This color-enhanced image, taken with scanning electron microscopy, shows huge amounts of SARS-CoV-2 particles (purple) that have burst from kidney cells (green) that the virus hijacked for replication. The domed, spherical cells in the upper right and lower left corners are distorted and about to burst from the virus particles inside and begin to self-destruct. (Image: NIAID Integrated Research Facility)
In February 2020, a trio of bio-imaging experts were sitting amiably around a dining table at a science conference in Washington, DC when the conversation shifted to a virus epidemic in China, which was then of great concern. Without foreseeing the impending global catastrophe, they wondered aloud how they could contribute to it.
Almost a year and a half later, these three scientists and their many collaborators in three national laboratories published a comprehensive study in the Biophysical Journal that – along with other new, complementary studies on coronavirus proteins and genetics – represents the first step in developing treatments for it Viral infection now burned into global consciousness as COVID-19.
Her foundational work focused on the protein-based machine that enables the SARS-CoV-2 virus to hijack the molecular machinery of our own cells in order to replicate in our bodies.
From structure to function to solutions
“It has been noticed that all organisms are just a means for DNA to make copies of itself, and nowhere is this more true than in the case of a virus,” said Greg Hura, a research fellow at the Lawrence Berkeley National Laboratory Lab). and one of the study’s lead authors. “The unique job of a virus is to make copies of its genetic material – unfortunately at our expense.”
Viruses and mammals, including humans, have been stuck in this battle for millions of years, he added, and over time the viruses have developed many tricks to copy their genes inside us while our bodies developed defense mechanisms. And while viruses often do a long list of other activities, their ability to harm us with infection really depends on whether they can replicate their genetic material (either RNA or DNA, depending on the species) to make more virus particles, and use our cells to translate their genetic code into proteins.
The protein-based machine responsible for RNA replication and translation in coronaviruses – and many other viruses – is known as the RNA transcription complex (RTC) and is a truly formidable piece of biological weaponry.
A depiction of the SARS-CoV-2 machinery illustrating its ability to quickly change structural arrangements – like a bicycle changing gears – to perform various tasks. (Photo credit: Greg Hura / Berkeley Lab)
In order to successfully duplicate viral RNA for new virus particles and to produce the many proteins of the new particles, the RTC must: distinguish between viral and host RNA, recognize and pair RNA bases instead of very similar DNA bases that are also in human cells are abundant, converting their RNA to mRNA (to fool human ribosomes into translating viral proteins), interface with copy-error-checking molecules, and transcribe specific sections of viral RNA to amplify certain proteins over others as needed – while at the same time trying to bypass the host’s immune system that will recognize it as a foreign protein.
As amazing as this may sound, every newly developed virus that is successful “must have incredibly sophisticated machines in order to overcome the mechanisms we developed,” explained Hura, who integrated the structural biology division into the molecular biophysics division Directs Berkeley Lab’s bioimaging division.
He and the other study leaders – Andrzej Joachimiak from Argonne National Laboratory and Hugh M. O’Neill from Oak Ridge National Laboratory – specialize in uncovering the atomic structure of proteins in order to understand how they function at the molecular level. So from the moment they first discussed COVID-19 at the dining table, the trio knew that studying the RTC would be a particular challenge, as multitasking protein machines like the RTC are not static or rigid, like molecular diagrams or balls -and stick models could suggest. They are flexible and have associated molecules, so-called non-structural and accessory proteins (Nsps), which, depending on the task, are available in a variety of quickly rearranged forms – similar to how a gear lever on a bicycle quickly adapts the vehicle to changing terrain.
Each of these Nsp arrangements gives insight into the different activities of the protein, and they also reveal different parts of the entire RTC surface that can be examined to find sites where potential drug molecules could bind and inhibit the whole machine.
After their chance meeting in Washington, the trio forged a plan to pool their knowledge and national laboratory resources to document the structure of as many RTC arrays as possible and identify how these shapes interact with other viral and human molecules.
Science during shutdown
The research relied on the combination of data from many advanced imaging techniques, as no approach alone can produce complete blueprints of infectious proteins in their natural state at the atomic level. They combined small-angle X-ray scattering (SAXS), X-ray crystallography, and small-angle neutron scattering (SANS) performed at Berkeley Lab’s Advanced Light Source, Argonne’s Advanced Photon Source, and Oak Ridge’s High Flux Isotope Reactor and Spallation Neutron source, respectively on samples of biosynthetically produced RTC.
“Aside from the complexity of the virus system, the work during the pandemic was very tough. But more than anything we have ever done, we were driven to this research by all the suffering families across the country and around the world experience. ”- Greg Hura, photographed working on the SAXS ALS beamline in June 2020 (Source: Thor Swift / Berkeley Lab)
Despite the extraordinary hurdles in conducting science under protective conditions, the collaboration was made possible thanks to funding for research and operation of the facility from the Department of Energy’s Office of Science National Virtual Biotechnology Laboratory (NVBL). During this time, the scientists collected detailed data on the main accessory proteins of the RTC and their interactions with RNA. All of their results were uploaded to the open access protein database prior to the publication of the journal article.
Among the many structural insights that will help in drug design, one notable discovery is that the assembly of the RTC subunits is incredibly precise. Again based on a mechanical metaphor, the scientists compare the assembly process with the assembly of a spring-based machine. You cannot insert a spring if the rest of the machine is already in place, you will have to compress the spring and place it at a certain assembly step, or the entire device will be inoperable. Likewise, the RTC-Nsps cannot move into their place in random or chaotic order; they have to follow a certain order of operations.
They also identified how one of the Nsps specifically recognizes the RNA molecules it is acting on and how to cut long strands of copied RNA into the correct length.
“Having the vaccines is certainly huge. But why are we only satisfied with this one line of defense? ”Said Hura. Joachimiak added: “This was a survey study and it has shown many directions that we and others should be following very closely. In order to fight this virus, we will need several ways to block its spread. “
“The combination of information from various structural techniques and calculations will be key to achieving this goal,” said O’Neill.
Because of the similarity of RTC proteins across virus strains, the team believes that any drugs designed to block RTC activity, in addition to any COVID-19 variants, could work in multiple viral infections.
Looking back at the beginning of their research trip, the scientists are amazed at the happy timing of it all. When we started talking, Hura said, “We had no idea that this epidemic was about to turn into a pandemic that would change a generation.”
This study was supported by the DOE Office of Science through the NVBL, a consortium of national DOE laboratories focused on responding to COVID-19, with funding from the Coronavirus CARES Act; and by the National Institutes of Health. The Advanced Light Source, Advanced Photon Source, High Flux Isotope Reactor and Spallation Neutron Source are user facilities of the DOE Office of Science.
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Lawrence Berkeley National Laboratory, founded in 1931, and its scientists have been awarded 14 Nobel Prizes. Today, researchers at the Berkeley Lab are developing sustainable energy and environmental solutions, developing useful new materials, pushing the boundaries of computer science and exploring the secrets of life, matter and the universe. Scientists from around the world rely on the laboratory’s facilities for their own discovery research. Berkeley Lab is a national multi-program laboratory run by the University of California for the US Department of Energy’s Office of Science.
The DOE’s Office of Science is the single largest funder of basic research in the physical sciences in the United States, working to address some of the most pressing challenges of our time. More information is available at energy.gov/science.
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