SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To exp...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-12-01
|
| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/12/12/1465 |
| _version_ | 1797544238485864448 |
|---|---|
| author | Katherine Elizabeth Havranek Ariana R. Jimenez Marissa Danielle Acciani Maria Fernanda Lay Mendoza Judith Mary Reyes Ballista Darren Austin Diaz Melinda Ann Brindley |
| author_facet | Katherine Elizabeth Havranek Ariana R. Jimenez Marissa Danielle Acciani Maria Fernanda Lay Mendoza Judith Mary Reyes Ballista Darren Austin Diaz Melinda Ann Brindley |
| author_sort | Katherine Elizabeth Havranek |
| collection | DOAJ |
| description | Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To explore S requirements for entry under BSL2 conditions, S has been pseudotyped onto vesicular stomatitis virus (VSV) or retroviral particles with varied success. Several alterations to S were demonstrated to improve pseudoparticle titers, but they have not been systematically compared. In this study, we produced pseudotyped VSV particles with multiple modifications to S, including truncation, mutation, and tagging strategies. The main objective of this study was to determine which modifications of the S protein optimize cell surface expression, incorporation into pseudotyped particles, and pseudoparticle entry. Removal of the last 19 residues of the cytoplasmic tail produced a hyper-fusogenic S, while removal of 21 residues increased S surface production and VSV incorporation. Additionally, we engineered a replication-competent VSV (rVSV) virus to produce the S-D614G variant with a truncated cytoplasmic tail. While the particles can be used to assess S entry requirements, the rVSV∆G/S<sub>Met1</sub>D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio). |
| first_indexed | 2024-03-10T13:57:35Z |
| format | Article |
| id | doaj.art-e1165f3257a2441d91d33821232aef02 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T13:57:35Z |
| publishDate | 2020-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-e1165f3257a2441d91d33821232aef022023-11-21T01:32:10ZengMDPI AGViruses1999-49152020-12-011212146510.3390/v12121465SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 VirusKatherine Elizabeth Havranek0Ariana R. Jimenez1Marissa Danielle Acciani2Maria Fernanda Lay Mendoza3Judith Mary Reyes Ballista4Darren Austin Diaz5Melinda Ann Brindley6Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USADepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USADepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USADepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USADepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USADepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USADepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USASevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To explore S requirements for entry under BSL2 conditions, S has been pseudotyped onto vesicular stomatitis virus (VSV) or retroviral particles with varied success. Several alterations to S were demonstrated to improve pseudoparticle titers, but they have not been systematically compared. In this study, we produced pseudotyped VSV particles with multiple modifications to S, including truncation, mutation, and tagging strategies. The main objective of this study was to determine which modifications of the S protein optimize cell surface expression, incorporation into pseudotyped particles, and pseudoparticle entry. Removal of the last 19 residues of the cytoplasmic tail produced a hyper-fusogenic S, while removal of 21 residues increased S surface production and VSV incorporation. Additionally, we engineered a replication-competent VSV (rVSV) virus to produce the S-D614G variant with a truncated cytoplasmic tail. While the particles can be used to assess S entry requirements, the rVSV∆G/S<sub>Met1</sub>D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio).https://www.mdpi.com/1999-4915/12/12/1465SARS-CoV-2spikefusionrecombinant VSV |
| spellingShingle | Katherine Elizabeth Havranek Ariana R. Jimenez Marissa Danielle Acciani Maria Fernanda Lay Mendoza Judith Mary Reyes Ballista Darren Austin Diaz Melinda Ann Brindley SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus Viruses SARS-CoV-2 spike fusion recombinant VSV |
| title | SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus |
| title_full | SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus |
| title_fullStr | SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus |
| title_full_unstemmed | SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus |
| title_short | SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus |
| title_sort | sars cov 2 spike alterations enhance pseudoparticle titers and replication competent vsv sars cov 2 virus |
| topic | SARS-CoV-2 spike fusion recombinant VSV |
| url | https://www.mdpi.com/1999-4915/12/12/1465 |
| work_keys_str_mv | AT katherineelizabethhavranek sarscov2spikealterationsenhancepseudoparticletitersandreplicationcompetentvsvsarscov2virus AT arianarjimenez sarscov2spikealterationsenhancepseudoparticletitersandreplicationcompetentvsvsarscov2virus AT marissadanielleacciani sarscov2spikealterationsenhancepseudoparticletitersandreplicationcompetentvsvsarscov2virus AT mariafernandalaymendoza sarscov2spikealterationsenhancepseudoparticletitersandreplicationcompetentvsvsarscov2virus AT judithmaryreyesballista sarscov2spikealterationsenhancepseudoparticletitersandreplicationcompetentvsvsarscov2virus AT darrenaustindiaz sarscov2spikealterationsenhancepseudoparticletitersandreplicationcompetentvsvsarscov2virus AT melindaannbrindley sarscov2spikealterationsenhancepseudoparticletitersandreplicationcompetentvsvsarscov2virus |