An Approach to Perform Cryo-EM Analysis of Glutaraldehyde Fixed BSL-3 Viruses in BSL-1/2 Conditions

Amar D Parvate, Purdue University

Abstract

Scientific investigations of biosafety level 3 (BSL-3) agents require special containment facilities and highly trained staff. The risks posed by BSL-3 virus handling restrict their investigations, especially in structural biology as large quantities of the pathogens are required for high resolution studies. Currently cryo-electron microscopy and tomography (cryo-EM and ET) have been used to solve the structures of viruses to 3-4 Å resolution. Cryo-EM inside a BSL-3 facility incur very high costs and require an extensive and cumbersome protocol for decontamination.^ A modified GraFix method was developed to inactivate BSL-3 viruses and facilitate their high resolution cryo-EM studies in BSL-1 conditions. Inactivation was performed by chemical fixation with 1% glutaraldehyde, which has been documented to completely inactivate viruses. The inactivation protocol was designed using Bunyamwera virus – a BSL-2 prototype for BSL-3 hantaviruses and tested by suing both cryo-EM and ET. To further prove the utility of this method in high resolution structural studies, single particle cryo-EM studies were performed on VEEV TC-83 - an icosahedral alphavirus. A structure with 7.5 Å resolution was obtained for TC-83 prepared using this protocol, which is the highest reported resolution for a virus inactivated with glutaraldehyde. In addition to being similar to previous cryo-EM structure of 4.4 Å resolution, our structure showed a third hitherto unknown glycosylation site on the E2 glycoprotein at Asn-212 of TC-83.^ The same approach was used in BSL-3 conditions to obtain inactivated samples of 3 Orthohantaviruses – Hantaan, Andes and Black Creek Canal Viruses. It was observed that a round morphology may be predominant in Old World viruses whereas New World viruses may show a predominantly tubular morphology. Cryo-tomography studies were performed on the purified and inactivated hantaviruses with a target resolution of 20 Å of the virus glycoprotein spike complex by sub-volume averaging. While the subvolume averaging failed to provide a noise free structure of the spike complex, it was observed that the RNP complex forms bends and kinks within the virus. Patches of naked membrane were observed on all Hantaviruses which may indicate that complete coating of viral surface with viral proteins is not absolutely essential for assembly.^ The protocol to prepare inactivated viruses was extended to Orthohantavirus infected mammalian cells. Our approach provided superior quality of images than the ones published in literature. The plasma membranes of HTNV infected cells were observed to send out plasma membrane projections to sequestered egressing viruses. Based on our images, New and Old World virus infections may progress through the cells at different rates. Our protocol is suitable to prepare electron tomography samples of Hantaan virus infected cells for future studies. The protocol was replicated successfully in multiple labs, on multiple viruses, at multiple times which indicate its robustness and flexibility. This could make many other hitherto challenging BSL-3/4 viruses amenable to cryo-EM analyses.^

Degree

Ph.D.

Advisors

Jason K. Lanman, Purdue University, Cynthia V. Stauffacher, Purdue University.

Subject Area

Virology|Biophysics

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