Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3′-Terminal Adenylyltransferase Activity

Tvarogová, Jana; Madhugiri, Ramakanth; Bylapudi, Ganesh; Ferguson, Lyndsey J.; Karl, Nadja; Ziebuhr, John

doi:10.1128/jvi.00291-19

Cited by 60 publications

(63 citation statements)

References 62 publications

(116 reference statements)

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“…They identified a conserved viral sequence on the positive-sense RNA that could be bound by host proteins, including cytoplasmic polyadenylation element binding protein 1 (CPEB1) (47), a protein that mediates polyadenylation. In addition, CoV nsp8 has been demonstrated to contain 3′ terminal adenylyltransferase (TAT) activity (48). Nsp8 can synthesize the polyA tail on the positive-sense RNA, and having a complement negative-sense RNA with a polyU extension greater than five uridines enhances the TAT activity.…”

Section: Discussionmentioning

confidence: 99%

Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors

Hackbart

Deng

Baker

2020

Proc. Natl. Acad. Sci. U.S.A.

269

265

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Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5′-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wildtype−infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.C oronaviruses (CoVs) are positive-sense RNA viruses that replicate in the cytoplasm of infected cells. The positivesense virion RNA is translated to generate the viral replication machinery, which then replicates the positive-sense RNA into negative-sense, genomic RNA and subgenomic RNAs (sgRNAs). The negative-sense RNAs then function as templates for synthesis of positive-sense genomic RNA and sgRNA (1, 2). This replication strategy can generate long double-stranded RNA (dsRNA) intermediates (3), that may act as pathogen-associated molecular patterns (PAMPs) recognized by cytoplasmic pattern recognition receptors (PRRs) (4, 5). The specific PRR that recognizes CoV RNA is MDA5, which can activate the type I interferon (IFN) response in macrophages (6). Other host dsRNA PRRs, such as PKR and OAS, are also activated and operate to limit CoV replication (7-11). CoVs encode multiple proteins that antagonize these innate immune responses, particularly the activation of the IFN response (9, 12-16), ultimately leading to a dysregulated immune response and increased immunopathogenesis (17,18). Understanding the mechanisms used by CoVs to delay IFN signaling may provide opportunities for the development of antivirals and live-attenuated vaccines to limit CoV infections.Here, we investigate the mechanism used by one CoV IFN antagonist, the nonstructural protein 15 (nsp15), which is an endoribonuclease designated EndoU. EndoU is highly conserved in all known CoVs (19,20). EndoU is similar to the cellular endoribonuclease XendoU, as revealed by bioinformatic analysis of the amino acid sequence (21). X-ray structures of EndoU revealed conserved endoribonuclease folds with catalytic histidine residues required for endoribonuclease activity (22)(23)(24)(25)(26). Purified EndoU was shown to cleave single-str...

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Section: Discussionmentioning

confidence: 99%

Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors

Hackbart

Deng

Baker

2020

Proc. Natl. Acad. Sci. U.S.A.

269

265

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“…Taken together with our results, this may suggest non-canonical sub-genomic RNAs with coding potential are produced during SARS-CoV-2 infection; however, this would require further validation. HCoV-299E non-structural protein 8 (nsp8) was recently shown to possess template-independent adenyltransferase activity [31]. Because poly-A tails play important roles in the stability and translation potential of canonical SARS-CoV-2 sgRNAs, it is interesting to speculate that coronaviruses might rely on the production of non-canonical, poly-adenylated sgRNAs to serve as decoys for cellular deadenylases.…”

Section: Discussionmentioning

confidence: 99%

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium

Ravindra

Alfajaro

Gasque

et al. 2020

Preprint

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SARS-CoV-2, the causative agent of COVID-19, has resulted in more than 3,000,000 infections and 200,000 deaths. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as the major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon stimulated genes in both infected and bystander cells. Here, we have conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and provide a detailed characterization of genes, cell types, and cell state changes associated with the infection.CoVs are enveloped viruses with positive-sense, single-stranded RNA genomes ranging from 26-30 kb [3]. Six human CoVs have been previously identified: HCoV-NL63 and HCoV-229E, which belong to the Alphacoronavirus genus; and HCoV-OC43, HCoV-HKU1, SARS-CoV, and Middle East Respiratory Syndrome CoV (MERS-CoV), which belong to the Betacoronavirus genus [4]. In the past two decades, CoVs have become a major public health concern due to potential zoonotic transmission, as revealed by the emergence of SARS-CoV in 2002, which infected 8, 000 people worldwide with a mortality rate of 10-15%, and MERS-CoV in 2012 and 2019, which infected 2, 500 people with a mortality rate of 35%, and now SARS-CoV-2 (WHO).Tissue and cell tropism are key determinants of viral pathogenesis. SARS-CoV-2 entry into cells depends on the binding of the viral spike (S) protein to its cognate receptor angiotensin-converting enzyme II (ACE2) on the cell surface [2]. ACE2 is also the receptor for SARS-CoV and HCoV-NL63, yet these viruses induce profoundly different morbidity and mortality suggesting unknown determinants of coronavirus pathogenesis [5,6]. Additionally, proteolytic priming of the S protein by host proteases is also critical for viral entry [7]. The cellular serine protease Type II transmembrane (TMPRSS2) is used by SARS-CoV-2 for S protein priming [8,7,9,10]. This is also used by SARS-CoV alongside the endosomal cysteine proteases cathepsin B and L [11,12]. Another host protease, furin, has been suggested to mediate SARS-CoV-2 pathogenesis; however, the precise role of host proteases in SARS-CoV-2 entry remains to be determined [13,10].SARS-CoV and MERS-CoV caused fatal pneumonia associated with rapid virus replication, elevation of proinflammatory cytokines, and immune cell infiltration [14]. These characteri...

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“…Reactions were quenched at indicated timepoints with 5X volume of FBD stop solution (formamide, 10 mM EDTA). To verify that activity was not due to either nsp7L8 or nsp8, which have been shown to contain primase-like noncanonical RdRp activities [40][41][42][43] assays were run in 310 the absence of nsp12. The NTP K d was estimated using the same setup described here, but with final (equimolar) concentration of ATP, GTP and UTP ranging from 0.78 -100 µM (2-fold dilution series).…”

Section: Steady-state Elongation Complex Reactionsmentioning

confidence: 99%

Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase

Shannon

Selisko

et al. 2020

Preprint

101

108

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The ongoing Corona Virus Disease 2019 pandemic, caused by severe 20 acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp 25 complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of 30 COVID-19.2 Coronaviruses (CoV) are large genome, positive-strand RNA viruses of the order 35 Nidovirales that have recently attracted global attention due to the ongoing COVID-19 pandemic. Despite significant efforts to control its spread, SARS-CoV-2 has caused substantial health and economic burden, emphasizing the immediate need for antiviral treatments. As with all positive strand RNA viruses, an RdRp lies at the core of the viral replication machinery and for CoVs this is the nsp12 protein. The pivotal role of nsp12 in the viral life-cycle, lack of host 40 homologues and high level of sequence and structural conservation makes it an optimal target for therapeutics. However, there has been remarkably little biochemical characterization of nsp12 and a lack of fundamental data to guide the design of antiviral therapeutics and study their mechanism of action (MoA). A promising class of RdRp inhibitors are nucleoside analogues (NAs), small molecule drugs that are metabolized intracellularly into their active ribonucleoside 45 5'-triphosphate (RTP) forms and incorporated into the nascent viral RNA by error-prone viral RdRps. This can disrupt RNA synthesis directly via chain termination, or can lead to the accumulation of deleterious mutations in the viral genome. For CoVs, the situation is complicated by the post-replicative repair capacity provided by the nsp14 exonuclease (ExoN) that is essential for maintaining the integrity of their large ~30 kb genomes 1-3 . Nsp14 has been 50 shown to remove certain NAs after insertion by the RdRp into the nascent RNA, thus reducing their antiviral effects 4-6 . Despite this, several NAs currently being used for the treatment of other viral infections have been identified as potential anti-CoV candidates 7-9 . Among these is the purine base analogue T-705 (Favipiravir, Avigan, Extended data Fig. 1a,b) that has broadspectrum activity against a number of RNA viruses and is currently licensed in Japan for use in 55

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Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3′-Terminal Adenylyltransferase Activity

Cited by 60 publications

References 62 publications

Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors

Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium

Favipiravir strikes the SARS-CoV-2 at its Achilles heel, the RNA polymerase

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