Caught red-handed

Researchers observe how a phenuivirus steals host mRNA caps

Viruses use the molecular infrastructure of infected host cells for their reproduction. While the viral polymerase replicates the genetic information of the virus, the host ribosome is needed to produce the viral proteins. However, the process by which a ribosome converts RNA into a protein – called translation – is tightly controlled and requires a so-called 5'RNA cap. Phenuiviruses cannot produce these RNA caps themselves, but steal them from their host. But how does this work? Researchers at the Bernhard Nocht Institute for Tropical Medicine (BNITM) and their collaborators have used cryo-electron microscopy to study and visualise different stages of this process on a molecular level. The results were published in the scientific journal Nucleic Acids Research.

©Chris Hoffmann | Harry Williams | Maria Rosenthal

Severe fever with thrombocytopenia syndrome virus (SFTS virus) is an RNA phenuivirus belonging to the group of Bunyaviruses. It causes an illness in humans that includes fever, muscle aches, diarrhea and a drop in blood platelets (thrombocytes). In severe cases, internal bleeding and multiple organ failure can also occur. Ticks transmit the SFTS virus and can infect a range of pets and livestock, including cats and cows, as well as humans. There are currently no effective drugs approved for use against the SFTS virus.

Hijacking the host protein factory

“Bunyaviruses need a kind of small key to gain access to the host cell's machinery,” explains Dr Maria Rosenthal, group leader of the BMBF junior research group Structural Virology at the BNITM. “These keys are the host's RNA caps. Viruses use this key to hijack the ribosome, which is the host's protein production factory.”

Dr Maria Rosenthal: a researcher with long hair and a blouse. The picture is in black and white. — Dr. Maria Rosenthal ©Martin Kunze

This is how proteins are made in the host cell: The ribosome recognises the RNA cap of a messenger RNA (mRNA) and begins to produce proteins. If there is no RNA cap on an RNA strand, usually no protein is made. Bunyavirus RNA does not contain such a cap. These viruses therefore need a strategy to get hold of the caps – i.e. the key. Some viruses, including SFTS virus, steal the RNA caps from the host cell by cutting them off the host RNA. The L protein of the SFTS virus was expected to be involved in this process. But how the process worked in detail was not known. In this study, researchers used cryo-electron microscopy (cryo-EM) to gain more insight into this process.

Ph.D. Harry Williams: A researcher smiles into the camera. He has short brown hair, a short beard and a checkered shirt. — Ph.D. Harry Williams ©M. Rosenthal

Structure determination by cryo-electron microscopy

First author Dr Harry M. Williams and his colleagues at the cryo-EM facility at the Centre for Structural Systems Biology (CSSB), led by Prof. Dr Kay Grünewald, used cryo-EM to take a series of images of the L protein. A single image is not enough to resolve the three-dimensional structure of the SFTS virus L protein for several reasons. Proteins are three-dimensional objects, so looking at the L protein from only direction would give the researchers only a partial understanding of its overall structure.

“Another limitation is that the contrast of images taken during cryo-EM experiments is typically very poor. To overcome this, we take many images of the L proteins during a single experiment. We then superimpose these images and subtract the background signal. However, we have to be careful that we only sum together images showing the L protein in similar shape. The L protein is large and has flexible domains that can point in different directions. So we often spend a lot of time trying to work out which images we can combine,” says Williams, describing the process.

Graphic shows the L protein of the SFTS virus in the ribbon model on the left and a zoom in on the cap binding domain with an RNA cap on the right. — Left: L protein of the SFTS virus. Right: Zoom in on the cap-binding domain of the L protein (turquoise), capped RNA is shown in pink. ©Williams et al. 2024

Viral L protein steals host RNA cap

Using the structural insights gained from cryo-EM and additional experiments, the research team was able to show how the viral L protein of the SFTS virus binds to host RNA caps and could cleave them from the rest of the RNA strand. They also showed that the virus uses the RNA caps as a starting point for the formation of a new viral mRNA strand. “While we think there certainly will be a role for host cell factors in perhaps delivering or handing off host mRNA to these L proteins, the process of cap-snatching itself is entirely driven by the L protein. The L protein alone is sufficient to recognise the host RNA, cut off the cap and synthesise new viral mRNA,” says Williams. In the next step, which the researchers have not yet investigated in this study, the host cell ribosome recognises the RNA cap on the newly formed viral mRNA strand and translates it into viral proteins.

The figure shows the modelled L protein of the SFTS virus in three different states of the transcription process. — Left: The viral genome of the SFTS virus is made up of RNA - the ends of this genetic information are shown as black strands on top. Cap binding domain (CBD) is shown in turqoise, endonuclease domain is shown in orange. Centre: The CBD recognises and binds the RNA cap (= capped primer shown in pink). The endonuclease cleaves the cap from the host RNA. The RNA polymerase of the L protein forms viral RNA in the centre of the molecule, using part of the stolen RNA (pink strand) as a starting point. Right: After elongating the viral RNA by two nucleotides, the structure remains stable: the RNA cap is still bound to the CBD and the endonuclease domain retains its position. ©Williams et al. 2024

Cap-snatching as a target for new drugs

Key steps in the life cycle of viruses can be targets for the development of inhibitory drugs. Researchers use structure determination methods such as cryo-EM to identify sites in the viral proteins where drugs can act. “If we could specifically inhibit the process of viral transcription, i.e. the formation of a new viral mRNA strand for protein synthesis, the life cycle of the virus would be interrupted," concludes group leader Rosenthal. In current and future studies, Rosenthal and her team are trying to find antiviral agents targeting virus-specific processes conducted by the L protein and including the described cap-snatching mechanism.