Nov 20, 2023

Public workspaceA validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells V.2

PLOS One
CheckPeer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.81wgb676qlpk/v2
  • Timothy K. Soh1,2,3,4,
  • Susanne Pfefferle5,6,
  • Stephanie Wurr5,7,
  • Ronald von Possel5,8,
  • Lisa Oestereich5,7,
  • Toni Rieger5,
  • Maria Rosenthal1,5,9,
  • Jens B. Bosse1,2,3,4
  • 1Centre for Structural Systems Biology, Hamburg, Germany;
  • 2Hannover Medical School, Institute of Virology, Hannover, Germany;
  • 3Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany;
  • 4Leibniz Institute of Virology (LIV), Hamburg, Germany;
  • 5Department of Virology, Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany;
  • 6University Center Hamburg-Eppendorf (UKE), Institute for Medical Microbiology, Virology and Hygiene, Hamburg, Germany;
  • 7DZIF German Center for Infection research, Partner site Hamburg-Lübeck-Borstel-Riems;
  • 8Department of Tropical Medicine and Infectious Diseases, Center for Internal Medicine, University of Rostock, Rostock, Germany;
  • 9Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Discovery Research ScreeningPort, Hamburg, Germany
  • Maria Rosenthal: rosenthal@bnitm.de;
  • Jens B. Bosse: jens.bosse@cssb-hamburg.de;
Jens B Bosse
Open access
DOI: dx.doi.org/10.17504/protocols.io.81wgb676qlpk/v2
External link: https://doi.org/10.1371/journal.pone.0274065
Protocol Citation: Timothy K. Soh, Susanne Pfefferle, Stephanie Wurr, Ronald von Possel, Lisa Oestereich, Toni Rieger, Maria Rosenthal, Jens B. Bosse 2023. A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb676qlpk/v2Version created by Jens B Bosse
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 15, 2023
Last Modified: November 20, 2023
Protocol Integer ID: 90986
Keywords: SARS-CoV-2, herpes, HSV-1, HCMV, UV crosslinking, UV inactivation, inactivation validation
Abstract
Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate diluted viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells.

The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.
Guidelines
This protocol was validated for 2x106 Vero E6 cells infected with SARS-CoV-2 at MOI 0.01, 5x105 Vero B4 cells infected with HSV-1 at MOI 3, and 2x105 HFF-1 cells infected with HCMV at MOI 3 in an individual 6-well.
Materials
phosphate-buffered saline (PBS)
Incidin Plus

infected cells
cell scraper
microcentrifuge tube
CryoELITE Tissue Vial (Wheaton, #W985100)

P1000 micropipette
microcentrifuge
UVP Crosslinker (CL-3000, Analytik Jena)
Safety warnings
Attention
Handle infectious materials within the appropriate containment facilities. Use UVP Crosslinkers in accordance with the manufacturer’s guidelines.
Before start
Begin with infected cells in a 6-well plate.
UV inactivate infected cells
UV inactivate infected cells
Wash each well with 1 mL PBS
1m
Scrape each well (2x106 cells) into 1 mL PBS
1m
Transfer cells into a 1.5 mL tube
1m
Pellet cells at Centrifigation16000 x g, 4°C, 00:01:00

1m
Resuspend cells in 200 μL PBS
1m
Transfer cells to a tissue vial
1m
254 nm UV irradiation of vials
24m
Irradiate vials with 2,500 mJ/cm2
5m
Mix the cell solution with a micropipette
1m
Repeat irradiation 3 additional times for a total of 10,000 mJ/cm2
18m
Screw the lids on the vials
1m
Disinfect the outside of the tissue vials by wiping with Incidin Plus
1m
Spotlight video
Spotlight video