Opening new windows into viruses inside the cell by electron cryo-tomography (cryo-ET)

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As the Caltech physicist Richard Feynman once explained, "It is very easy to answer many fundamental biological questions; you just look at the thing!". If we could simply look at a virus inside a eukaryotic cell and observe all the host and virus molecules interacting with one another in their native state, we would vastly improve our understanding of the virus life cycle and the cellular innate immune responses. In fact, several key historical breakthroughs in virology and cellular biology have been made through advances in imaging technologies. The development of traditional electron microscopy (EM) led to the first detailed pictures of a virus and later the fine ultrastructure of cellular organelles such as the Golgi apparatus and endoplasmic reticulum. Successive technological advances have brought us to the point that we are able to image viruses at close to atomic resolution, a feat recognized by the 2017 Nobel Prize in Chemistry 'for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution". With recent electron cryo-tomography (cryo-ET) technology developments, we are in touching distance of visualizing any virus in its native state and context within the cell. For the first time, all stages of a viral replication cycle from attachment and entry through genome replication to morphogenesis and egress may be visualized at macromolecular resolution. This is an exceedingly exciting moment to work in this field. This new technology affords us the opportunity to solve the structure of viral proteins in their natural habitat with unprecedented resolution. To understand virus-host interactions using cryo-ET technology I am proposing to address a number of important biological questions targeting the virus order Bunyavirales. Viruses in this order are representative of many emerging viruses which pose a high-risk to human and animal health. In cryo-ET, samples are plunged into a cryogen (liquid ethane, or a mixture of ethane and propane), preserving them in a frozen-hydrated, near-native state. The frozen samples are then imaged in a transmission electron microscope, and a series of 2-D projection images are recorded as the sample is rotated incrementally around an axis. This so-called "tilt-series" is then reconstructed into a 3-D "tomogram", with typical resolution sufficient to make out the shapes and arrangement of large macromolecules (~5 nm). If the tomogram, or a set of tomograms, contains structurally homogeneous copies of an object of interest, "tomogram subvolumes" containing the objects can be computationally extracted, aligned and combined, a process we call "subtomogram averaging" (STA) to improve the signal-to-noise ratio and clarify details, typically improving the resolution to ~2-3 nm. For exceptionally favorable samples such as pseudo-crystalline protein coats on cells or viruses, the resolution of STA can be pushed to even 3.1A, sufficient to build atomic models de novo. To learn about important aspects of Bunyavirales biology, my research will apply state of the art technologies, such as cryo-CLEM, cryo-FIB milling, cryo-ET and STA to visualize infection of the related viruses TOSV and RVFV (Phenuiviridae) in situ at high-resolution. Using these techniques, I will look for novel structural aspects of both the pro- and antiviral processes that take place during the immune response and the triggering of the antiviral innate immune response of the cell. More specifically I will investigate (i) restriction of the virus RNPs by the MxA restriction factor, and (ii) the architecture of TOSV and RVFV host response antagonist NSs filaments inside the nucleus. Identifying new structures and key interactions between the virus and the host will be a step change in understanding fundamental aspects of viral replication and the host antiviral responses and might even allow for new insights into host targeted therapeutics.