What Viruses Can Tell Us About RNA Processing: Q&A with the Weitzman Lab

Viruses can teach us a lot about cell biology. Because these miniscule microbes take over a cell’s function by inserting their genetic material into a host cell, scientists use viruses as valuable tools and models for studying processes like DNA replication, RNA transcription, protein translation, splicing, and more. But in the lab of Matthew Weitzman, PhD, at Children’s Hospital of Philadelphia, researchers are making discoveries that challenge the dogma of what we know about a particular virus, adenovirus, and how it manipulates cell function. In this Q&A, we sit down with Dr. Weitzman, co-director of the Division of Protective Immunity, and Alexander Price, PhD, a senior scientist in the Weitzman Lab, to take a magnified look at the microscopic world of viruses and RNA processing.

Dr. Weitzman, can you give us a little background about your lab?

Dr. Weitzman: I am a professor of pathology and I've been at CHOP for ten years. I run a basic science lab. My lab works on virus-host interactions and we’re interested in using viruses as tools to learn about how the cell works, how they are relevant to disease, and how they are used as delivery systems for putting genes into cells. We work on a range of human DNA viruses and I have a lab of around 10 to 12 people that work with me.

Can you give us some background on DNA, RNA, and RNA processing as it relates to your work?

Dr. Weitzman: So, DNA makes RNA and RNA makes protein. We study DNA viruses, so these viruses have a DNA genome and they use the infected cell’s own machinery to make RNA. The RNA gets processed into what's called messenger RNA (mRNA), and that mRNA is translated into proteins. The process of turning that long strand of RNA into a messenger RNA to make an appropriate protein is called splicing. But we don't know about how RNA processing is regulated in viral infection. Alex has a number of studies that are emerging, two of them published and two that are going to be published, that show how a virus manipulates and takes control of RNA processing pathways. This is important because it teaches us about the cell and about host responses, and it points to regulatory pathways that control this fundamental process of RNA splicing.

Dr. Price, can you tell us more about your research?

Dr. Price: The research that I’m working on has two major directions that have been in two different studies. The first one we published was a Nature Communications article on how RNA modifications are co-opted by a virus and mediate processing. Just like DNA and proteins can be modified after they're produced to change their form and function, so can RNA. In a similar way that epigenetics can regulate the DNA genome and control whether it's read or accessible, these RNA modifications can do the same thing. This is a mark that can be added to or removed from RNA after it's already been produced, and then change how that RNA would function in the cell.

For this research, you focused on a particular virus, adenovirus. Why?

Dr. Price: It’s always been thought that adenovirus is a great model for understanding how cells produce RNA. Many of the key functions in the process of going from DNA to RNA were actually discovered or characterized using this virus, including RNA capping and RNA splicing – things that are critically important for making human mRNA and all of the mRNA vaccines that are being used right now. But where our research diverges from this is that while it was always thought that these virus-derived RNAs look exactly like human RNAs, we're finding caveats based on how the viral genome is organized that make its RNA processing especially difficult for the virus as it needs to be very highly regulated.

Tell us more about what you discovered in this first study?

Dr. Price: Well, the first question we set out to answer with RNA modifications was how can adenovirus encode for over a hundred different proteins to make the fully functional virus all from a genome the average size of a single human gene? We knew the virus compacts all this information into a tiny space using RNA splicing, but to a level that cellular genes don't have to deal with. We found that a way to make RNA splicing very efficient was to mark its messages with these RNA modifications called m⁶A. This modification increased the splicing efficiency of these genes such that one DNA piece can make tens to hundreds of RNAs.

Tell us about your second study.

Dr. Price: The second angle, which was recently published as a breakthrough article at Nucleic Acids Research, was asking the flip side of the same question. This virus needs RNA processing to be efficient to make this vast amount information from a small amount of space. But what happens if that RNA processing is inefficient or can't occur properly?

It was always thought that during infection, DNA viruses make something called double-stranded RNA and therefore would be sensed by the cell’s immune system. We really challenged this dogma. We used imaging by microscopy with two independent monoclonal antibodies to establish whether adenovirus produces double-stranded RNA and found no detectable double-stranded RNA at all. This was due to the fact that adenovirus was able to splice its RNA very efficiently. We were able to perturb this in a number of ways, either by directly acting on the virus’ ability to splice or by using mutant viruses that are very poor at splicing. And when this happens, they now produced an abundant amount of this double-stranded RNA, and these RNAs were in the nucleus as opposed to the cytoplasm as we would have expected. And then we saw the immune system now acting against this molecule in the nucleus. So this really changed the idea of when and where the innate immune system would be able to detect a DNA virus, and also introduced an entirely new mechanism by which adenovirus might be able to avoid detection from the immune system in the first place.

What excites you the most about this line of research?

Dr. Price: In general, I've always thought viruses are great tools for teaching us about the cell. And most of this was because at the beginning of this era of molecular biology, viruses were genetically amenable and they worked really well. They took over cellular processes. So we were able to study the cell by using the virus. But when we really look with the closer lens, we 're finding out that the virus has to overcome hurdles that a typical cellular gene expression program doesn't have to face. And basically anything that a virus does that's different than what a human does is how a human can tell that it's infected by a virus. And so I think this really opened up a whole new area of where to look for differences in viruses and how a human cell might be able to detect something that's very minuscule and different.

These studies also allow us to ask very broad questions about other human diseases. It’s known that when some of these RNA processing pathways go awry in a human, even absent of a virus, can lead to double-stranded RNA production. These errors make the cell think it is infected by a virus and can lead to autoimmune diseases, as well as being dysregulated in some cancers. And so we think that this virus might be shedding a light on a way of which the cell surveys its own nucleus to prevent very different diseases. So I think that’s my most exciting future direction.

Dr. Weitzman: For me, the dogma was that the reason you have this small viral associated RNA is because the virus causes double stranded RNA and we discovered that no, it doesn't. So rewriting this dogma opens up new questions and challenges. It’s those new challenges that are unexpected and excite me about how viruses continue to provide us insights into fundamental processes in the human cell.