SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus.

Katherine Havranek,Judith Reyes Ballista,Marissa Acciani,Melinda Brindley,Darren Diaz,Ariana Jimenez,Maria Lay Mendoza

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/SMet1D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio).

Highlights

The causative agent responsible for the global COVID-19 pandemic is the novel beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
The SMet1 D614G∆21 construct was implemented because the emergence of this prominent SNP (D614G)
Establishing a BSL2 model for SARS-CoV-2 replication is critical for rapidly progressing our understanding of this virus and managing the ongoing pandemic, as these models exponentially increase the number of facilities and researchers that can contribute

Summary

Introduction

The causative agent responsible for the global COVID-19 pandemic is the novel beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to their ability to mutate rapidly and undergo genetic recombination, coronaviruses are capable of adapting to new ecological niches and infecting a wide variety of animals including humans [1]. A decade later, MERS-CoV emerged in the Middle East, resulting in over a thousand cases with a mortality rate approaching 35% [5]. In 2019, SARS-CoV-2 was identified in Wuhan, Hubei province, China and has since infected tens of millions of people across the globe, resulting in more than a million deaths [6]. The breadth and scale of the current COVID-19 pandemic are unprecedented

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