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Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins.
Viruses. 2020 12 17; 12(12)V

Abstract

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

Authors+Show Affiliations

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

33348746

Citation

Lay Mendoza, Maria Fernanda, et al. "Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins." Viruses, vol. 12, no. 12, 2020.
Lay Mendoza MF, Acciani MD, Levit CN, et al. Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins. Viruses. 2020;12(12).
Lay Mendoza, M. F., Acciani, M. D., Levit, C. N., Santa Maria, C., & Brindley, M. A. (2020). Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins. Viruses, 12(12). https://doi.org/10.3390/v12121457
Lay Mendoza MF, et al. Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins. Viruses. 2020 12 17;12(12) PubMed PMID: 33348746.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins. AU - Lay Mendoza,Maria Fernanda, AU - Acciani,Marissa Danielle, AU - Levit,Courtney Nina, AU - Santa Maria,Christopher, AU - Brindley,Melinda Ann, Y1 - 2020/12/17/ PY - 2020/11/24/received PY - 2020/12/11/revised PY - 2020/12/15/accepted PY - 2020/12/22/entrez PY - 2020/12/23/pubmed PY - 2021/1/5/medline KW - chikungunya KW - coronavirus KW - ebola KW - entry KW - kinetics KW - lassa KW - live assay KW - luciferase KW - real-time KW - vesicular stomatitis virus JF - Viruses JO - Viruses VL - 12 IS - 12 N2 - Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements. SN - 1999-4915 UR - https://www.unboundmedicine.com/medline/citation/33348746/Monitoring_Viral_Entry_in_Real_Time_Using_a_Luciferase_Recombinant_Vesicular_Stomatitis_Virus_Producing_SARS_CoV_2_EBOV_LASV_CHIKV_and_VSV_Glycoproteins_ L2 - https://www.mdpi.com/resolver?pii=v12121457 DB - PRIME DP - Unbound Medicine ER -