In a nutshell:
First complete structure of one of the flu virus’ key machines: its polymerase
Could prove instrumental in designing new drugs
The structure, obtained by scientists at the European Molecular Biology Laboratory (EMBL) in Grenoble, France, allows researchers to finally understand how the machine works as a whole. Published today in two papers in Nature, the work could prove instrumental in designing new drugs to treat serious flu infections and combat flu pandemics.
The machine in question, the influenza virus polymerase, carries out two vital tasks for the virus. It makes copies of the virus’ genetic material – the viral RNA – to package into new viruses that can infect other cells; and it reads out the instructions in that genetic material to make viral messenger RNA, which directs the infected cell to produce the proteins the virus needs. Scientists – including Stephen Cusack, head of EMBL Grenoble, who led the current study, and collaborators – had been able to determine the structure of several parts of the polymerase in the past. But how those parts came together to function as a whole, and how viral RNA being fed in to the polymerase could be treated in two different ways remained a mystery.
“The flu polymerase was discovered 40 years ago, so there are hundreds of papers out there trying to fathom how it works. But only now that we have the complete structure can we really begin to understand it,” says Stephen Cusack, head of EMBL Grenoble, who led the work.
Using X-ray crystallography, performed at the European Synchrotron Radiation Facility (ESRF) in Grenoble, Cusack and colleagues were able to determine the atomic structure of the whole polymerase from two strains of influenza: influenza B, one of the strains that cause seasonal flu in humans, but which evolves slowly and therefore isn’t considered a pandemic threat; and the strain of influenza A – the fast-evolving strain that affects humans, birds and other animals and can cause pandemics – that infects bats.
“The high-intensity X-ray beamlines at the ESRF, equipped with state-of-the-art Dectris detectors, were crucial for getting high quality crystallographic data from the weakly diffracting and radiation sensitive crystals of the large polymerase complex,” says Cusack. “We couldn’t have got the data at such a good resolution without them”.
The structures reveal how the polymerase specifically recognises and binds to the viral RNA, rather than just any available RNA, and how that binding activates the machine. They also show that the three component proteins that make up the polymerase are very intertwined, which explains why it has been very difficult to piece together how this machine works based on structures of individual parts.
Although the structures of both viruses’ polymerases were very similar, the scientists found one key difference, which showed that one part of the machine can swivel around to a large degree. That ability to swivel explains exactly how the polymerase uses host cell RNA to kick-start the production of viral proteins. The swivelling component takes the necessary piece of host cell RNA and directs it into a slot leading to the machine’s heart, where it triggers the production of viral messenger RNA.
Now that they know exactly where each atom fits in this key viral machine, researchers aiming to design drugs to stop influenza in its tracks have a much wider range of potential targets at their disposal – like would-be saboteurs who gain access to the whole production plant instead of just sneaking looks through the windows. And because this is such a fundamental piece of the viral machinery, not only are the versions in the different influenza strains very similar to each other, but they also hold many similarities to their counterparts in related viruses such as lassa, hanta, rabies or ebola.
The EMBL scientists aim to explore the new insights this structure provides for drug design, as well as continuing to try to determine the structure of the human version of influenza A, because although the bat version is close enough that it already provides remarkable insights, ultimately fine-tuning drugs for treating people would benefit from/require knowledge of the version of the virus that infects humans. And, since this viral machine has to be flexible and change shape to carry out its different tasks, Cusack and colleagues also want to get further snapshots of the polymerase in different states.
“This doesn’t mean we now have all the answers,” says Cusack, “In fact, we have as many new questions as answers, but at least now we have a solid basis on which to probe further.”
The work was carried out on the ESRF’s ID23-1 beamline. The study was conducted within the joint Unit of Virus-Host Cell Interactions (UVHCI), a collaboration between EMBL, the Centre National de la Recherche Scientifique (CNRS) and the Grenoble University Joseph Fourier. The work was funded by an Advanced Investigator grant from the European Research Council (ERC) to Stephen Cusack and by the EU-funded project FluPHARM.
Pflug, Guilligay, Reich & Cusack. The crystal structure of influenza A virus polymerase in complex with the viral RNA promoter. Nature, 19 November 2014.
Reich, Guilligay, Pflug et al.
Structural insights into the mechanisms of cap-dependent transcription and unprimed replication by influenza virus polymerase. Nature, 19 November 2014.
The influenza virus genome comprises eight segments of negative sense, single stranded viral RNA (vRNA), which are packaged in individual ribonucleoprotein particles (RNPs). RNPs contain one heterotrimeric viral RNA-dependent RNA polymerase, which is bound to the conserved 3′ and 5′ extremities of the vRNA promoter, and multiple copies of nucleoprotein. The viral polymerase both transcribes each genome segment, using a unique cap-snatching mechanism to acquire the 5′ cap-structure, and replicates them via a full-length complimentary intermediate (cRNA). Here we present the crystal structure at 2.7 Å resolution of the entire influenza A polymerase bound to the promoter, the first such structure of any negative strand RNA virus. The three polymerase subunits, PA, PB1 and PB2 mutually stabilize each other through extensive and intricate interfaces. PB1 has a canonical polymerase fold, including a priming loop, with a large, enclosed catalytic and RNA binding active cavity connected to solvent by three channels, the NTP entrance, the template entrance and template/product exit. The domains involved in cap-snatching, the PA endonuclease domain and the PB2 cap-binding domain, form protrusions which face each other across a solvent channel. Ten nucleotides at the extreme 5′ end of the promoter, which acts as an allosteric regulator of polymerase function, form a compact stem-loop structure (‘hook’) which is bound in a pocket formed by PB1 and PA close to the active site. The next four 5′ end nucleotides form canonical base-pairs with the distal part of the promoter 3′ end. The 3′ end proximal nucleotides interact with all three subunits and are directed towards the template entrance to the polymerase active site. This structure not only lays the basis for an atomic-level mechanistic understanding of the multiple functions of influenza polymerase but will also shed light on host-specific polymerase variants and open new opportunities for anti-influenza drug design.
The heterotrimeric influenza virus RNA polymerase transcribes and replicates the segmented viral single-stranded RNA genome. To transcribe, a short capped oligomer, derived by ‘cap-snatching’ from host pre-mRNA, is used to prime mRNA synthesis, whereas replication is unprimed and generates full-length copies of the template. Here we use crystal structures of influenza A (FluA) and B (FluB) polymerases, both complexed with the vRNA promoter, together with known structures of other viral RNA polymerases, to give the first detailed mechanistic insight into both these processes. In the FluA structure, an ordered priming loop suggests that influenza initiates unprimed template replication by a similar mechanism to that described for Ø6 bacteriophage and Flaviviridae such as hepatitis C. Comparing the FluA and FluB polymerase structures suggests a mechanism for cap-dependent transcription since they differ in the orientation of the PB2 cap-binding domain. In the FluA structure the cap-binding site faces the PA endonuclease active site, a configuration compatible with cap-snatching. However in the FluB structure the cap-binding domain has rotated in situ by 70° and RNA-like residual extra electron density descends part way from the cap binding site towards the PB1 active site. Modelling this as RNA its prolongation is compatible with the primer strand in known structures of other RNA polymerases bound to primer-template duplexes. The tight binding of the 5′ hook of the viral promoter to the polymerase is compatible with the role of this element in allosterically activating polymerase functions and in stalling transcription at the 5′ proximal oligo-U stretch leading to poly(A)-tail generation. Finally the structure needs to undergo significant conformational changes to convert the observed pre-initiation state into the initiation and elongation states, requiring relocation of the 3′ into the polymerase active site and movement of elements of PB2 that block the exit of the template strand.
Sonia Furtado Neves
EMBL Press Officer, Meyerhofstraße 1, 69117 Heidelberg, Germany
|Tel:||+49 6221 387-8263|