SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies

Abstract

Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019-2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restrictive biosafety containments laboratories. We developed two effective and rapid purification methods followed by UV inactivation that allow easy downstream handling of the virus. We monitored the purification through titering, sequencing, mass spectrometry and electron cryogenic microscopy. Although pelleting through a sucrose cushion, followed by gentle resuspension overnight gave the best particle recovery, infectivity decreased, and the purity was significantly worse than if using the size exclusion resin Capto Core. Capto Core can be used in batch mode, and was seven times faster than the pelleting method, obviating the need for ultracentrifugation in the containment laboratory, but resulting in a dilute virus. UV inactivation was readily optimized to allow handling of the inactivated samples under standard operating conditions. When containment laboratory space is limited, we recommend the use of Capto Core for purification and UV for inactivation as a simple, rapid workflow prior, for instance, to electron cryogenic microscopy or cell activation experiments.

Keywords: SARS-CoV-2; UV inactivation; purification; size exclusion; structural studies.

Figure 1

Virus yield and purity at different purification steps. (a) Virus titers; (b) SDS-PAGE of total protein content and WB of S and N in samples. Molecular weight markers are indicated on the left; (c) Total spectral counts of peptides detected in samples; (d) Relative protein amount in samples normalized to ORF9b. (a–d) pre-cleared stock (PC), Capto Core 1× (CC1), Capto Core 2× (CC2); pelleting through 20% sucrose (SP).

Figure 2

Inactivation of SARS-CoV-2 infectivity by UV. Titers of SARS-CoV-2 after treatment with indicated amounts of UV energy are represented with bars. Bar plot represents data from three independent experiments.

Figure 3

Morphology of inactivated SARS-CoV-2 particles. (a) Representative micrographs of SP SARS-CoV-2. (b) Representative micrographs of CC2 purified and concentrated SARS-CoV-2. (a,b) Images taken at −6 µm defocus; Intact SARS-CoV-2 particles (full white arrows), deformed particles (empty white arrows), and contaminating cellular derivatives (empty black arrows) are indicated. (c) Class averages of 100 particles picked from the imaged SP micrographs in the top row, and from 100 particles picked from the imaged CC2 concentrated micrographs in the bottom row. The percentage of the particles in the class is shown inside each box, and the average diameter of each class is shown below the box. The box size is 140.25 nm × 140.25 nm. (d) A section through a tomogram of concentrated Capto Core 2× purified SARS-CoV-2. Pre-fusion spikes are indicated by full black arrows. Tomogram was collected at −3 µm defocus.