Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D−/−ESCs) as expected. Surprisingly, D−/−ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D−/−ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D−/−ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D−/−ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D−/−ESCs. Treatment of D−/−ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.

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Recent studies have demonstrated that pluripotent stem cells (PSCs), including human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have underdeveloped antiviral innate immunity. Specifically, they fail to express IFN in response to viral stimuli. This finding has led to the hypothesis that the lack of IFN response is an intrinsic property of PSCs. It has also inspired the investigation of the underlying molecular mechanisms. In an effort to conduct a holistic analysis of antiviral responses in ESCs, we have developed an assay that can simultaneously measure RNA‐mediated gene expression and RNA‐induced antiviral responses. In this system, GFP is expressed from functionalized synthetic GFP‐mRNA and is used to measure the cellular translation activity, whereas dsRNA and 5’‐triphosphate‐ssRNA act as viral RNA analogs. Transfection of ESCs with these synthetic RNAs can potently activate all common antiviral signaling pathways, including RNA interference (RNAi), dsRNA activated protein kinase (PKR), OAS‐RNase L, and IFN responses. Our data revealed that RNAi activity was detected in ESCs, but not in Dicer knockout ESCs (D‐/‐ESCs) as expected since Dicer is a key enzyme in miRNA and siRNA processing. The antiviral responses mediated by PKR, OAS‐RNase L, and IFN were either not detected or detected at low levels in ESCs, but they increased dramatically in D‐/‐ESCs, as indicated by increased activity of PKR (measured by low level GFP expression and PKR phosphorylation) and RNase L (determined by RNA degradation). Furthermore, the components of the IFN signaling pathway are expressed at higher basal levels in D‐/‐ESCs than in normal ESCs and are further up‐regulated by RNA transfection. The most notable effect of RNA transfection into D‐/‐ESCs is the induction of cell death and decreased cell proliferation, which is at least partly attributed to PKR activation and increased expression of the cell cycle inhibitors p21 and p19. Our findings suggest that ESCs are exquisitely sensitive to cellular damages caused by antiviral response and that Dicer acts as a repressor of antiviral responses in ESCs under normal conditions. Support or Funding Information This work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).

A mild, efficient, and environmentally friendly method for the selective protection of secondary hydroxyl groups is described. The method involves the protection of both primary and secondary hydroxyl groups as tert-butyldimethylsilyl (TBDMS) ethers and selective deprotection of the primary TBDMS group with formic acid in acetonitrile/water. The rates of desilylation of primary and secondary TBDMS ethers by different concentrations of formic acid are determined. Formic acid of 5–20% concentration is found to selectively deprotect primary TBDMS ethers while keeping more than 95% of their secondary counterparts intact.