Distantly related viruses share a common machinery for replication

Credit: Gerlach et al. / Cell 2015

The vRNA template (blue line strand) enters the polymerase active site along a tunnel (green) and then exits through another tunnel.
The template is used to make a mRNA or vRNA copy (yellow strand) by adding on RNA building blocks (red) which enter through a third tunnel.
The new copy (product) exits via a fourth tunnel.

Credit: Gerlach et al. / Cell 2015

In a nutshell:

  • 3D structure of the La Crosse orthobunyavirus (LACV) polymerase seen in unprecedented detail
  • The structure of the polymerase is likely to be similar for all viruses within the same group as the influenza virus
  • Finding opens up new avenues for treatment of diseases caused by this group of viruses

Grenoble, 21 May 2015 – In an article published today in Cell, researchers unveil the first detailed 3D-structure of the replication machinery – polymerase – of the La Crosse orthobunyavirus (LACV), a virus which can cause human encephalitis. LACV is in the same broad group of viruses as influenza and the findings show that the LACV polymerase has striking similarities to influenza virus polymerase whose atomic structure was previously determined by the same team.

Stephen Cusack and Juan Reguera from EMBL Grenoble, who jointly led the research, now believe that all viruses within this group are likely to have a very similar polymerase. This opens up the possibility of quicker routes to developing treatments for the diseases they cause.

Many dangerous human viruses, including influenza, Ebola and rabies viruses use RNA as their genetic material instead of DNA; these viruses have a special machine, known as the polymerase, to replicate their RNA genomes. The polymerase carries out two distinct functions: it makes copies of the virus’ genetic material, or viral RNA (vRNA), destined to be packaged into progeny viruses; and it makes viral messenger RNA (mRNA), which  are used to make viral proteins by hijacking the host cell protein making machinery.

LACV and influenza viruses belong to the so-called segmented negative-strand RNA viruses, whose members cause diseases including Lassa fever, Crimean Congo haemorrhagic fever, as well as all forms of influenza.

This group of viruses has a very specific way of making their mRNA – they ‘snatch’ a ‘cap’ of genetic material from the host cell mRNA and insert it into their own. This sets them apart from other groups of viruses, but also means that the complex machinery required to do this is likely to be common to them all.

Piotr Gerlach, who carried out this work for his PhD, explains: “These viruses have evolved separately over millions of years, so they are genetically quite different. Yet their polymerases maintain the same structural architecture and carry out replication and transcription of the genome in the same fashion.”

For both processes, vRNA needs to pass through the polymerase so that it can be copied. Using X-ray crystallography and cryo-electron microscopy, the team were able to identify that both ends of the vRNA attach to the polymerase to create a circle, and then sections of vRNA thread themselves through the polymerase before rejoining the circle again. Depending on whether polymerase is in replication or transcription mode, the vRNA is used as a template to create copies of itself, or – with the addition of the ‘cap’ snatched from the host cell – to create mRNA coding for viral proteins.

The structure of the polymerase clearly shows that the exit and entry points for the template vRNA are close together, allowing it to easily pass through the polymerase and back out again. It also shows that the exit for whatever is produced – whether vRNA copies or mRNA – is on the opposite site of the polymerase, ensuring that no mistaken interactions between template and product occur.

This level of detail has allowed the team to deduce how the polymerase functions in LACV and also give new insight into the influenza polymerase. Work is already underway to develop anti-viral drugs that inhibit the influenza virus polymerase. The team hope that by understanding structural and functional similarities across the whole group, promising compounds could be adapted to other related viruses.

Source article:

Gerlach, Malet, Cusack & Reguera. Cell, 21 May 2015. doi: 10.1016/j.cell.2015.05.006

Article abstract

Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, respectively, apo- and vRNA bound La Crosse orthobunyavirus (LACV) polymerase that give atomic-resolution insight into how such RNPs perform RNA synthesis. The complementary 30 and 50 vRNA extremities are sequence specifically bound in separate sites on the polymerase. The 50 end binds as a stem-loop, allosterically structuring functionally important polymerase active site loops. Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis. These results will aid development of replication inhibitors of diverse, serious human pathogenic viruses.

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