Periodic Reporting for period 1 - Entero3D (In situ 3D structures of viral replication complexes: cryo-electron tomography of enterovirus-infected cells)
Reporting period: 2019-01-01 to 2020-12-31
Viruses are not autonomous, they depend on the cell they infect, to replicate their genome and form new viral particles capable of infecting neighboring cells. To this end, many viruses will drastically change a cell’s internal structure and metabolic activity within hours of infection. One group of viruses causing rapid structural alterations to cells are enteroviruses. They represent a major genus of positive-sense single-stranded RNA viruses and include human pathogens such as Poliovirus, Coxsackieviruses and Rhinoviruses. They are thus responsible for a wide variety of diseases such as poliomyelitis, myocarditis, gastroenteritis and respiratory infections. Enteroviruses reorganize the internal membranes of a cell, generating replication complexes, which are the sites of viral genome replication. Specific mechanism of how cellular membranes are rearranged into viral replication complexes and how this is connected to the viral RNA replication and packaging into new virions are unknown. In this project, I study the interior of the infected cells using cryo-electron tomography. Cryo-electron tomography provides extraordinary structural details into biological processes that were before unreachable. This will help us to determine the first high-resolution structures of viral enterovirus replication complexes in cells, providing critical insights into the genome replication of these highly medically relevant viruses, and potentially generating new concepts for virus inhibitor design.
During the first year of the project, I developed and optimised a robust workflow to study enterovirus-infected cells with cryo-electron tomography (cryo-ET). First, I established a protocol to grow and infect human cells on the electron microscopy grids. I used different indicators (e.g. viral RNA and protein expression) to select time-points post infection at which to plunge-freeze (unfixed, unstained) cells to study corresponding structural organization of the viral replication complexes by cryo-ET. I improved the conditions for plunge freezing of the infected cells to obtain a homogenous thin film of ice. The frozen samples are then transferred to the FEI Scios FIB/SEM for cryo-focus ion-beam milling. The focused ion beam is used to trim away bulk material from the interior of the infected cells, leaving a slab of 150-300 nm thickness (thinner for higher resolution, thicker for more cellular context). Such thinned regions (lamellas) represent a cutout view of the cytoplasm and I used them for subsequent imaging by cryo-ET (Titan Krios). In the second half of the year, cell density, virus infection, plunge freezing and milling have been optimised as such I could obtain several tomograms of enterovirus-infected cells and observe virus related events in all milled cells, with high resolution. 3D tomograms of infected cells showed striking links between replication membranes, including autophagy membranes, virion assembly and genome packaging. At this point, we decided to investigate more the role of cellular autophagy in virus particle formation. End of the first year (2019) and beginning of the second year (2020), we have used several chemical drugs to block autophagy as well as CRISPR KO cells for autophagy proteins (LC3 and GABARAP), this way we showed that autophagy activation is critical for both virus assembly and release. Through these two years, I attended several seminars and conferences in which I have presented our preliminary findings and the progress of my work. This helped me to cumulate several technical advices and scientific feedbacks, which helped the project to move forward. The last six months of the grant were dedicated to the data processing, including subtomogram averaging of two distinct repetitive structures observed in the infected cells. Currently we are preparing the manuscript that we plan to submit by the end of March this year (2021). I believe that our findings provide insights into novel mechanisms of enterovirus genome replication and enterovirus-host interaction, including the interplay between host autophagy and virus replication
Viruses evolve due to constantly changing survival environments. Most importantly, the natural evolution of the viruses tends to be faster than their hosts, such as humans. That makes us vulnerable to new virus variants as we are facing currently with the recent outbreak of a new coronavirus, SARS-CoV-2, that sadly led to worldwide large number of lost human lives. The spread of the virus resulted in unprecedented health and socio-economic crisis. Enteroviruses are some of the most common human viral pathogens, and, similarly to coronaviruses, enteroviruses are positive-sense, single-stranded RNA viruses capable of causing a wide spectrum of illness, including hand-foot-and-mouth disease, conjunctivitis, hepatitis, myocarditis, meningitis, encephalitis. In 2014, outbreaks of severe respiratory illness associated with enterovirus D68 occurred among children in the North America. Except poliovirus, we lack vaccines and antivirals therapies for many of the enteroviruses. This is partly due to high mutation rates of enteroviruses and likelihood of acquired resistance. That is why there is an urge to understand the mechanisms these viruses are using when infecting human cells, how they cause disease, or how our bodies react to infections. To answer such an important biological question, one must provide a strong biological answer and bridge the space between fundamental research and applicable research in designing new therapies. In many cases, structures of isolated enterovirus particles been studied in detail. However, the structure and mechanism of their replication complexes, the main intracellular manifestation of the viruses, is still poorly understood. The approach I propose here will be a unique attempt to reveal the structural basis of how cellular membranes are disrupted and reshaped to support enterovirus infection. Using novel cryo-focused ion beam milling technology combined with cryo-electron tomography, we could visualize and identify molecular assemblies in enterovirus-infected cells, not visible in conventional EM. This ultimately helped us to characterize further how the virus hijacks the host cellular machinery for its optimum replication. The main impact of this project will thus be structural and molecular insights on how enteroviral proteins can coordinate a remodeling of cellular membranes and create a localized viral genome factory. This will be among the first high-resolution structural study of viral replication complexes in cells and will thus serve as a paradigm for molecular understanding of pathogen-induced membrane remodeling. Thus, the proposed project has the potential to provide solid scientific benefits and potentially also new concepts for therapeutics.