ABSTRACT
Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3'-untranslated region (3'-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3'UTR of SERINC5 mRNA, reducing SERINC5 expression in vitro. Moreover, we found that an anti-svRNA treatment to Vero E6 cells before SARS-CoV-2 infection recovered the levels of SERINC5 and reduced the levels of N and S viral proteins. Finally, we showed that SERINC5 positively controls the levels of Mitochondrial Antiviral Signalling (MAVS) protein in Vero E6. These results highlight the therapeutic potential of targeting svRNAs based on their action on key proteins of the innate immune response during SARS-CoV-2 viral infection.
ABSTRACT
Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.