Astroviruses are small, non-enveloped RNA viruses that infect the small intestine, where they cause gastroenteritis that can be particularly severe in young children, the elderly, and immunocompromised patients. Despite their clinical importance, fundamental aspects of the astrovirus replication cycle have remained poorly resolved, including the deceptively simple question of how newly assembled viral particles leave the cells that produce them. The research team, led by Natalie Pedicino and colleagues, reasoned that the answer might lie in a distinctive feature of astrovirus tropism: these viruses preferentially infect goblet cells, the specialized epithelial cells of the intestinal lining whose entire existence is organized around the production and secretion of mucus.

Goblet cells are the mucus factories of the gut. They synthesize enormous quantities of mucins, the heavily glycosylated proteins that give mucus its gel-like protective properties, and store them in membrane-bound secretory granules packed within the cytoplasm. When appropriately stimulated, goblet cells undergo regulated exocytosis, fusing these granules with the plasma membrane and expelling their mucin cargo into the intestinal lumen. This process, known as mucus secretion, is a fundamental component of intestinal barrier function, and it is driven in large part by intracellular calcium signals that trigger granule fusion. Because astroviruses infect precisely the cells that perform this function, the researchers hypothesized that the viruses might have evolved to exploit the secretory pathway itself, packaging themselves into the same cellular cargo that goblet cells are built to release.

To test this hypothesis, the team turned to the murine astrovirus model, a well-established experimental system that permits the study of astrovirus infection in its natural intestinal context. Examining infected intestinal tissue, the researchers made a striking observation: the release of virus from infected cells co-occurred almost exclusively with episodes of mucus secretion from goblet cells. Wherever virus was being shed, mucus was being expelled, and the two events appeared to be tightly coupled rather than merely coincidental. This spatial and temporal association suggested that viral egress was not an independent process but was physically and mechanistically linked to the secretory activity of the infected goblet cells.

The correlation extended beyond individual cells to the level of the whole tissue. When the researchers tracked infection kinetics over time, they found that mucus secretion rates rose and fell in parallel with viral load. As the infection progressed and the immune system began to clear the virus, mucus secretion returned toward baseline levels, mirroring the decline in viral shedding. This coordinated pattern reinforced the idea that the secretory state of the tissue and the propagation of the virus were intimately connected throughout the course of infection, not just at a single snapshot in time.

Correlation alone, however, cannot establish causation, so the researchers next manipulated mucus secretion directly and asked what happened to viral release. When they disrupted mucin biosynthesis, thereby impairing the goblet cells’ ability to produce and package their mucus cargo, viral shedding from infected tissue was reduced. Conversely, when they stimulated mucus secretion pharmacologically using carbachol, a drug that activates muscarinic receptors and triggers the calcium-dependent secretory pathway, viral release increased. The bidirectional nature of this effect, in which dampening secretion reduced virus release and enhancing secretion boosted it, provided strong functional evidence that the mucus secretory pathway is not merely a passive correlate of infection but an active route of viral egress.

To determine whether this mechanism extends to human astroviruses, which are clinically the most relevant, the team needed an appropriate cell culture system. They established a new infection model using LS174T cells, a human cell line derived from a colonic adenocarcinoma that retains goblet-cell-like properties, including the capacity to produce and secrete the major intestinal mucin MUC2. In these infected goblet-like cells, the researchers observed that human astrovirus particles colocalized with MUC2 within the cell, suggesting that the virus and the mucin cargo occupy the same intracellular compartments during the secretory process. The association was further confirmed using purified mucin granules, with viral particles found in association with the isolated granules themselves.

The pharmacological experiments in the human cell model recapitulated and extended the findings from the murine system. Stimulating LS174T cells with carbachol enhanced the release of both mucus and virus, and this dual effect could be blocked by the addition of a muscarinic receptor antagonist, which prevents the receptor-mediated activation of the secretory pathway. The researchers also probed the intracellular signal that drives granule exocytosis: calcium. When they blocked intracellular calcium signaling, the trigger that normally causes mucin granules to fuse with the plasma membrane, virus release was reduced. Together, these experiments traced the egress pathway from receptor activation through calcium signaling to granule exocytosis, showing that human astrovirus exploits each step of the canonical mucus secretion machinery.

The significance of these findings lies in what they reveal about both the astrovirus replication cycle and the broader biology of non-enveloped enteric viruses. Egress is the step of the viral life cycle that determines how efficiently a virus spreads from cell to cell and, ultimately, from host to host. By demonstrating that astroviruses use mucus secretion as a non-lytic form of egress, the study resolves a longstanding gap in understanding how these viruses complete their replication cycle. It also suggests a possible explanation for aspects of astrovirus pathogenesis: a virus that exits the cell by hitching a ride on mucus is delivered directly into the luminal mucus layer of the intestine, positioning itself for transmission while potentially modulating the barrier and immune functions of the mucus itself. The tight coupling between viral shedding and secretory activity may therefore have consequences that extend beyond viral spread to the diarrhea and intestinal dysfunction that characterize astrovirus disease.

More broadly, the work adds mucus secretion to the repertoire of host pathways that non-enveloped viruses can commandeer for exit, joining mechanisms such as extracellular vesicle release and cell-to-cell spread that have been described for other enteric pathogens. It also highlights the value of studying viruses in the context of the specific cell types they naturally infect. The connection between astroviruses and goblet cells was the key insight that made this discovery possible, and it serves as a reminder that viral strategies are often best understood not in generic cell culture systems but in the specialized cellular environments where infection actually occurs. As researchers continue to probe how this novel egress pathway shapes astrovirus transmission and disease, the findings open new avenues for therapeutic intervention, since drugs or interventions that modulate mucus secretion could, in principle, influence viral shedding and the course of infection.

Subject of Research: Non-lytic egress of astroviruses through mucus secretion from intestinal goblet cells

Article Title: Astroviruses use mucus secretion as a novel form of viral egress

Article References: Pedicino, N., Kirkpatrick, M. G., Pablo, M. A., Gulman, J., Heredia-Osuna, N., & Cortez, V. (2026). Astroviruses use mucus secretion as a novel form of viral egress. PLOS Pathogens, 22(10), e1014683. https://doi.org/10.1371/journal.ppat.1014683

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014683

Keywords: astrovirus, viral egress, goblet cells, mucus secretion, MUC2, mucin granules, pediatric diarrhea, non-enveloped viruses, calcium signaling, carbachol, LS174T cells, murine astrovirus

The study, published September 10 in the journal Science, was led by senior author Stephen Harrison, the Giovanni Armenise-Harvard Professor of Basic Medical Sciences at Harvard Medical School, a Howard Hughes Medical Institute Investigator, and a professor of pediatrics at Boston Children’s Hospital. First author Marilina de Sautu, a research fellow in the Harrison Lab, carried out the work in close collaboration with Simon Jenni, an HMS research scientist in biological chemistry and molecular pharmacology. The team used a strain of rotavirus, a pathogen best known for causing severe diarrheal disease in young children, as a model system for probing how non-enveloped viruses deliver their packaged genomes across a membrane barrier.

Previous work from the Harrison Lab and other groups had established the early stages of rotavirus entry. The virus first attaches to the cell membrane and is then taken up by the cell, becoming engulfed within a membrane-bound compartment inside the cytoplasm. At that point, the virus faces a fundamental problem: its genetic material, packaged inside a protein shell, must cross the membrane of that compartment to reach the cell interior, where it can hijack the host’s molecular machinery and begin replicating. What remained unknown was the identity of the molecular agent responsible for perforating the membrane and the precise mechanism by which it operates.

The new study answers both questions. The researchers showed that the process begins when virus protein 5, known as VP5, makes the surrounding membrane permeable to calcium ions. The resulting loss of calcium triggers a cascade: virus protein 7, or VP7, dissociates from the virus particle and then forms a pore in the membrane. Through that pore, the virus can eject its RNA into the cell. The finding resolves a long-running debate about which of the viral proteins performs the decisive membrane-perforating step, and it reveals an unexpected division of labor between two structural proteins that were already known to play roles in the virus’s architecture.

For Harrison, the outcome carried a personal surprise. He had long assumed that VP5, which had been implicated in membrane interactions, would be the protein that punched the hole. Instead, the evidence pointed to VP7. He noted that earlier work by Philip Dormitzer, a former colleague at Harvard Medical School and Boston Children’s Hospital who is now a biopharmaceutical executive, and by their former student Shane Trask, had hinted that VP7 might be the responsible agent. The confirmation, Harrison said, represents a particularly satisfying form of hole punching, one that no one had directly visualized before.

The technical breakthrough that made the discovery possible is cryo-electron tomography, or cryo-ET, a relatively new extension of cryo-electron microscopy. In cryo-ET, an ultrathin biological sample is flash-frozen in a glass-like state of vitreous ice, preserving its native structure without chemical fixation or staining. The microscope then records high-resolution images from many angles as the sample tilts in the beam, and computational reconstruction assembles these projections into a three-dimensional model of the specimen. Harrison likens the approach to a CT scan of a patient, with the key difference that in cryo-ET the microscope remains stationary while the sample rotates through a range of tilt angles.

Applied to rotavirus, the technique delivered what Harrison describes as a molecular movie of infection. The tomographic reconstructions captured each stage of the entry process: the virus attaching to the cell membrane, being engulfed into the intracellular compartment, perforating the compartmental membrane, and ejecting its RNA genome into the cytoplasm. The imaging was conducted at the Harvard Cryo-EM Center for Structural Biology at Harvard Medical School, where Harrison serves as faculty director, with the help of managing director Richard Walsh and study co-author Conny Leistner. Harrison emphasized that such experiments simply would not have been feasible a few years ago, and he pointed to parallel work by HMS professor Alan Brown on cilia, published online in Science the same day, as further evidence of the technology’s power to reveal basic biology and address biomedical problems.

Critical support for the structural findings came from the laboratory of co-author Tomas Kirchhausen, professor of cell biology at Harvard Medical School and professor of pediatrics at Boston Children’s Hospital. Live-cell and single-molecule imaging experiments from his group were essential both for motivating the study and for validating the cryo-ET observations, ensuring that the snapshots of membrane perforation reflected events occurring in living cells rather than artifacts of sample preparation. This combination of in situ structural imaging with dynamic cellular imaging represents an increasingly influential strategy in modern cell biology, allowing researchers to connect molecular-scale architecture with the behavior of intact, functioning systems.

Beyond its significance for basic virology, the work carries substantial implications for biotechnology and medicine. Harrison framed the study as addressing a general question: what mechanisms exist for delivering large cargo into cells? Non-enveloped viruses have evolved elegant solutions to transporting relatively bulky particles across membranes without destroying the cell, and understanding those solutions could inspire new methods for delivering gene therapies and other large therapeutic agents into cells. Kirchhausen has already begun laying the groundwork for imaging the entry steps of adeno-associated viruses, or AAVs, which are among the most widely used vectors for gene therapy in biotech and pharmaceutical applications. If researchers can learn how evolution solved the delivery problem for one virus, Harrison argues, they gain a step ahead in engineering delivery systems for therapeutic purposes.

Important questions remain. Harrison noted that a further set of experiments is still needed to confirm the specifics of how VP7 forms the pore and executes membrane perforation. De Sautu, for her part, plans to continue investigating other aspects of viral entry as she completes her postdoctoral research and establishes her own laboratory, and the team intends to extend its analysis to the corresponding entry mechanisms of other non-enveloped viruses. For Harrison, who has spent decades working to close the knowledge gap between enveloped and non-enveloped virus entry, the study fulfills a career-long ambition of seeing infection unfold step by step. His motivation, he said, is fundamentally curiosity about how evolution solved the problem of moving a large particle into a cell without harming it, but the prospect that the findings will inspire biotechnologists developing new ways to treat and prevent disease adds a rewarding practical dimension to the discovery.

Subject of Research: Mechanism of membrane perforation during rotavirus cell entry

Article Title: ‘Molecular movie’ fills in blanks of how non-enveloped viruses infect cells

Article References: ‘Molecular movie’ fills in blanks of how non-enveloped viruses infect cells. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: rotavirus, cryo-electron tomography, non-enveloped viruses, viral entry, VP5, VP7, membrane perforation, structural biology, gene therapy delivery, Harvard Medical School, Science journal, virology