In the early days of the COVID-19 pandemic, as Diana Bautista and others in her UC Berkeley lab suffered sneezing and coughing fits and achy bodies, she recognized an interesting scientific question: How, exactly, do viruses cause the noxious symptoms we're all familiar with from bouts with colds, the flu and COVID?
Five years later - and after a detour to prove to scientific reviewers that her experimental subjects, mice, do sneeze - she has the beginnings of an answer. She now devotes more than half of her energy and lab staff to investigating the wiles of coronaviruses like SARS-CoV-2 and how they interact with the nervous system and the immune system to give us a headache - literally.
In the Nov. 13 issue of the journal Neuron, she and her colleagues report that SARS-CoV-2 proteins stimulate sensory nerves directly, triggering sneezing, coughing and pain. The prevailing view was that these symptoms come from inflammatory mediators released by infected epithelial and immune cells acting on sensory neurons. This study shows there is another route by which viruses trigger sneezing, coughing and respiratory dysfunction.
This is the first evidence that viral proteins can directly activate sensory nerves to promote viral symptoms, and could lead to therapeutic interventions. This direct activation may also help explain some of the chronic conditions associated with long COVID, including headache, shortness of breath, persistent cough, sore throat and chest pain.
The specific viral protein that triggers the sensory nerves is a known protease called PLpro, for papain-like protease. It and another protease, called Mpro - for main protease - are produced in cells infected with the virus, and cuts the long viral polyproteins into the individual proteins crucial to replication. After the virus kills these cells, the contents - including the viral proteases - are released into the body and stimulate a subset of sensory nerves called nociceptors, which are found throughout the airways and are particularly sensitive to noxious stimulants.
While the drug Paxlovid targets Mpro and alleviates some symptoms of COVID-19, targeting PLpro might also have benefits. Since the pandemic began, numerous drugs targeting PLpro have been developed, though none were approved for COVID.
"We think that these protease inhibitors that people spent a lot of time and money developing could be important not just for preventing viral replication," said Bautista, a Berkeley professor of molecular and cell biology and a Howard Hughes Medical Institute investigator. "They could also help treat symptoms like coughing, which could limit viral spread, and potentially prevent severe COVID-19 in individuals who are susceptible."
The researchers also found that Mpro directly triggers sensory neurons, though it triggers different reactions than does PLpro. They're finding that other viruses, like the influenza virus, trigger symptoms in still other ways.
"There are many modes by which viral and bacterial infections can act in our bodies - there are distinct molecular and cellular routes of triggering this type of airway hyper-reactivity," she said. "With so many emerging pathogens, we're just starting to understand how they work. So far, it looks like they can drive symptoms through both the immune system and the nervous system, using different molecular mechanisms. I think we have a lot to figure out."
That raises the question: Are symptoms like coughing and sneezing primarily protecting the host by expelling the virus, or are they helping the virus reach more people?
"Who is it helping?" she said. "Maybe it's a little bit of both in the end. If it's early enough, then it's beneficial to get out as much of that virus as possible before it can infect other cells. So it's good having these symptoms early on to sneeze out mucus containing the virus."
Do mice sneeze?
Bautista specializes in studying the sensory neurons involved in touch, itch and pain, and in earlier work identified the specific receptor, called TRPA1, that responds to chemicals in wasabi and garlic. She has also conducted research on how chemicals affect the trigeminal nerves that innervate the face - nerves that can get inflamed and become painful during a COVID infection.
Diana Bautista lab/UC Berkeley
In a sense, research on how coronaviruses interact with sensory nerves was a natural extension of this earlier work. In the current paper, Bautista and her colleagues show that PLpro reacts with something on the surface of sensory neurons to activate the TRPA1 ion channel - the same channel activated by wasabi and garlic - to trigger pain, sneezing, cough and airway inflammation. TRPA1 essentially causes the release of neuropeptides that go on to trigger reflexes and other physiological consequences.
To demonstrate the role of PLpro, she and her team purified the protein and showed that the protein alone was sufficient to activate cultured sensory neurons. When the enzymatic activity of the protease was eliminated through inactivation of the protein's catalytic site, it had no effect on sensory neurons.
The researchers then dabbed the noses of mice with 2 microliters of purified PLpro. While water alone might trigger a single sneeze, PLpro triggered up to 30 sneezes per minute.
Despite audio and video recordings of mice sneezing, some reviewers of the paper were not convinced that mice do, in fact, sneeze. So she purchased a $75,000 plethysmograph to measure the lung spasms that accompany the sneeze, which turned out to be similar to those in other sneezing animals, like guinea pigs. Her team even dyed the liquid containing PLpro blue so that they could see the expelled snot and measure the amount in each sneeze.
They also discovered that PLpro triggered mechanical hypersensitivity and pain, suggesting that direct activation of sensory nerves might be a mechanism that triggers the aches and pains associated with viral infection.
PLpro from two other varieties of coronavirus, SARS-CoV-1 and MERS, also triggered sensory neurons.
Cailin Hoang/UC Berkeley
While the new paper answers some questions about how viruses interact with the nervous system - until now, an open question in biology - her lab has much more work to do.
"This opened up lots of areas of research," said Bautista. "If just two viral proteins can have such a dramatic consequence on the nervous system, what else are viruses and other viral proteins doing? What happens when the neurons themselves get infected, which we know can happen? How are the neurons contributing to the antiviral response? There are many questions.
"I guess that's why so many people in my lab want to work on this, because, from a biological standpoint, it's just fascinating. How did we not think about this before?"
The work was funded by grants from the National Institutes of Health (OD TR01 NS116992) and the Howard Hughes Medical Institute. Co-authors of the paper are Sonali Mali, Ricardo Silva, Zhongyan Gong, Michael Cronce, Uyen Vo, Cliff Vuong and Jeffery Cox of UC Berkeley and Yalda Moayedi of the New York University College of Dentistry. Bautista is a member of Berkeley's Helen Wills Neuroscience Institute.
