they are going to use adenovirus, which causes the common cold. Adenovirus was chosen because it is a non-enveloped virus, which means that its capsid not protected from exposure to antibodies prior to infection.
Since TRIM21 binds antibodies, the authors first needed to ask whether antibodies even entered the cytosol during viral infection. To test this, adenovirus particles were pre-incubated with antibodies, and then used to infect cultured cells. Cells were then co-stained for the antibody-bound virus, and TRIM21.
If antibody-bound virus is able to get inside the cells, then we'll see fluorescence inside the cell, rather than outside. Additionally, if TRIM21 is binding to these antibodies, it will co-localize with the virus.
If you’ll direct your attention to the top panel, the first image shows the stained nucleus, adenovirus is stained in red, TRIM21 in green, and the merged image shown last. Remember that we’re detecting the virus by staining the antibodies that are bound to it, so we can conclude from these first two panels that antibody-bound virus is able to infect cells.
If you’ll look here in the second panel, you’ll see the same images of virus, TRIM21, and the merge at a higher magnification. The large blobs of TRIM21 here co-localize with the virus particles in the merged image, so we can conclude that TRIM21 is being recruited to the site of the virus.
I want to add here that the infection was only 30 minutes long, which may explain why TRIM21 still looks pretty diffuse, though it is forming what we call puncta here.
Since TRIM21 is being recruited to the site of infection, the investigators asked what the effect might be on the number of virus particles.
To do this, they used a virus that carries a green fluorescent protein, so that they could quantify levels of infection by flow cytometry. They infected cells using this virus, and then knocked down the levels of TRIM21 using siRNA to measure the effect on virus infection.
If TRIM21 is reducing the number of virus particles inside the cell, then fluorescence will decrease in the presence of Control siRNA, but not TRIM21 siRNA.
I am not going to tell you about the interferon treatment, so please ignore the two dotted lines here. The authors measured the decrease in infection due to the addition of antibody, increasing along the x-axis, and by % of fluorescent cells. In the absence of antibody, the virus infected roughly 50% of cells, normalized here to be 100%. If you’ll allow your eyes to follow this Control siRNA line, you can see that the percentage of infected cells decreased rapidly with the addition of increasing concentrations of antibody, such that at 400 ng/mL antibody, infection was reduced roughly 60-fold.
The previous experiments demonstrate that there is an intracellular immune response mediated by TRIM21 and antibodies, and that this response is capable of reducing levels of viral infection.
Next they examine the mechanism by which TRIM21 targets antibody, and how the virus is neutralized.
To begin investigating the fate of the virus particles after TRIM21 is recruited to the site of infection, the authors performed viral infection experiments in the presence of a proteasome inhibitor, since the proteasome is a common pathway to degradation inside a cell. If the proteasome is involved, the inhibitor will reverse the effect of TRIM21 on levels of infection.
That is indeed what the authors saw. The black bars indicate percentage of fluorescent cells in the presence of both antibody and TRIM21, and the grey bars are a negative control in this case, showing the percentage of fluorescent cells in the absence of antibody. MG132 here is the proteasome inhibitor, and you can see that compared to non-treated cells infected with this virus and antibody, the decrease in infection is significantly reversed. Not shown here are data demonstrating that this is indeed dependent on the activity of TRIM21.
So from these experiments, we can conclude that TRIM21 and proteasome function are essential components in the same pathway.
At this point, it becomes necessary to tell you that the TRIM21 protein has a domain containing ubiquitin-ligase activity. Knowing this, the authors hypothesized that ubiquitination might be necessary to target the antibody-bound virus to the proteasome.
So, they tested the full-length protein, as well as a mutant protein, missing the ubiquitin-ligase domain. Protein was incubated with the fluorescent antibody-coated virus particles, and allowed to infect cells that had been depleted of endogenous TRIM21. The percentage of cells containing virus was quantified using flow cytometry.
If ubiquitination is indeed necessary for the targeting of the virus to the proteasome, then deletion mutant will be unable to mediate neutralization of the virus.
If you’ll focus here on the top figure, levels of infection are restored nearly to control levels here with the mutant TRIM21, which is missing ubiquitin-ligase activity. They also provide co-localization experiments to show that ubiquitin is co-localizing with both TRIM21 and antibody-bound virus, thereby demonstrated that the TRIM21-associated viral particle is ubiquitinated. This would imply that no direct viral interactions are needed.
After experiments showing that TRIM21 does indeed autoubiquitinate, the authors transfected cells with latex beads coated in antibody, and stained for TRIM21 and ubiquitin. If TRIM21 does not require direct interaction with the virus in order to target the antibody-bound virus to the proteasome, then TRIM21, ubiquitin, and the latex beads will co-localize.
The top panel shows the colors and labels, but I really want to focus on the bottom panel here, which shows colocalization inside the cell of latex bead, TRIM21, and ubiquitin, suggesting that direct interaction with an invading viral particle may not be required to recruit TRIM21, and the virus may not be the target of ubiquitination.
To summarize what these authors have shown:
• There is an intracellular immune response mediated by TRIM21 and antibodies
• This response is capable of reducing levels of viral infection
• TRIM21 targets the antibody-bound virus to the proteasome via ubiquitination
• This response does not require direct viral interaction
Conclusions and future directions:
• Discovered a system of intracellular immunity in which antibodies and TRIM21 mediate the neutralization of virus
• The mechanism by which TRIM21 mediates neutralization of virus makes it difficult for viruses to evolve and escape
• TRIM21 may be effective against other non-enveloped viruses
With that, I will take any questions.