SARS-CoV-2 RNAs are processed into 22-nt vsRNAs in Vero cells

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic, resulting in great fatalities around the world. Although the antiviral roles of RNA interference (RNAi) have been well studied in plants, nematodes and insects, the antiviral roles of RNAi in mammalians are still debating as RNAi effect is suspected to be suppressed by interferon (IFN) signaling pathways in most cell types. To determine the role of RNAi in mammalian resistance to SARS-CoV-2, we studied the profiling of host small RNAs and SARS-CoV-2 virus-derived small RNAs (vsRNAs) in the early infection stages of Vero cells, an IFN-deficient cell line. We found that host microRNAs (miRNAs) were dysregulated upon SARS-CoV-2 infection, resulting in downregulation of microRNAs playing antiviral functions and upregulation of microRNAs facilitating viral proliferations. Moreover, vsRNA peaked at 22 nt at negative strand but not the positive strand of SARS-CoV-2 and formed successive Dicer-spliced pattern at both strands. Similar characteristics of vsRNAs were observed in IFN-deficient cell lines infected with Sindbis and Zika viruses. Together, these findings indicate that host cell may deploy RNAi pathway to combat SARS-CoV-2 infection in IFN-deficient cells, informing the alternative antiviral strategies to be developed for patients or tissues with IFN deficiency.

SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression

“…We next examined the impact of NSP2 on the repression activity of miRNAs on a reporter mRNA that contains miRNA binding sites. A recent analysis of dysregulated host miRNAs revealed miR-20a as one of the most significantly upregulated miRNAs in SARS-CoV-2 infected cells [23], likely due to its immunosuppressive effects [24,25]. We used a dual luciferase reporter system in which the Firefly Luciferase (FL) mRNA contains 3x miR-20abinding sites within its 3´ UTR (miR-20 WT; Fig.…”

“…However, in principle NSP2 could also enhance the silencing mediated by antiviral miRNAs [39]. To avoid the potential harmful effects of NSP2-induced repression by antiviral miRNAs, the virus can manipulate the expression pattern of antiviral miRNAs [23,[40][41][42][43]. Analysis of the expression of 128 human miRNAs with potential to target the SARS-CoV-2 genome revealed their very low expression in lung epithelia [43], which likely allows the virus to avoid the effects of antiviral miRNAs and replicate in these cells.…”

“…Hitherto, our data have revealed that NSP2 augments miR-20-mediated repression. To investigate whether NSP2 also augments the translational repression by other miRNAs, we used two other abundant miRNAs: let-7a, which is also upregulated in SARS-CoV-2 infected cells [27], and miR-92, the expression of which is not known to be modulated by SARS-CoV-2 [23]. Like the miR-20 reporter (Fig.…”

The SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression

“…We next examined the impact of NSP2 on the repression activity of miRNAs on a reporter mRNA that contains miRNA-binding sites. A recent analysis of dysregulated host miRNAs revealed miR-20a as one of the most significantly upregulated miRNAs in SARS-CoV-2-infected cells ( Liu et al, 2022 ), likely due to its immunosuppressive effects ( Cox et al, 2010 ; Reddycherla et al, 2015 ). We used a dual luciferase reporter system in which the firefly luciferase ( FL ) mRNA contains 3× miR-20a -binding sites within its 3′ UTR (miR-20 WT; Fig.…”

“…Hitherto, our data have revealed that NSP2 augments miR-20a -mediated repression. To investigate whether NSP2 also augments the translational repression by other miRNAs, we used two other abundant miRNAs: let-7a , which is also upregulated in SARS-CoV-2-infected cells ( Nersisyan et al, 2020 ), and miR-92 , the expression of which is not known to be modulated by SARS-CoV-2 ( Liu et al, 2022 ). Similar to the miR-20 reporter ( Fig.…”