#weekendusers: Finding out how viruses package the genome

21-10-2016

Scientists strive to better understand the mechanisms behind virus replication.

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Viruses are ubiquitous pathogens in all types of life forms. They cause major public health issues and create serious economic concerns worldwide. These infectious agents replicate inside the living cells of other organisms by selectively capturing in the host cell the right segments of nucleic acids needed for them to self-assemble. The ability of viruses to self-assemble still defies theoretical understanding. There are currently no physical models that can reliably account for the dynamic pathways along which the hundreds of molecular building blocks making up a virus fit into the final structure with pinpoint accuracy.

This is the question that Dr Guillaume Tresset and his team from the Université Paris Sud are trying to answer this week. They are using time-resolved small-angle X-ray scattering (TR-SAXS) techniques on the ESRF’s ID02 beamline. “We want to elucidate the mechanisms of genome packaging and understand how these viruses manage to select their genome efficiently”, says Dr Tresset.

The research team will be studying single-stranded (ss) RNA plant viruses. This category of virus also includes hepatitis C and Zika, plus human norovirus, which is responsible for causing severe gastroenteritis.

Most viruses consist of nucleic acids which encode, transmit and express the genetic code. These viral components are protected by a nanometre-scale protein shell called the capsid. For some viruses, the capsid is wrapped in a lipid envelope. During viral replication in the host cell, the capsid proteins and nucleic acids are synthesized and have to self-assemble into a fully infectious viral particle. In particular, viral nucleic acids have to be compacted and packaged into the capsid. This step is crucial for the survival of the virus and must be carried out in a rapid and error-free manner. In particular, the capsid must uptake only the viral genome and not the nucleic acids coming from the host cell, which suggests some selectivity on the part of the capsid proteins.

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All set to work through the weekend, the team members take a short break to pose in front of ID02 experimental hutch. From left to right, front row: Johannes Moeller, ESRF post-doc and local contact for the experiment, Maelenn Chevreuil, Doru Constantin (LPS Orsay); middle row: Stéphane Bressanelli, Thibault Tubiana (I2BC, Gif sur Yvette), Jingzhi Chen (LPS Orsay); back row: Guillaume Tresset, Mehdi Zeghal (LPS Orsay).

The self-assembly of viral particles can be performed in vitro from purified capsid proteins and ssRNA. The process is multiscale, with events occurring over milliseconds up to several hours and with molecules sized in the nanometre scale (from a few to tens of nanometres).  “TR-SAXS is an ideal technique of investigation. Because the scattering intensity of molecular species is quite low, only synchrotron sources are able to probe the processes with accuracy. The ESRF’s ID02 beamline is capable of achieving a time resolution of a few milliseconds, which is crucial to capture the early events”, says Guillaume Tresset.

Single-stranded (ss)RNA viruses often follow a simple architecture with an icosahedral capsid containing the genome in the form of ssRNA.

The team is expecting to answer a long-standing question in the field: do capsid proteins bind first to the genome prior to forming an icosahedral shell or is the icosahedral shell built up simultaneously with the uptake of the genome?

The scientists are from two laboratories in the Paris region, the LPS (Laboratoire de Physique des Solides) in Orsay, and the I2BC (Institut de Biologie Intégrative de la Cellule) in Gif sur Yvette.

Top image: Members of the team (L to R), Mehdi Zeghal, St├ęphane Bressanelli and Guillaume Tresset examine the set-up in the experimental hutch of ID02. Credit: ESRF/C. Argoud