Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

Abstract

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient (DORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of DORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the DORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while DORF7a, DORF7b and DORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19.

Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses

“…Importantly, this 2A approach does not remove any viral gene from the viral genome. Although recent data from our laboratory (28) suggest that ORF7a is not essential for SARS-CoV-2 replication in vitro and in vivo (29), it is still largely unknown whether the lack of ORF7a affects some unknown aspects of SARS-CoV-2 pathogenesis. In addition, this also increases the instability concern regarding the reporter gene, as shown in a recent study where a reporter gene fused into the C terminus of ORF7a was not stable (30).…”

Visualization of SARS-CoV-2 infection dynamic

“…Importantly, this new 2A approach does not remove any viral gene from the viral genome. Although recent data from our laboratory (Silvas et al, 2021) suggest that ORF7a is not essential for SARS-CoV-2 replication in vitro and in vivo , it is still largely unknown whether the lack of ORF7a affects some unknown aspects of SARS-CoV-2 infections. In addition, this also increases the instability concern of reporter gene, as shown in a recent study where a reporter gene fused into the C-terminus of ORF7a was not stable (Rihn et al, 2021).…”

Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy

“…Moreover, elucidation of the major FcγR(s) (FcγRI, FcγRIII and/or FcγRIV) engaged by NAbs will help design ultrapotent SARS-CoV-2 Nab therapies (Smith et al, 2012). Here we have taken a step in this direction by introducing GASDALIE mutations to augment FcγR interactions and enhance in vivo potency of CV3-25.…”

“…SARS-CoV-2-nLuc closely mimics the wildtype virus replication kinetics 108 and stably maintains the nLuc reporter over five generations in vitro. Further, ORF7a deletion was 109 recently shown not to affect pathology of the wildtype virus (Silvas et al, 2021). In vivo BLI revealed that the virus spreads from the nasal cavity to lungs to establish infection.…”