RSV particles require a starter kit for successful infection

RSV is a so-called single-stranded RNA virus. This means its genetic information is stored in a single strand of viral RNA, packaged in a layer of proteins and surrounded by an envelope. The virus primarily infects cells in the airways. Once RSV enters such a cell, it attempts to copy its genetic information and produce new virus particles.

To do this efficiently, the virus builds specialized structures inside the cell known as viral factories. These are assembly sites where viral proteins and viral RNA come together. Here, the virus’s genetic information is copied and converted into mRNA, which can later be translated into protein. Without these factories, RSV cannot replicate.

A mystery: how do viral factories form?

Viral factories consist of viral proteins that bind to one another. They have no membrane, but are held together by many protein-protein interactions. Such a structure can only form when the concentration of viral proteins is high enough to allow many of these protein-protein interactions to occur.

This raised a puzzle. To make large amounts of viral proteins, viral factories are needed. But to form viral factories, large amounts of viral proteins are required. This is a classic “chicken-and-egg problem.” Until now, it was unclear how RSV solves this problem at the very start of an infection.

Watching viruses live

To investigate this, the researchers developed a new advanced microscopy technique. Using this method, they were able for the first time to track individual virus particles in living cells, starting from the moment the virus enters the cell. They developed a fluorescent protein that binds strongly to the viral RNA, which causes it to light up as soon it enters the cel. This allowed the researchers to visualize individual viral RNA molecules within seconds after infection.

Infection begins with a viral particle entering the cel, after which the virus’s genetic information is copied. In the case of a successful infection (left), the viral RNA will grow into a viral factory by attracting viral proteins and fusing with other viral RNAs. In the case of an unsuccessful infection (right), the viral factory is not formed, and no viral proteins are produced. Credit: Dhanushika Ratnayake. Copyright: Hubrecht Institute.   

RSV enters the cell prepared

Using this technique, the researchers made a surprising discovery. Some RSV particles already carry a small “starter kit” when they enter the cell. This starter kit consists of a group of viral proteins that are already assembled together around the viral RNA. The researchers call this a Pre-Replication Center, or PRC.

Such a starter kit immediately creates a high local concentration of viral proteins, allowing a viral factory to form rapidly. The virus therefore does not have to start from scratch, but instead enters the cell already prepared. From this small starting point, viral factories continue to grow by attracting additional viral proteins.

The starter kit makes the difference

Virus particles with such a starter kit turned out to be far more successful. They began copying their genetic information more quickly and produced more viral proteins. Virus particles without a starter kit were unable to do this: they produced hardly any viral proteins and did not form viral factories. Infection by these particles simply stalled.

Of the many of RSV particles a person is exposed to, only a few actually manage to enter cells in the airways. And of those few, only a small fraction carry a starter kit. It is therefore likely that someone only becomes ill when such a “well-prepared” virus particle enters a cell. Analyses of RSV particles obtained from the airways of people infected with the RS virus, obtained via a collaboration with the UMC Utrecht, similarly showed that only a subset of virus particles carry a starter kit. This confirms the relevance of this starter kit for disease.

Is the starter kit a general mechanism for other viruses?

This discovery highlights how crucial the very first moments of an infection are. By better understanding how viral factories form, researchers may eventually find ways to block this process. If the starter kit cannot function, the virus never gets going. These new insights could, in the long term, contribute to improving treatment of severe viral infections.

RSV is not the only virus that depends on viral factories. Other major pathogens, such as the viruses that cause measles and mumps, also rely on the formation of similar viral factories. It is possible that these viruses likewise make use of pre-assembled starter kits. Further research will need to determine whether this is a general mechanism used by many viruses.

Publication
Pre-assembly of biomolecular condensate seeds drives RSV replication. Dhanushika Ratnayake, Marie Galloux*, Sanne Boersma*, Marko Noerenberg*, Christina Sizun, Carlos Sacristan, Julien Sourimant, Anke J. Lakerveld, Anne T. Gelderloos, Leonie Apperloo, Yana Demyanenko, Matthijs J. D. Baars, Rupa Banerjee, Birgit Dreier, Sven Furler, Natalie I. Mazur, Louis J. Bont, Shabaz Mohammed, Andreas Plückthun, Jean-François Éléouët, Geert J. P. L. Kops, Alfredo Castello, Puck B. van Kasteren, Marie-Anne Rameix-Welti & Marvin E. Tanenbaum. Nature, 2025.

* Authors contributed equally