Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. ...Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.
Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding ...to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized
HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the
locus may play an important role for HCMV replication.
Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.
The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T ...cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell-keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.
Abstract
RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in ...unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA–RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.
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Abstract
RNA-protein interactions determine the cellular fate of RNA and are central to regulating gene expression outcomes in health and disease. To date, no method exists that is able to identify ...proteins that interact with specific regions within endogenous RNAs in live cells. Here, we develop SHIFTR (Selective RNase H-mediated interactome framing for target RNA regions), an efficient and scalable approach to identify proteins bound to selected regions within endogenous RNAs using mass spectrometry. Compared to state-of-the-art techniques, SHIFTR is superior in accuracy, captures minimal background interactions and requires orders of magnitude lower input material. We establish SHIFTR workflows for targeting RNA classes of different length and abundance, including short and long non-coding RNAs, as well as mRNAs and demonstrate that SHIFTR is compatible with sequentially mapping interactomes for multiple target RNAs in a single experiment. Using SHIFTR, we comprehensively identify interactions of cis-regulatory elements located at the 5′ and 3′-terminal regions of authentic SARS-CoV-2 RNAs in infected cells and accurately recover known and novel interactions linked to the function of these viral RNA elements. SHIFTR enables the systematic mapping of region-resolved RNA interactomes for any RNA in any cell type and has the potential to revolutionize our understanding of transcriptomes and their regulation.
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Graphical Abstract
The recent pandemic has reminded the public that basic research in virology is pivotal for human health. Understanding the mechanisms of successful viral replication and the role of host factors can ...help to combat viral infections and prevent future pandemics.
Our lab has published the first SARS-CoV-2 RNA-protein interaction atlas, laying the foundation to investigate the interplay between viral RNA and host RNA binding proteins (RBP). Based on this, my project created the largest collection of binding profiles of host and viral RBPs on SARS-CoV-2 RNA to date. This revealed the host protein SND1 as the first human RBP that specifically binds negative sense viral RNA at the 5´ end, a region associated with viral transcription initiation. The binding profile shares similarities with the viral RBP nsp9, which binds the 5´ ends of positive and negative sense SARS-CoV-2 RNA. Depletion of SND1 shows reduced levels of viral RNA revealing it as a proviral host factor. To decode the underlying molecular mechanism, I characterized the protein-protein interactions of SND1 in SARS-CoV-2 infected and uninfected cells. Infection remodels the protein interactors of SND1 from general RNA biology to membrane association and viral RNA synthesis. Upon infection, SND1 specifically interacts with nsp9, the RBP that shares the same binding region on the negative strand of SARS-CoV-2 RNA. Recent work demonstrates that nsp9 is NMPylated in vitro suggesting a functional role of nsp9 in priming of viral RNA synthesis. I was able to show that nsp9 is covalently linked to the 5´ ends of SARS-CoV-2 RNA during infection of human cells. Analysing the covalent bond of nsp9 with the viral RNA on nucleotide level shows close proximity to the initiation sites of viral RNA synthesis, suggesting that nsp9 acts as a protein-primer of SARS-CoV-2 RNA synthesis. SND1 modulates the distribution of nsp9 on the viral RNA, since depletion of SND1 results in imbalanced occupancy of nsp9 at the 5´ends of viral RNA.
This study is the first to provide evidence for the priming mechanism of SARS-CoV-2 in authentic viral replication and further reveals how this mechanism is modulated by the host RBP SND1. Detailed knowledge about priming of viral RNA synthesis can help to find targeted antivirals that could be used to fight coronaviral infections.
Die letzte Pandemie zeigte erneut, das Grundlagenforschung im Bereich der Virologie essentiell für die Gesundheit des Menschen ist. Das Wissen über Schlüsselelemente erfolgreicher viraler Replikation und der Relevanz humaner Proteine darin kann helfen Infektionen zu bekämpfen und künftige Pandemien zu verhindern.
Unser Labor publizierte das erste SARS-CoV-2 RNA Protein-Interaktom und legte dabei den Grundstein für die Forschung am Zwischenspiel viraler RNA und humanen RNA Bindeproteinen (RBPs). Basierend darauf, generierte mein Projekt die bislang größte Sammlung an Bindeprofilen humaner sowie viraler RBPs auf der SARS-CoV-2 RNA. Dabei zeigte sich der Wirtsfaktor SND1 als das erste human RBP das in der Lage ist den Negativstrang der viral RNA zu binden, spezifisch an dessen 5´ Ende welches mit der Transkriptionsinitiierung assoziiert ist. Diese Bindestelle ist ähnlich zu dem viralen RBP nsp9, welches die 5´ Enden der positiv und negativ RNA bindet. Das Fehlen von SND1 in der Wirtszelle führt zu reduzierten Mengen viraler RNA und impliziert daher einen proviralen Einfluss von SND1. Um den zugrundeliegenden molekularen Mechanismus zu verstehen, betrachtete ich die Protein-Protein Interaktionen von SND1 in SARS-CoV-2 infizierten und uninfizierten Zellen. Dabei zeigte sich, dass durch die Infektion die Interaktionspartner von SND1 von genereller RNA Biologie zu Membranassoziierung sowie viraler RNA Synthese verschiebt. Mit Infektion der Zelle interagiert SND1 spezifisch mit nsp9, das RBP welches dieselbe Binderegion am Negativstrang mit SND1 auf der SARS-CoV-2 RNA teilt. Neuste in vitro Studien zeigen, dass nsp9 NMPyliert wird und deuten damit eine Relevanz von nsp9 in Priming an. Ich konnte im Kontext authentischer viraler Replikation zeigen, dass nsp9 kovalent an die 5´ Enden der SARS-CoV-2 RNA gebunden ist. Bei genauerer Untersuchung der kovalenten Bindung von nsp9 an der viralen RNA auf Nukleotidebene zeigt, dass diese Nahe der Initiationsstelle der Transkription liegen, was eine Relevanz von nsp9 als Protein-Primer in der SARS-CoV-2 RNA Synthese impliziert. Die Richtige Verteilung von nsp9 auf der viralen RNA wird von SND1 moduliert, da Abwesenheit von SND1 zu einem Ungleichgewicht von nsp9 an den 5´ Enden führt.
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Diese Studie ist die Erste, die Evidenzen für den Primingmechanismus von SARS-CoV-2 in authentischer viraler Replikation zeigt und wie diese durch SND1 moduliert wird. Detailliertes Wissen über das Priming viraler RNA Synthese kann dabei helfen gezielte nach antiviralen Substanzen zu suchen, die dabei helfen könnten Infektionen durch Coronaviren zu bekämpfen.
Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins ...bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5′ end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5′ ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
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•SND1 is required for nascent SARS-CoV-2 RNA synthesis early during infection•SND1 directly interacts with NSP9 and both proteins bind negative-sense viral RNA•NSP9 is covalently linked to viral RNA at initiation sites, indicating protein priming•SND1 modulates the covalent linkage of NSP9 to positive- and negative-sense viral RNA
Mapping of subgenome-resolved SARS-CoV-2 RNA-protein interactions reveals that the host protein SND1 binds negative-sense SARS-CoV-2 RNA and promotes viral RNA synthesis by recruiting NSP9, which likely serves as a protein primer for RNA production.
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