Chengjin Ye, Kevin Chiem, Jun-Gyu Park, Jesus A. Silvas, Desarey Morales Vasquez, Julien Sourimant, Michelle J. Lin, Alexander L. Greninger, Richard K. Plemper, Jordi B. Torrelles, James J. Kobie, Mark R. Walter, Juan Carlos de la Torre, and Luis Martinez-Sobrido

PNAS October 12, 2021 118 (41) e2111593118

Significance

To date, due to the insufficient expression level of reporter genes from previous recombinant (r)SARS-CoV-2, in which the viral open reading frame (ORF) 7a gene was replaced by reporter genes, tracking the SARS-CoV-2 infection dynamic has been challenging. Here, we engineered rSARS-CoV-2 expressing fluorescent (Venus) or luciferase (Nano luciferase) reporter genes from the viral nucleocapsid (N) locus, without deleting any viral gene. These novel reporter-expressing rSARS-CoV-2, which give a higher expression level of reporter genes, allow us to monitor SARS-CoV-2 replication dynamic both in vitro and in vivo. These new reporter-expressing rSARS-CoVs-2 represent an excellent option to assess viral replication, tropisms, and pathogenicity as well as the rapid in vivo evaluation of effective countermeasures for the treatment of SARS-CoV-2 infection.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

See: https://www.pnas.org/content/118/41/e2111593118

Fig. 1.

Generation of an rSARS-CoV-2 expressing Venus-2A. (A) Schematic representation of the BAC for generation of rSARS-CoV-2/Venus-2A. The sequence encoding the fusion construct Venus-2A was inserted in the viral genome of SARS-CoV-2 in the BAC. The viral ORF8, the intergenic sequences between ORF8 and N (ACAAACTAAA), Venus (green), the PTV-1 2A (light blue), and the viral N (dark blue) are indicated. (B) Vero E6 cells were mock infected or infected with rBAC-SARS-CoV-2/WT or rSARS-CoV-2/Venus-2A for 48 h, fixed, and immunostained with a MAb against the viral N protein (1C7C7). Cell nuclei were stained with DAPI. Representative images are shown. (Scale bars, 100 μm.) (C) Whole cell lysates from Vero E6 cells mock infected or infected with rSARS-CoV-2 WT or Venus-2A for 48 h were subjected to Western blot analysis using antibodies against Venus and the viral N protein (1C7C7). β-actin was used as a loading control. (D) Total cellular RNA from Vero E6 cells mock infected or infected with WT or Venus-2A rSARS-CoV-2 was isolated at 48 hpi. RT-PCR was used to amplify Venus (Top) or the region between the ORF8 and N proteins (Bottom), and the products were separated on a 0.7% agarose gel.