Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.
Highlights
The current pandemic of coronavirus disease 2019 (COVID19) caused by the newly emerged coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 100 million infections and 2.44 million deaths as of February 18, 2021 [1]
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cellbased virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions
The replicon was linked to a T7 minimal promoter upstream of the 5′ untranslated region (UTR), and a cassette containing a 26-nt poly(A), a hepatitis D virus ribozyme (Rbz), and a T7 terminator at the 3′ end to yield precise viral genome terminus (Fig. 1A)
Summary
The current pandemic of coronavirus disease 2019 (COVID19) caused by the newly emerged coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 100 million infections and 2.44 million deaths as of February 18, 2021 [1]. Deletion of the structural genes rendered the replicon defective in producing progeny virions, thereby enabling safe handling under BSL-2 conditions Another advantage of the transient replicon system over traditional live virus-based models is that the replicon RNA can be introduced to a variety of cell lines, allowing for assessment of viral replication under more physiologically relevant conditions. Most SARS-CoV-2 studies require biosafety level 3 facilities, which are in high demand, costly, and difficult to access To overcome these limitations, we engineered a SARS-CoV-2 replicon, which is a modified virus subgenome capable of self-replicating without producing infectious virus, allowing the viral replication to be studied in a conventional biomedical laboratory setting. Successful implementation of the technology will accelerate the development of effective treatment for SARS-CoV-2 infection
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