Stress Decreases Host Viral Resistance and Increases Covid Susceptibility in Embryonic Stem Cells.

Stress-induced changes in viral receptor and susceptibility gene expression were measured in embryonic stem cells (ESC) and differentiated progeny. Rex1 promoter-Red Fluorescence Protein reporter ESC were tested by RNAseq after 72hr exposures to control stress hyperosmotic sorbitol under stemness culture (NS) to quantify stress-forced differentiation (SFD) transcriptomic programs. Control ESC cultured with stemness factor removal produced normal differentiation (ND). Bulk RNAseq transcriptomic analysis showed significant upregulation of two genes involved in Covid-19 cell uptake, Vimentin (VIM) and Transmembrane Serine Protease 2 (TMPRSS2). SFD increased the hepatitis A virus receptor (Havcr1) and the transplacental Herpes simplex 1 (HSV1) virus receptor (Pvrl1) compared with ESC undergoing ND. Several other coronavirus receptors, Glutamyl Aminopeptidase (ENPEP) and Dipeptidyl Peptidase 4 (DPP4) were upregulated significantly in SFD>ND. Although stressed ESC are more susceptible to infection due to increased expression of viral receptors and decreased resistance, the necessary Covid-19 receptor, angiotensin converting enzyme (ACE)2, was not expressed in our experiments. TMPRSS2, ENPEP, and DPP4 mediate Coronavirus uptake, but are also markers of extra-embryonic endoderm (XEN), which arise from ESC undergoing ND or SFD. Mouse and human ESCs differentiated to XEN increase TMPRSS2 and other Covid-19 uptake-mediating gene expression, but only some lines express ACE2. Covid-19 susceptibility appears to be genotype-specific and not ubiquitous. Of the 30 gene ontology (GO) groups for viral susceptibility, 15 underwent significant stress-forced changes. Of these, 4 GO groups mediated negative viral regulation and most genes in these increase in ND and decrease with SFD, thus suggesting that stress increases ESC viral susceptibility. Taken together, the data suggest that a control hyperosmotic stress can increase Covid-19 susceptibility and decrease viral host resistance in mouse ESC. However, this limited pilot study should be followed with studies in human ESC, tests of environmental, hormonal, and pharmaceutical stressors and direct tests for infection of stressed, cultured ESC and embryos by Covid-19.

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation.

The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

The genetic material inside cells is often packaged into thread-like structures called chromosomes. In humans, mice and other mammals, a pair of sex chromosomes determines the genetic or chromosomal sex of each individual. Those who inherit two "X" chromosomes are said to be chromosomally female, while chromosomal males have one "X" and one "Y" chromosome. This means females have twice as many copies of genes on the X chromosome as a male does, which turns out to be double the number that the body needs. To solve this problem, mammals have developed a strategy known as dosage compensation. The second X chromosome in females becomes "silent": its DNA remains unchanged, but none of the genes are active. A long noncoding RNA molecule called Xist is responsible for switching off the extra X genes in female cells. It does this by coating the entirety of the second X chromosome. Normally, RNA molecules transmit the coded instructions in genes to the cellular machinery that manufactures proteins. "Noncoding" RNAs like Xist, however, are RNAs that have taken on different jobs inside the cell. Researchers believe that the ancestral Xist gene may have once encoded a protein but changed over time to produce only a noncoding RNA. Carter, Xu et al. therefore set out to find out how exactly this might have happened, and also how Xist might have acquired its ability to switch genes off. Initial experiments used mouse cells grown in the laboratory, in which a protein called Spen was deleted. Spen is known to help Xist silence the X chromosome. In female cells lacking Spen, the second X chromosome remained active. Other chromosomes in male and female cells also had stretches of DNA that became active upon Spen's removal. These DNA sequences, termed endogenous retroviruses, were remnants of ancestral viral infections. In other words, Spen normally acted as an antiviral defense. Analysis of genetic sequences showed that Spen recognized endogenous retrovirus sequences resembling a key region in Xist, a region which was needed for Xist to work properly. Inserting fragments of endogenous retroviruses into a defective version of Xist lacking this region also partially restored its ability to inactivate genes, suggesting that X chromosome silencing might work by hijacking cellular defenses against viruses. That is, female cells essentially 'pretend' there is a viral infection on the second X chromosome by coating it with Xist (which mimics endogenous retroviruses), thus directing Spen to shut it down. This research is an important step towards understanding how female cells carry out dosage compensation in mammals. More broadly, it sheds new light on how ancient viruses may have shaped the evolution of noncoding RNAs in the human genome.

Template Activating Factor-I α Regulates Retroviral Silencing during Reprogramming.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.

Histone demethylase Kdm2a regulates germ cell genes and endogenous retroviruses in embryonic stem cells.

Aim: To investigate the function of Kdm2a in embryonic stem cells (ESCs). Materials & methods: Expression profile analysis after Kdm2a knockout. Analysis of Kdm2a, H3K4me3 and H3K27me3 ChIP-seq data in ESCs. qPCR analysis and ChIP-qPCR analysis of epigenetic changes after Kdm2a loss. Results:Kdm2a was dispensable for pluripotency maintenance in ESCs. Kdm2a genomic binding profile was positively correlated with that of H3K4me3, Zfx and Tet1. Kdm2a directly regulated germ cell genes in primordial germ cell-like cells. Kdm2a loss led to the reduced expression of endogenous retrovirus IAPEy and resulted in the gain of H3K36me2 and loss of H3K4me3 on IAPEy. Conclusion: Kdm2a regulates germ cell genes and endogenous retroviruses in ESCs possibly through demethylating H3K36me2 and influencing H3K4me3 deposition.

A DNA-sensing-independent role of a nuclear RNA helicase, DHX9, in stimulation of NF-κB-mediated innate immunity against DNA virus infection.

DExD/H-box helicase 9 (DHX9), or RNA helicase A (RHA), is an abundant multifunctional nuclear protein. Although it was previously reported to act as a cytosolic DNA sensor in plasmacytoid dendritic cells (pDCs), the role and molecular mechanisms of action of DHX9 in cells that are not pDCs during DNA virus infection are not clear. Here, a macrophage-specific knockout and a fibroblast-specific knockdown of DHX9 impaired antiviral innate immunity against DNA viruses, leading to increased virus replication. DHX9 enhanced NF-κB-mediated transactivation in the nucleus, which required its ATPase-dependent helicase (ATPase/helicase) domain, but not the cytosolic DNA-sensing domain. In addition, DNA virus infection did not induce cytoplasmic translocation of nuclear DHX9 in macrophages and fibroblasts. Nuclear DHX9 was associated with a multiprotein complex including both NF-κB p65 and RNA polymerase II (RNAPII) in chromatin containing NF-κB-binding sites. DHX9 was essential for the recruitment of RNAPII rather than NF-κB p65, to the corresponding promoters; this function also required its ATPase/helicase activity. Taken together, our results show a critical role of nuclear DHX9 (as a transcription coactivator) in the stimulation of NF-κB-mediated innate immunity against DNA virus infection, independently of DHX9's DNA-sensing function.

A CRISPR knockout screen identifies SETDB1-target retroelement silencing factors in embryonic stem cells.

In mouse embryonic stem cells (mESCs), the expression of provirus and endogenous retroelements is epigenetically repressed. Although many cellular factors involved in retroelement silencing have been identified, the complete molecular mechanism remains elusive. In this study, we performed a genome-wide CRISPR screen to advance our understanding of retroelement silencing in mESCs. The Moloney murine leukemia virus (MLV)-based retroviral vector MSCV-GFP, which is repressed by the SETDB1/TRIM28 pathway in mESCs, was used as a reporter provirus, and we identified more than 80 genes involved in this process. In particular, ATF7IP and the BAF complex components are linked with the repression of most of the SETDB1 targets. We characterized two factors, MORC2A and RESF1, of which RESF1 is a novel molecule in retroelement silencing. Although both factors are recruited to repress provirus, their roles in repression are different. MORC2A appears to function dependent on repressive epigenetic modifications, while RESF1 regulates repressive epigenetic modifications associated with SETDB1. Our genome-wide CRISPR screen cataloged genes which function at different levels in silencing of SETDB1-target retroelements and provides a useful resource for further molecular studies.

Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor.

Replication of the murine leukemia viruses is strongly suppressed in mouse embryonic stem (ES) cells. Proviral DNAs are formed normally but are then silenced by a large complex bound to DNA by the ES cell-specific zinc-finger protein ZFP809. We show here that ZFP809 expression is not regulated by transcription but rather by protein turnover: ZFP809 protein is stable in embryonic cells but highly unstable in differentiated cells. The protein is heavily modified by the accumulation of polyubiquitin chains in differentiated cells and stabilized by the proteasome inhibitor MG132. A short sequence of amino acids at the C terminus of ZFP809, including a single lysine residue (K391), is required for the rapid turnover of the protein. The silencing cofactor TRIM28 was found to promote the degradation of ZFP809 in differentiated cells. These findings suggest that the stem cell state is established not only by an unusual transcriptional profile but also by unusual regulation of protein levels through the proteasomal degradation pathway.

Retrovirus Integration: Some Assembly Required?

Integration is a key feature of the retroviral life cycle. This process involves packaging of the viral genome into chromatin, which is often assumed to occur as a post-integration step. In this issue of Cell Host & Microbe, Wang and colleagues (Wang et al., 2016) show that chromatinization occurs before integration, raising new questions about the role of histones in retroviral integration and transcription.

Development of Antiviral Innate Immunity During In Vitro Differentiation of Mouse Embryonic Stem Cells.

The innate immunity of embryonic stem cells (ESCs) has recently emerged as an important issue in ESC biology and in ESC-based regenerative medicine. We have recently reported that mouse ESCs (mESCs) do not have a functional type I interferon (IFN)-based antiviral innate immunity. They are deficient in expressing IFN in response to viral infection and have limited ability to respond to IFN. Using fibroblasts (FBs) as a cell model, the current study investigated the development of antiviral mechanisms during in vitro differentiation of mESCs. We demonstrate that mESC-differentiated FBs (mESC-FBs) share extensive similarities with naturally differentiated FBs in morphology, marker expression, and growth pattern, but their development of antiviral mechanisms lags behind. Nonetheless, the antiviral mechanisms are inducible during mESC differentiation as demonstrated by the transition of nuclear factor kappa B (NFκB), a key transcription factor for IFN expression, from its inactive state in mESCs to its active state in mESC-FBs and by increased responses of mESC-FBs to viral stimuli and IFN during their continued in vitro propagation. Together with our previously published study, the current data provide important insights into molecular basis for the deficiency of IFN expression in mESCs and the development of antiviral innate immunity during mESC differentiation.

IFIT1 Differentially Interferes with Translation and Replication of Alphavirus Genomes and Promotes Induction of Type I Interferon.

Alphaviruses are a group of widely distributed human and animal pathogens. It is well established that their replication is sensitive to type I IFN treatment, but the mechanism of IFN inhibitory function remains poorly understood. Using a new experimental system, we demonstrate that in the presence of IFN-ß, activation of interferon-stimulated genes (ISGs) does not interfere with either attachment of alphavirus virions to the cells, or their entry and nucleocapsid disassembly. However, it strongly affects translation of the virion-delivered virus-specific RNAs. One of the ISG products, IFIT1 protein, plays a major role in this translation block, although an IFIT1-independent mechanism is also involved. The 5'UTRs of the alphavirus genomes were found to differ significantly in their ability to drive translation in the presence of increased concentration of IFIT1. Prior studies have shown that adaptation of naturally circulating alphaviruses to replication in tissue culture results in accumulation of mutations in the 5'UTR, which increase the efficiency of the promoter located in the 5'end of the genome. Here, we show that these mutations also decrease resistance of viral RNA to IFIT1-induced translation inhibition. In the presence of higher levels of IFIT1, alphaviruses with wt 5'UTRs became potent inducers of type I IFN, suggesting a new mechanism of type I IFN induction. We applied this knowledge of IFIT1 interaction with alphaviruses to develop new attenuated variants of Venezuelan equine encephalitis and chikungunya viruses that are more sensitive to the antiviral effects of IFIT1, and thus could serve as novel vaccine candidates.

High-Throughput Differentiation and Screening of a Library of Mutant Stem Cell Clones Defines New Host-Based Genes Involved in Rabies Virus Infection.

We used a genomic library of mutant murine embryonic stem cells (ESCs) and report the methodology required to simultaneously culture, differentiate, and screen more than 3,200 heterozygous mutant clones to identify host-based genes involved in both sensitivity and resistance to rabies virus infection. Established neuronal differentiation protocols were miniaturized such that many clones could be handled simultaneously, and molecular markers were used to show that the resultant cultures were pan-neuronal. Next, we used a green fluorescent protein (GFP) labeled rabies virus to develop, validate, and implement one of the first host-based, high-content, high-throughput screens for rabies virus. Undifferentiated cell and neuron cultures were infected with GFP-rabies and live imaging was used to evaluate GFP intensity at time points corresponding to initial infection/uptake and early and late replication. Furthermore, supernatants were used to evaluate viral shedding potential. After repeated testing, 63 genes involved in either sensitivity or resistance to rabies infection were identified. To further explore hits, we used a completely independent system (siRNA) to show that reduction in target gene expression leads to the observed phenotype. We validated the immune modulatory gene Unc13d and the dynein adapter gene Bbs4 by treating wild-type ESCs and primary neurons with siRNA; treated cultures were resistant to rabies infection/replication. Overall, the potential of such in vitro functional genomics screens in stem cells adds additional value to other libraries of stem cells. This technique is applicable to any bacterial or virus interactome and any cell or tissue types that can be differentiated from ESCs.