Cryo-ET Captures Molecular Movie of Rotavirus Punching Into Cells

For decades, structural biologists have understood the entry strategies of enveloped viruses far better than those of their non-enveloped counterparts. Viruses such as influenza, HIV, herpesvirus, and SARS-CoV-2 carry an outer lipid membrane that fuses with host cell membranes, a process that has been dissected in exquisite molecular detail. Non-enveloped viruses, including poliovirus, human papillomavirus, adenovirus, and rotavirus, lack this membrane and must instead breach a cellular membrane without the benefit of fusion machinery. How they accomplish this feat has remained one of the more stubborn gaps in virology. Now, a team at Harvard Medical School has filled in a crucial piece of that puzzle, capturing step-by-step images of how rotavirus, a virus less than 100 nanometers across, punches a hole through the membrane of the compartment that engulfs it and delivers its genetic core into the cell.

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