Exosomes/microvesicles target SARS-CoV-2 via innate and RNA-induced immunity with PIWI-piRNA system

Murine neural stem cells (NSCs) were recently shown to release piRNA-containing exosomes/microvesicles (Ex/Mv) for exerting antiviral immunity, but it remains unknown if these Ex/Mv could target SARS-CoV-2 and whether the PIWI-piRNA system is important for these antiviral actions. Here, using in vitro infection models, researchers from the Albert Einstein College of Medicine show that hypothalamic NSCs (htNSCs) Ex/Mv provided an innate immunity protection against SARS-CoV-2. Importantly, enhanced antiviral actions were achieved by using induced Ex/Mv that were derived from induced htNSCs through twice being exposed to several RNA fragments of SARS-CoV-2 genome, a process that was designed not to involve protein translation of these RNA fragments. The increased antiviral effects of these induced Ex/Mv were associated with increased expression of piRNA species some of which could predictably target SARS-CoV-2 genome. Knockout of piRNA-interacting protein PIWIL2 in htNSCs led to reductions in both innate and induced antiviral effects of Ex/Mv in targeting SARS-CoV-2. Taken together, this study demonstrates a case suggesting Ex/Mv from certain cell types have innate and adaptive immunity against SARS-CoV-2, and the PIWI-piRNA system is important for these antiviral actions.

Piwil2 knockout in htNSCs leading to decreased Ex/Mv piRNAs

(A, B) Knockout of Piwil2 gene in htNSCs was done using mammalian CRISPR lentiviral transduction followed by blasticidin selection to create stable htNSC-PIWIL2 KO cell lines. These htNSC-PIWIL2 KO cells were evaluated in comparison with control htNSCs using (A) Western blot for Piwil2 protein with β-actin as a technical control, and (B) Piwil2 immunostaining (yellow) of neurospheres, whereas DAPI nuclear staining (blue) was used as a reference. Scale bars, 50 μm. (C, D, E, F) Equal amount of Ex/Mv from htNSC-PIWIL2 KO (labelled as PIWIL2 KO Ex/Mv) versus control htNSCs (labelled as Control Ex/Mv) were analyzed for (C) total small RNAs at the size of 6–150 nt, including (D) small RNA subpopulations at the size of 10–70 nt or 22–32 nt via small RNA analysis assay using 2100 bioanalyzer, (E) total protein levels via BCA, and (F) a list of piRNAs via qPCR. AU, arbitrary unit. (F) Labels of piRNA species corresponded to a region in SARS-CoV-2 genome including regions for making spike protein (S), membrane protein (M) and nucleocapsid (N), open reading frames (O), UTR (U), and gap sequences which link some of these regions (G).