tetano
Editor, Senior Moderator
Viruses
. 2020 Dec 18;12(12):E1465.
doi: 10.3390/v12121465.
SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus
Katherine Elizabeth Havranek[SUP] 1 [/SUP], Ariana R Jimenez[SUP] 1 [/SUP], Marissa Danielle Acciani[SUP] 1 [/SUP], Maria Fernanda Lay Mendoza[SUP] 1 [/SUP], Judith Mary Reyes Ballista[SUP] 1 [/SUP], Darren Austin Diaz[SUP] 1 [/SUP], Melinda Ann Brindley[SUP] 1 2 [/SUP]
Affiliations
Abstract
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).
Keywords: SARS-CoV-2; fusion; recombinant VSV; spike.
. 2020 Dec 18;12(12):E1465.
doi: 10.3390/v12121465.
SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus
Katherine Elizabeth Havranek[SUP] 1 [/SUP], Ariana R Jimenez[SUP] 1 [/SUP], Marissa Danielle Acciani[SUP] 1 [/SUP], Maria Fernanda Lay Mendoza[SUP] 1 [/SUP], Judith Mary Reyes Ballista[SUP] 1 [/SUP], Darren Austin Diaz[SUP] 1 [/SUP], Melinda Ann Brindley[SUP] 1 2 [/SUP]
Affiliations
- PMID: 33353101
- DOI: 10.3390/v12121465
Abstract
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).
Keywords: SARS-CoV-2; fusion; recombinant VSV; spike.