Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development. © 2022 by the authors.

Study of the inhibitory effect of STAT1 on PDCoV infection.

Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT, STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.

Applications of the versatile CRISPR-Cas13 RNA targeting system.

CRISPR-Cas are adaptable natural prokaryotic defense systems that act against invading viruses and plasmids. Among the six currently known major CRISPR-Cas types, the type VI CRISPR-Cas13 is the only one known to exclusively bind and cleave foreign RNA. Within the last couple of years, this system has been adapted to serve numerous, and sometimes not obvious, applications, including some that might be developed as effective molecular therapies. Indeed, Cas13 has been adapted to kill antibiotic-resistant bacteria. In a cell-free environment, Cas13 has been used in the development of highly specific, sensitive, multiplexing-capable, and field-adaptable detection tools. Importantly, Cas13 can be reprogrammed and applied to eukaryotes to either combat pathogenic RNA viruses or in the regulation of gene expression, facilitating the knockdown of mRNA, circular RNA, and noncoding RNA. Furthermore, Cas13 has been harnessed for in vivo RNA modifications including programmable regulation of alternative splicing, A-to-I and C to U editing, and m6A modifications. Finally, approaches allowing for the detection and characterization of RNA-interacting proteins have also been demonstrated. Here, we provide a comprehensive overview of the applications utilizing CRISPR-Cas13 that illustrate its versatility. We also discuss the most important limitations of the CRISPR-Cas13-based technologies, and controversies regarding them. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.