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SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs. Supplementary Information The online version contains supplementary material available at 10.1007/s12250-021-00385-9.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially described to target the respiratory system and now has been reported to infect a variety of cell types, including cardiomyocytes, neurons, hepatocytes, and gut enterocytes. However, it remains unclear whether the virus can directly infect human embryonic stem cells (hESCs) or early embryos. Herein, we sought to investigate this question in a cell-culture system of hESCs. Both the RNA and S protein of SARS-CoV-2 were detected in the infected hESCs and the formation of syncytium was observed. The increased level of subgenomic viral RNA and the presence of dsRNA indicate active replication of SARS-CoV-2 in hESCs. The increase of viral titers in the supernatants revealed virion release, further indicating the successful life cycle of SARS-CoV-2 in hESCs. Remarkably, immunofluorescence microscopy showed that only a small portion of hESCs were infected, which may reflect low expression of SARS-CoV-2 receptors. By setting |log2 (fold change)| > 0.5 as the threshold, a total of 1,566 genes were differentially expressed in SARS-CoV-2-infected hESCs, among which 17 interferon-stimulated genes (ISGs) were significantly upregulated. Altogether, our results provide novel evidence to support the ability of SARS-CoV-2 to infect and replicate in hESCs.

SARS-CoV-2 is evolving with mutations throughout the genome all the time and a number of major variants emerged, including several variants of concern (VOC), such as Delta and Omicron variants. In this study, we demonstrated that mutations in the regions corresponding to the sequences of the probes and 3’-end of primers have a significant impact on qPCR detection efficiency. We also found that the G28916T mutation of the N gene accounts for 78.78% sequenced genomes of Delta variant. It was found that detection sensitivity of G28916T mutant was 2.35 and 1.74 times less than that of the wt sequence and detection limit was reduced from 1 copy/μl to 10 copies/μl for the commercially available CP3 and CP4 primer/probe sets. These results indicate that the detection probes and primers should be optimized to keep maximal detection efficiency in response to the emergence of new variants.

Interferon-inducible p200 family protein IFI204 was reported to be involved in DNA sensing, and subsequently induces the production of type I interferons and proinflammatory mediators. However, its function in the regulation of antiviral innate immune signaling pathway remains unclear. Here we reported a novel role of IFI204 that specifically inhibits the IRF7-mediated type I interferons response during viral infection. IFI204 and other p200 family proteins are highly expressed in mouse hepatitis coronavirus-infected bone marrow-derived dendritic cells. The abundant IFI204 could significantly interact with IRF7 in nucleus by its HIN domain and prevent the binding of IRF7 with its corresponding promoter. Moreover, other p200 family proteins that possess HIN domain could also inhibit the IRF7-mediated type I interferons. These results reveal that, besides the positive regulation function in type I interferon response at the early stage of DNA virus infection, the interferon-inducible p200 family proteins such as IFI204 could also negatively regulate the IRF7-mediated type I interferon response after RNA virus infection to avoid unnecessary host damage from hyper-inflammatory responses., Author summary The regulation of type I interferon signaling pathway is dynamic sequential processes and must be tightly regulated to keep balance between antiviral immune and hyper-inflammatory responses. The precise regulation mechanisms of the innate immune signaling pathway are still worth studying. Here, we found a novel role of the interferon-inducible p200 family protein IFI204 that specifically inhibits the IRF7-mediated type I interferon production by negative control of the transcriptional activity of IRF7 in the nucleus at the late stage of RNA virus infection. Previous studies showed that IFI204 is involved in DNA sensing during DNA virus infection to initiate antiviral immune responses. We demonstrate that IFI204 can inhibit IRF7-mediated activation of type I IFN responses induced by RNA virus infection, which is in contrast with its role in IRF3 activation in cGAS-STING DNA sensing pathway during DNA virus infection. Such negative regulation may help to avoid hyper-inflammatory responses induced by the over-activated IRF7-mediated type I interferons at late stage of the viral infection. Thus, the current study sheds light on the regulation roles of p200 family proteins and the accurate regulation system of type I interferons signaling pathway.

RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans , while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.