Unpacking SARS-CoV-2

Faculty teaches graduate student about cryo-EM system, backlit and opened.

Less than a year after he joined the Department of Biochemistry, Robert Kirchdoerfer BS’06 and his nascent coronavirus research program were thrust into the spotlight. The new assistant professor was quickly becoming known around the UW–Madison campus as “the coronavirus guy,” a linchpin of efforts at the university to understand the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19.

Kirchdoerfer is an expert in using cryo-electron microscopy and other advanced techniques to characterize viral proteins and complexes. As a graduate student and then as a postdoctoral researcher at The Scripps Research Institute in Southern California, he was a member of one of the first teams to stabilize spike proteins, notoriously tricky proteins that rapidly change configurations to adopt a shape that’s not relevant for recognition by antibodies. By keeping spike proteins in a single configuration, scientists could use cryo-electron microscopy (cryo-EM), an imaging technique used to visualize molecules on sub-nanometer scales, to study the spike protein’s structure, investigate the transitions the protein takes to recognize host protein receptors, and design vaccines with it as an active ingredient.

Today at UW–Madison, Kirchdoerfer and his colleagues are studying the structures and functions of proteins and RNA synthesis complexes from SARS-CoV-2. Their research, which synthesizes expertise and results from several disciplines and state-of-the-art technologies, demonstrates just how much of the “life cycle” of SARS-CoV-2 remains to be discovered—every research study, every experiment, informs another, and lives hang in the balance.

Complementary Data

In spring 2020, scientists at the National Magnetic Resonance Facility at Madison (NMRFAM), a campus-wide and national facility housed in the biochemistry department, were looking for ways to contribute to the fight against COVID-19. Nuclear magnetic resonance (NMR) spectroscopy could complement Kirchdoerfer’s cryo-EM research, said biochemistry professor and NMRFAM co-director Katherine Henzler-Wildman, by providing new insights into parts of the virus that are too small to study individually with cryo-EM.

Henzler-Wildman and her NMRFAM co-director, biochemistry professor Chad Rienstra, decided to study the membrane protein and two nonstructural proteins, nsp7 and nsp8, as part of an international consortium called the COVID19-NMR Project. Improved knowledge of nonstructural proteins, parts of the RNA synthesis machinery responsible for replicating and transcribing the viral genome after a virus infects a host cell, can lead to antiviral drugs that halt a virus’ replication process.

The scientists’ NMR experiments, performed using protein produced by Kirchdoerfer’s lab, confirmed that SARS-CoV-2 nsp7 is spectroscopically and structurally similar to the nsp7 in the original SARS virus, SARS-CoV. In an ideal world, their studies on nsp8 and the membrane protein would also be straightforward. But that isn’t how research often progresses.

“We can’t say much more right now, but our results for nsp8 aren’t what we expected,” remarks Henzler-Wildman. “We thought that nsp8, which joins with other nonstructural proteins like nsp7 to form the larger complexes necessary for replicating the virus’ genetic material, would be dynamic with multiple conformations in solution. It actually has concentration-dependent oligomerization.” Now, the scientists must consider a different set of potential dynamics, including what nsp8 does in solution and how it ends up in its various conformations.

Graphic of SARS-CoV-2 genome and proteins.
Illustration of SARS-CoV-2 genome and proteins. Department of Biochemistry scientists have improved our knowledge of the spike protein, membrane protein, nsp7, nsp8, nsp12 and more. Used with permission from RCSB PDB-101.

The NMRFAM team faced a different challenge with the membrane protein: it had never been reliably produced in a laboratory. So, while scientists believe the protein plays an important role in viral “budding,” a process viruses can use to exit host cells, the protein remains understudied—and, some experts say, underutilized—in the fight against COVID-19.

By fall 2021, the UW–Madison team was making significant progress at purifying the membrane protein and was pondering their next steps. Data they collect may be especially important: research being conducted a few feet away suggests that this protein may be an active ingredient in the next SARS-CoV-2 vaccine.

The Next Vaccine Candidate?

In a collaboration with researchers at UW–Madison’s School of Medicine and Public Health, preeminent virologist Ann Palmenberg had been identifying molecular interactions between rhinovirus-C, a virus closely linked to wheezing and asthma, and its cellular receptor when the pandemic hit.

“Basically, we’re dissecting components of the immune system down to the biochemical level of, what are the antibodies you want to induce, what are the antibodies you don’t want to induce, and why does one work but not another,” Palmenberg, a biochemistry professor and Institute for Molecular Virology affiliate, explains. “We were just about to make the next batch of chips and collect data [on rhinoviruses]…when COVID-19 came. We said, you know what, instead of designing the rhinovirus sequences on this chip, let’s put coronavirus sequences on it.”

The peptide array technology Palmenberg used was a brainchild from UW–Madison scientists including biochemistry professor and Biotechnology Center affiliate Michael Sussman (this technology was transferred to Roche, a Swiss multinational healthcare company). Each chip contains the entire genome of a virus in the form of peptides, or fragments of proteins. By identifying where antibodies stick on these fragments, and by comparing this information to viral structures from cryo-EM, scientists can pinpoint how to kill a virus.

Experimental workflow graphic.
Tiling peptides on peptide binding arrays. Image from Irene Ong, an assistant professor in the School of Medicine and Public Health, an example of serum reactivity against a rhinovirus (RV) capsid peptide array.

Palmenberg and her collaborators decided to pivot their long-standing rhinovirus-C project to study how protein snippets from SARS-CoV-2 and the six other coronaviruses known to infect humans responded to plasma samples from two groups of people—patients with COVID-19 and individuals who hadn’t been exposed to the virus. Nimble Therapeutics, a Madison-based company spun out of Roche Sequencing Solutions in 2019, built the chips at a substantial discount, and Kirchdoerfer, an Institute for Molecular Virology affiliate, helped the scientists match antibody-sequence pairs from the protein chips to structures from cryo-EM.

Their results demonstrate that humans mount strong, broad antibody responses to the spike, membrane, and nucleocapsid proteins. Because the immunogenicity of spike-based mRNA vaccines is variable, and because not all individuals who get COVID-19 produce detectable antibodies against the spike or nucleocapsid proteins, the scientists suggest that membrane proteins could be a promising target for future SARS-CoV-2 diagnostics, vaccines, and therapeutics.

Signal from Noise

Though his role may not always be obvious, Kirchdoerfer has played a part in many projects that aim to understand SARS-CoV-2. He’s had a hand, for example, in devising new strategies to characterize the activity of enzymes, substances that act as catalysts in biological processes. This project, led by Michael Sussman, is expected to be important for rapid, timely characterization of enzymatic activity in SARS-CoV-2.

Kirchdoerfer’s own ongoing research using cryo-EM may also provide insights into the workings of the SARS-CoV-2 RNA synthesis complex and lead to new antiviral drugs that could help treat patients with COVID-19.

“Rob works on a number of fronts surrounding how coronaviruses function, from isolated components to intact viruses. To investigate how SARS-CoV-2 replicates, he has started by assembling and examining the structure and function of components of the virus’ replication complex,” says biochemistry professor and Morgridge Institute for Research affiliate Elizabeth Wright.

Spike protein structures
Spike proteins from SARS-CoV (left) and SARS-CoV-2 (right). Only the SARS-CoV is shown bound to angiotensin converting enzyme 2 (ACE2), orange. Image courtesy of Robert Kirchdoerfer.

“He does the fundamental molecular biology, protein expression, and functional assays in his lab to determine if samples are of sufficient quality for cryo-EM imaging. We at the Cryo-Electron Microscopy Research Center then support him during the sample preparation, imaging, and initial data processing steps,” Wright says. The center, which Wright directs, provides services to UW investigators who are working on SARS-CoV-2 and other projects.

Kirchdoerfer brings technologies and techniques from multiple disciplines—virology, structural biology, cell biology, and biochemistry, among them—together to explore the function of enigmatic SARS-CoV-2 proteins. But cryo-EM remains a mainstay of his work.

“A lot of the strengths for looking at SARS-CoV-2 with cryo-EM are the general strengths of the technique,” he says. “You don’t need a crystal. It’s great for larger complexes. We also have different moving parts in this machine…With cryo-EM, so much of the data is handled computationally to address that movement that we can access even those moving regions.”

But understanding SARS-CoV-2 isn’t his end goal—it’s just the beginning.

“During an outbreak, there’s intense scientific interest, but as soon as that outbreak ends, interest also ebbs. What I would like to do with my lab is more pandemic preparedness—looking for the next virus that’s going to cause a pandemic.”

To that end, Kirchdoerfer is studying other coronaviruses and other virus families, and he’s kicking off projects on viral entry—how viruses recognize cells, enter cells, and how virus evolution tunes the spike protein to undergo fusion with host cells—in collaboration with classical virologists, veterinary biologists, and epidemiologists at UW–Madison.

Beyond Science

Kirchdoerfer and his colleagues are just now getting back to research they were working on before the pandemic started. But their pandemic-related outreach, service and teaching activities continue.

Biochemistry professor Paul Friesen PhD’83 has taught biochemistry courses for majors and non-majors for nearly two decades. His teaching philosophy—getting students to understand not only the what but also the why of diseases and disease prevention—hasn’t changed during the pandemic. Though he jokes that he didn’t have gray hair before the week that courses went online, he’s more certain than ever about the importance of the university’s connection to students and the rest of the community.

“I like to talk about general principles so students can go back and advocate, to their parents, to their neighbors, to whomever, that science is important. Now, that has taken on a more dramatic role,” reflects Friesen.

When he wasn’t teaching, Friesen was thinking of ways to keep his students engaged and connected. Since most of his students were off campus, he brought campus to them by taking photos of familiar sights and sharing them during lectures. To re-create the in-classroom experience, he even recorded student lectures in a lecture hall, and he emphasized SARS-CoV-2 in all his classes.

Friesen, who’s director of the Institute for Molecular Virology, has also responded to public queries about the virus—whenever, wherever. He recalls a time early in the pandemic when he shared what he knew about viruses, and SARS-CoV-2 in particular, to commuters on a packed Madison Metro bus. Palmenberg, who has spent much of the pandemic reviewing grant applications so that scientists can acquire funding to study SARS-CoV-2, likewise fielded countless phone calls and emails. And since January 2020, Kirchdoerfer has participated in 24 interviews and panels (not including conversations for this story) and counting.

Whether they are studying a new virus, sharing knowledge with inquisitive minds or engaging communities throughout Wisconsin, scientists in the Department of Biochemistry manifest one exceptional quality: creativity.

“Often people think of scientists as being incredibly objective and precise, and that’s very true. But it really comes down to creativity, I think, in trying to bring together pieces of data that on the surface might not appear to talk to one another. It’s a little bit of an art form—but then, an art form you go back to test,” Kirchdoerfer says.

Written by Catherine Steffel, Ph.D. Please direct media inquiries and questions about this work to communications@biochem.wisc.edu.