Tracking unspliced RNA: Professor Rumlová studies how retroviruses “hack” cells

Could you describe your project and its main objectives?

Retroviruses, such as HIV, are viruses that carry genetic information in the form of RNA; however, after entering a cell, they are able to reverse transcribe it into DNA and integrate it into the host genome. In order to replicate, they must transport their genetic information—in the form of unspliced RNA—from the nucleus into the cytosol. There, it serves both as a template for the production of viral proteins and as the genome of new viral particles. This is highly unusual, as the cell very carefully controls which RNA molecules are allowed to leave the nucleus. Normal protein-coding cellular RNAs, known as messenger RNAs (mRNAs), must undergo splicing—a processing step in which certain segments are removed—before being exported. When this process fails, mRNA that retains these segments is held back in the nucleus as part of quality control and subsequently degraded. Retroviruses have managed to outwit this cellular control machinery, allowing their unspliced RNA to pass into the cytoplasm.

Our project focuses on identifying the cellular proteins that assist retroviruses in this step and on the mechanism by which protein complexes bound to viral RNA are remodelled during RNA transport. The goal is to understand how the virus uses cellular mechanisms to its advantage and how this pathway is linked to the subsequent fate of the RNA, i.e. to its translation and packaging into new viral particles.

Why is this export of unspliced ​​RNA so important for retroviral replication?

Because this RNA has a dual role for the virus. On the one hand, it serves as a template for synthesizing viral proteins in the cytoplasm; on the other, it represents the genetic material itself, which must be packaged into new viral particles. The virus must package its RNA genome in its unspliced form, as only this RNA contains essential sequences without which further viral replication would be impossible.

Simply put: the virus needs to get a complete copy of its genetic instructions out of the nucleus. Once the viral RNA is in the cytoplasm, it can be used as a template for synthesizing viral proteins; and a portion of these RNA molecules is then selected and packaged into a new particle as a genome. We are interested in how the virus forces the cell to allow viral RNA that is, from the cell’s perspective, “unfinished,” that is unspliced, to leave the nucleus. We are also investigating how the cell and the virus together - albeit unintentionally from the cell’s perspective - determine which RNA molecule will be translated into proteins and which will be packaged as the genome. This is one of the key, yet still not fully understood, steps of retroviral replication.

What do you think it was about your project that impressed the Ministry of Education, Youth and Sports’ jury the most?

I believe the decisive factor was a combination of several elements. First, the reviewers were primarily interested in the scientific problem itself. We are investigating a mechanism that is essential for retroviruses, but still not sufficiently understood, which may also have broader implications for the regulation of cellular processes. In addition, the quality of the team, our strong collaboration with the NIH and Rockefeller University, and the use of modern methods that will allow us to track viral RNA in great detail also played a role. It was also important that the project has clear objectives and a realistic plan.

How will this research contribute to the development of new treatments for retroviral infections?

It is important to say that this is primarily basic research. Rather than developing a new drug directly, we are trying to understand, at the molecular level, the step that the virus cannot do without. Only when we fully understand the mechanism of these cellular processes and identify the essential factors for the export of unspliced ​​RNA and how they function, will we be able to better determine which interactions or mechanisms represent potentially vulnerable points. Similar mechanisms associated with the export of unspliced ​​mRNA also appear in some diseases, such as cancer, so I  see the benefit of our research mainly in that it will refine our understanding of these pathological processes in the cell, and retroviruses help us do so. Therefore, our work on retroviruses not only shows us how the virus works, but also how the cell itself works.

What responsibilities does your UCT Prague team have? How does the collaboration with your American partners work?

At UCT Prague, we are conducting the project’s primary experimental work. Our research involves cell cultures, viral models, and various biochemical techniques ranging from immunochemistry to microscopy that allow us to track viral RNA and identify its cellular partners. In collaboration with colleagues from NIH, Frederick, we are developing single-particle tracking methods to observe viral RNA movement in living cells across both space and time. Furthermore, we are working with colleagues from Rockefeller University to employ iCLIP methods for the precise mapping of protein-binding sites on RNA. Both collaborations are valuable to us, because they are not just about data sharing, but also about the transfer of expertise and detailed know-how.

Who else is collaborating on the project?

At UCT Prague, we collaborate closely with the Department of Biotechnology and the Department of Biochemistry and Microbiology. Externally, we work with colleagues from the Institute of Organic Chemistry and Biochemistry of the CAS (IOCB Prague) and the Central European Institute of Technology in Brno (CEITEC). Our research naturally necessitates the integration of diverse fields of expertise; it requires combining virology, cell biology, and biochemistry with advanced imaging and modern approaches to study RNA-protein interactions. This is exactly what makes our project capable of addressing complex questions that a single laboratory cannot tackle on its own.

Are there plans to continue the project? Have you brought on new collaborators, and are you looking to welcome new colleagues to the team?

Yes, definitely. In basic research, it often happens that one answered question opens up several more. High-quality, properly interpreted data provide a foundation you can build on — and that is what moves scientists forward. It is already clear that the topic of retroviral RNA export and packaging has broader implications and may reveal more general principles of how RNA functions within the cell. We also hope the project will be attractive to students, as it connects classical molecular biology, biochemistry, and virology with cutting-edge biotechnological methods. Students are already involved in the project as part of their bachelor’s and master’s theses.

What was the personal turning point that motivated you to pursue a long-term investigation into retroviral particles?

I first encountered retroviruses in the early 1990s during my diploma thesis, when I was working at IOCB Prague on HIV-1 protease inhibitors. At that time, AIDS could not yet be effectively treated, and retrovirus research had a completely different dimension. From the very beginning, I was fascinated by how such a relatively small and simple virus can outsmart evolutionarily highly optimized cellular processes and turn them to its own advantage. And that has never stopped fascinating me. From a scientific point of view, retroviruses are remarkable; just consider one of their enzymes, reverse transcriptase, without which no diagnostic or research laboratory in the world could function today. Without it, it would be impossible to diagnose RNA viruses like SARS-CoV-2, influenza, or HIV using RT-PCR tests, or, for example to produce recombinant human proteins in bacterial cells.