Biologists have used crystallography conducted at Berkeley Lab’s advanced light source to reveal the unusual protein structure of the new virus.
A team of HIV researchers, cell biologists and biophysicists who collaborated together to support the science of COVID-19 has identified the atomic structure of a coronavirus protein that is believed to help the pathogen evade and inhibit the human immune cell response. The Skeleton Map – which is now published in the journal PNAS, But has been open to the scientific community since August – it laid the groundwork for new antiviral therapies specifically designed for SARS-CoV-2, and enabled further investigations into how the new virus was destroying the human body.
“Using X-ray crystallography, we constructed an atomic model of ORF8, highlighting two unique regions: one only present in SARS-CoV-2 and its direct predecessor, and the other absent from any other coronavirus,” said lead author James Hurley, a professor at the University California at Berkeley and former faculty scientist at Lawrence Berkeley National Laboratory (Berkeley Laboratory). These regions fix the protein – a secreted protein that is not bound to a membrane like the virus’s characteristic spike proteins – and create interfaces between new molecules. We, and others in the research community, believe that these interfaces are involved in the reactions that make SARS-CoV-2 more infectious than the strains that evolved from them.
Structural Biology in the Spotlight
Generating maps of protein structure is always labor intensive, as scientists have to engineer a bacterium that can pump large amounts of the molecule, manipulate the particles into pure crystal form, and then take many X-ray diffraction images of the crystals. These images – which are produced when X-ray beams bounce off the atoms in the crystals and pass them through gaps in the lattice, creating a pattern of spots – are collected and analyzed via a special program to locate each individual atom. This painstaking process can take years, depending on the complexity of the protein.
For many proteins, the map-building process is aided by comparing the structure of the unsolved molecule to other proteins with similar amino acid sequences that have already been mapped, allowing scientists to make informed guesses about how the protein is folded into its 3D shape.
But for ORF8, the team had to start from scratch. The amino acid sequence of ORF8 is completely different from any other protein in that scientists had no reference to its general shape, and it is the three-dimensional shape of the protein that determines its function.
Hurley and colleagues at UC Berkeley, with expertise in the structural analysis of HIV proteins, worked with Mark Alier, a biophysicist and crystallography expert at the Berkeley Center for Structural Biology, located in the Advanced Light Source (ALS) in the Berkeley Lab. The team worked together to increase the velocity for six months – Hurley’s lab produced crystal samples and sent them to Allaire, who would use X-ray lines for ALS to capture diffraction images. It took hundreds of crystals with multiple copies of the protein and thousands of diffraction images analyzed by special computer algorithms to solve the puzzle of ORF8 structure together.
Coronaviruses develop differently from viruses such as influenza or HIV, which rapidly accumulate many small changes through a process called hypermobility. In coronaviruses, large parts of nucleic acids sometimes move through recombination, ”Hurley explained. When this happens, large and new regions of proteins can appear. Genetic analyzes conducted very early in the SARS-CoV-2 pandemic revealed that This new strain evolved from the Corona virus that infects bats, and that an important recombinant mutation occurred in the genome region that codes for a protein called ORF7, found in many corona viruses. The new form of ORF7, called ORF8, quickly attracted the attention of virologists and epidemiologists because Important genetic variation events such as those seen in ORF8 are often the cause of the virulence of a new strain.
“Essentially, this mutation caused the protein to double in size, and the substance that duplicated was not associated with any known fold,” Hurley added. “There is a nucleus of about half of it attached to a known fold type in a structure resolved from previous coronaviruses, but the other half was brand new.”
Answer the call
Like many scientists who work on COVID-19 research, Hurley and his colleagues chose to share their findings before publishing the data in a peer-reviewed journal, allowing others to initiate impactful follow-up studies months before the traditional publication process. Allowed. As Alier explained, the hands-on crisis caused by the pandemic has turned everyone in the research community into a pragmatic mindset. Instead of worrying about who got something done first, or sticking to the boundaries of their specific fields of study, scientists shared data early and often, and took on new projects when they had the necessary resources and experience.
In this case, Hurley’s UC Berkeley co-authors had viral protein expertise and crystallography, and Allaire, a longtime collaborator, was also up the hill with expertise in crystallography, and most importantly, the ray line was still working. The ALS received private CARES Act funding to keep running for COVID-19 investigations. The team learned through a review of the SARS-CoV-2 genetic analysis published in January that ORF8 was an important piece of the pandemic puzzle (which was more dangerous at the time), so they got to work.
The authors have since moved on to other projects and are satisfied that they have laid the groundwork for other groups to study ORF8 in more detail. (Currently, there are several investigations underway focusing on how ORF8 interacts with cell receptors and how it interacts with antibodies, as affected individuals appear to produce antibodies that bind to ORF8 in addition to antibodies specific to the surface proteins of the virus.)
“When we started this, other projects were put on hold, and we had this unique opportunity to stick together and solve a pressing problem,” said Aller, part of the division of molecular biophysics and integrated bioimaging at Berkeley Lab. “We worked very closely, with a lot of back and forth, until we made it through. It was really one of the best collaborations of my career.”
Lawrence Berkeley National Laboratory and its scientists were founded in 1931 on the belief that the best scientific challenge is to engage with teams, and they have won 14 Nobel Prizes. Today, Berkeley Lab researchers develop environmental and sustainable energy solutions, create useful new materials, push the frontiers of computing, and probe the secrets of life, matter and the universe. Scientists from all over the world rely on laboratory facilities for their Discovery Science. Berkeley Lab is a national multi-program laboratory, administered by the University of California for the US Department of Energy’s Science Office.
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