How do viruses multiply?

December 12, 2019

For the first time in 3D and atomic resolution: Researchers from the Department of Molecular Biology at the Max Planck Institute for Biophysical Chemistry, in cooperation with colleagues from Würzburg, have succeeded in presenting the propagation strategy of Vaccinia viruses. These viruses also serve as vaccines against human smallpox diseases and as the basis for new cancer therapies. (Cell, December 12, 2019)

For viruses to multiply, they usually need support of the cells they infect. Only in their host´s nucleus can they find the machines, proteins, and building blocks with which they can copy their genetic material before infecting other cells.

But not all viruses find their way into the cell nucleus. Some remain outside the cytoplasm and have to double their genetic material without help. To do so, they carry the necessary “machinery” with them. A special nanomachine combined with various subunits – RNA polymerase – plays an important role in this process. This cellular copying machine reads the genetic information from the virus´ genome and translates it into messenger RNA – that serves as a blueprint for the proteins encoded in the genome. This process is called transcription.

Scientists led by Patrick Cramer, Director and Head of the Department of Molecular Biology at the Max Planck Institute (MPI) for Biophysical Chemistry, and Utz Fischer of the Julius Maximilian University (JMU) in Würzburg have now succeeded, for the first time, to solve the structure of these nanomachines from poxviruses three-dimensionally and in atomic resolution. Henning Urlaub, Research Group Leader at the MPI for Biophysical Chemistry, was also involved in the analyses. The Scientists worked with Vaccinia, a DNA virus. This pathogen, which is completely harmless to humans, is not only the basis for all vaccines against smallpox infections. It is also tested in oncolytic virotherapy to combat cancer. 

A molecular clamp that holds everything together

“The RNA polymerase of the Vaccinia virus exists essentially in two forms: the actual core enzyme and an even larger complex, which has further specific functionalities thanks to the addition of subunits,” Fischer explains. The core enzyme is similar to the molecular copy machine that occurs naturally in living cells and is the subject of intensive research by the Cramer Department: RNA polymerase II. The second complex of Vaccinia RNA polymerase is an all-rounder. It consists of numerous subunits and carries out the entire transcription process for the virus. This enables the virus to multiply.

The Vaccinia RNA polymerase complex at work.

It can be seen how the Vaccinia RNA polymerase complex changes during copying (transcription).

The complex is held together by a molecule that the virus hijacks from its host cell: a so-called transfer RNA (tRNA). This type of molecule does not normally play a role in transcription, but provides the amino acid building blocks for protein production. If this host tRNA were not involved, the huge complex would likely fall apart. 

Structures of Vaccinia transcription complexes purified from infected cells.

A) Cartoon representation of the core Vaccinia RNA polymerase (RNAP). The individual subunits are shown in different colors.

B) Surface representation of the complete Vaccinia RNA polymerase complex. In addition to the core Vaccinia RNA polymerase, it contains viral transcription factors required for the different steps of the transcription cycle as well as a host tRNA molecule (shown as cartoon).

To find out how the viral RNA polymerase works, the researchers determined – under the participation of Henning Urlaub, Research Group Leader at the MPI for Biophysical Chemistry – its three-dimensional structure during different transcription steps. “With the new findings, we can now understand the entire process of virus replication. It's like watching a film to see how this nanomachine functions at the atomic level and how the individual processes are choreographed,” Cramer says. His colleague, structural biologist Hauke Hillen, adds: “What´s amazing is how the building blocks of the machine rearrange themselves after the start of transcription to drive the synthesis of the RNA product – this complex is really very dynamic”.

Structure of actively transcribing Vaccinia RNA polymerase.
The complete Vaccinia RNA polymerase (RNAP) complex (left) undergoes large conformational rearrangements upon binding of DNA and formation of the actively transcribing co-transcriptional capping complex (right).

A supermicroscope provides the necessary data

The necessary data is provided by a device that has revolutionized structural analysis in recent years – the cryo-electron microscope. It provides images with a resolution on the atomic scale. For their images, the researchers shock-froze and “photographed” the RNA polymerase in different phases of transcription. In this way, millions of snapshots of the nanomachine were taken during its work, which the researchers then assembled into an overall picture. Hillen and his colleague from Würzburg, Clemens Grimm, spent about six months with hard computer work until they developed spatial models of the polymerase complexes from several terabytes of data. Using 3D glasses, anyone can now look at the complex, rotate it around as they like and dissect it into its subunits. 

Among other things, the new findings offer the possibility of influencing the viral reproduction cycle, which has therapeutic potential. Studies using Vaccinia viruses in the fight against cancer are currently underway worldwide. The company Genelux has already been able to show in animal experiments and in patients that specially optimized Vaccinia viruses can even reduce tumours and detect minute metastases.

Modified from a press release of the University of Würzburg/is

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