Mammalian viral suppressors of RNA interference.

The antiviral defense directed by the RNAi pathway employs distinct specificity and effector mechanisms compared with other immune responses. The specificity of antiviral RNAi is programmed by siRNAs processed from virus-derived double-stranded RNA by Dicer endonuclease. Argonaute-containing RNA-induced silencing complex loaded with the viral siRNAs acts as the effector to mediate specific virus clearance by RNAi. Recent studies have provided evidence for the production and antiviral function of virus-derived siRNAs in both undifferentiated and differentiated mammalian cells infected with a range of RNA viruses when the cognate virus-encoded suppressor of RNAi (VSR) is rendered nonfunctional. In this review, we discuss the function, mechanism, and evolutionary origin of the validated mammalian VSRs and cell culture assays for their identification.

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, we 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.

AGO CLIP-based imputation of potent siRNA sequences targeting SARS-CoV-2 with antifibrotic miRNA-like activity.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2-8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-ß signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online ( http://clip.korea.ac.kr/covid19 ).

Upping the ante on mammalian antiviral RNA interference.

SARS-CoV-2 has mutually illuminated our collective knowledge and knowledge gaps, particularly in antiviral defense and therapeutic strategies. A recent study in Science (Poirier et al., 2021) uncovers an ancient antiviral mechanism that mammals utilize to suppress viruses, including SARS-CoV-2 and Zika virus, that could have broad implications for therapeutic strategies.

Designing an effective therapeutic siRNA to silence RdRp gene of SARS-CoV-2.

The devastating outbreak of COVID-19 has spread all over the world and has become a global health concern. There is no specific therapeutics to encounter the COVID-19. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control human viral infections employing post-transcriptional gene silencing (PTGS) through neutralizing target complementary mRNA. RNA-dependent RNA polymerase (RdRp) encoded by the viral RdRp gene as a part of the replication-transcription complex can be adopted as an acceptable target for controlling SARS-CoV-2 mediated infection. Therefore, in the current study, accessible siRNA designing tools, including significant algorithms and parameters, were rationally used to design the candidate siRNAs against SARS-COV-2 encoded RdRp. The designed siRNA molecules possessed adequate nucleotide-based and other features for potent gene silencing. The targets of the designed siRNAs revealed no significant matches within the whole human genome, ruling out any possibilities for off-target silencing by the siRNAs. Characterization with different potential parameters of efficacy allowed selecting the finest siRNA among all the designed siRNA molecules. Further, validation assessment and target site accessibility prediction also rationalized the suitability of this siRNA molecule. Molecular docking study between the selected siRNA molecule and component of RNA interference (RNAi) pathway gave an excellent outcome. Molecular dynamics of two complexes: siRNA and argonaute complex, guide RNA, and target protein complex, have shown structural stability of these proteins. Therefore, the designed siRNA molecule might act as an effective therapeutic agent against the SARS-CoV-2 at the genome level and can prevent further outbreaks of COVID-19 in humans.