Compared to other organisms, viruses have very small genomes. Their genetic material is not sufficient to maintain their own metabolism, produce proteins or reproduce independently. Therefore, they hijack the biological processes of their host cell.

A central step in viral replication is transcription – the precise ‘overwriting’ of viral genes in messenger RNA (mRNA). While most DNA viruses smuggle their genetic information into the cell nucleus – the logistical control centre of the host cell – in order to use the machinery there, poxviruses pursue a different strategy.

They remain in the cytoplasm and act there independently of the cell nucleus. To do this, they bring their own highly specialised mini-factories, including a viral transcription apparatus. This autonomy requires its own control tools to activate the viral genes at the right time.

A study by the University of Würzburg, now published in the journal Nature Communications, shows for the first time the mechanical elegance with which the viral protein VITF-3 controls this process. Vaccinia viruses, the most widely studied model viruses from the poxvirus family, were examined at the molecular level by a research group led by Utz Fischer, holder of the Chair of Biochemistry 1. Stefan Jungwirth, Clemens Grimm and Julia Bartuli carried out the central work in the laboratory.

A kink in the genetic material

In the study, the team was able to show that VITF-3 acts as a molecular clamp. This factor consists of two building blocks that together form a closed ring structure. What is special about it is that: "VITF-3 alone is completely unreactive towards DNA. The ring is so stably closed that it cannot attach itself to the genetic material," explains Utz Fischer.

Only with the help of a second player can viral transcription begin. Viral RNA polymerase (vRNAP) – the virus's actual copying tool – plays a central role in this process. “Contact with the polymerase opens the VITF-3 ring and places it precisely around the DNA like a cuff,” explains Stefan Jungwirth, who conducted extensive experiments with the viral complexes for the project.

Closing the clamp anchors the entire machinery at the starting point. This intervention breaks the DNA double helix, creating a kink of about 90 degrees in the genetic material, and the DNA is literally forced into the throat of the copying machine (cleft). The sharp kink is crucial: it exposes the DNA strands so that the polymerase can begin copying.

Detective work at atomic level

With the help of cryo-electron microscopy, the team has succeeded in solving this molecular puzzle. “With this technique, the protein complexes are flash-frozen at minus 196 degrees Celsius to stop them in their natural motion. An electron beam and magnetic lenses then provide us with a greatly magnified image,” explains Clemens Grimm, who was responsible for elucidating the structure.

The team analysed around nine million individual molecules in this way. Using the data set obtained in this way, it was able to reconstruct a model with a resolution of 2.4 Ångström. To put this into perspective, one Ångström is about the size of the diameter of a hydrogen atom or, in other words, one ten-millionth of a millimetre. At this scale, the researchers were able to identify the molecular details of the viral motor and every turn of the DNA helix.

The most important findings:

The structural analysis of VITF-3 revealed an architecture that is completely atypical for this protein family, as the related proteins in humans or yeast do not interact with each other on their own. In contrast, the ring observed in vaccinia is already locked in place in its free state.

The atomic structural analysis also revealed the role of the so-called capping enzyme. It is stably integrated into the complex and ensures that the newly formed viral mRNA is immediately provided with a kind of protective cap. Thus camouflaged, the host cell does not recognise the foreign code as a threat and begins to produce viral proteins.

The electron microscope data also show that direct physical contact with VITF-3 also positions the polymerase on the DNA. This interaction enables the machinery to recognise the specific start signal of viral genes on the DNA with extreme accuracy. Smallpox viruses thus prove to be highly efficient specialists that achieve maximum results with a minimum number of factors.

The study also suggests a fascinating dynamic at the end of the process: as soon as the newly formed mRNA reaches a length of about twelve nucleotides, it physically collides with an extension of VITF-3. This collision may cause the polymerase to detach from the clamp and allow the mRNA production phase to begin.

Possible approach for new active substances

Deciphering this unusual mechanism not only provides fundamental insights into the evolution of gene control, but also opens up new avenues for antiviral therapies. Since it is specific to the Poxviridae family – which includes not only the vaccinia virus but also the mpox virus and variola viruses, the pathogens that cause deadly smallpox – it offers a target for new drugs. Future drugs could, for example, prevent the VITF-3 ring from closing, thereby nipping viral replication in the bud.

Furthermore, the study highlights the impressive adaptability of viruses, which have developed highly efficient tools over the course of evolution to repurpose the complex processes of life for their own replication.