scholarly journals MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

2021 ◽  
Author(s):  
Courtney Comar ◽  
Clayton Otter ◽  
Jessica Pfannenstiel ◽  
Ethan Doerger ◽  
David Renner ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Innate Immune ◽  
Genome Replication ◽  
Accessory Proteins ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Recombinant Viruses ◽  
Dsrna Binding Protein ◽  
Immune Pathways

Middle East respiratory syndrome coronavirus (MERS CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS CoV is adept at antagonizing early innate immune pathways; these include interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L), all activated in response to viral double stranded (ds)RNA generated during genome replication. This is in contrast to SARS CoV 2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA induced innate immunity during MERS CoV infection in order to optimize viral replication.

scholarly journals Antagonism of dsRNA-Induced Innate Immune Pathways by NS4a and NS4b Accessory Proteins during MERS Coronavirus Infection

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Courtney E. Comar ◽  
Stephen A. Goldstein ◽  
Yize Li ◽  
Boyd Yount ◽  
Ralph S. Baric ◽  
...  
Keyword(s):  
Middle East ◽  
Respiratory Disease ◽  
Innate Immune ◽  
Middle East Respiratory Syndrome ◽  
Nuclear Localization Sequence ◽  
Accessory Proteins ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Human Airway ◽  
Immune Pathways

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in 2012 as a novel etiological agent of severe respiratory disease in humans. As during infection by other viruses, host sensing of viral double-stranded RNA (dsRNA) induces several antiviral pathways. These include interferon (IFN), oligoadenylate synthetase (OAS)-RNase L, and protein kinase R (PKR). Coronaviruses, including MERS-CoV, potently suppress the activation of these pathways, inducing only modest host responses. Our study describes the functions of two accessory proteins unique to MERS-CoV and related viruses, NS4a and NS4b, during infection in human airway epithelium-derived A549 cells. NS4a has been previously characterized as a dsRNA binding protein, while NS4b is a 2′,5′-phosphodiesterase with structural and enzymatic similarity to NS2 encoded by mouse hepatitis virus (MHV). We found that deletion of NS4a results in increased interferon lambda (IFNL1) expression, as does mutation of either the catalytic site or nuclear localization sequence of NS4b. All of the mutant viruses we tested exhibited slight decreases in replication. We previously reported that, like MHV NS2, NS4b antagonizes OAS-RNase L, but suppression of IFN is a previously unidentified function for viral phosphodiesterases. Unexpectedly, deletion of NS4a does not result in robust activation of the PKR or OAS-RNase L pathways. Therefore, MERS-CoV likely encodes other proteins that contribute to suppression or evasion of these antiviral innate immune pathways that should be an important focus of future work. This study provides additional insight into the complex interactions between MERS-CoV and the host immune response. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is the second novel zoonotic coronavirus to emerge in the 21st century and cause outbreaks of severe respiratory disease. More than 2,200 cases and 800 deaths have been reported to date, yet there are no licensed vaccines or treatments. Coronaviruses encode unique accessory proteins that are not required for replication but most likely play roles in immune antagonism and/or pathogenesis. Our study describes the functions of MERS-CoV accessory proteins NS4a and NS4b during infection of a human airway-derived cell line. Loss of these accessory proteins during MERS-CoV infection leads to host antiviral activation and modestly attenuates replication. In the case of both NS4a and NS4b, we have identified roles during infection not previously described, yet the lack of robust activation suggests much remains to be learned about the interactions between MERS-CoV and the infected host.

scholarly journals SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes

2021 ◽  
Vol 118 (16) ◽  
pp. e2022643118
Author(s):  
Yize Li ◽  
David M. Renner ◽  
Courtney E. Comar ◽  
Jillian N. Whelan ◽  
Hanako M. Reyes ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Innate Immune ◽  
Alveolar Type ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Induced Pluripotent ◽  
Host Virus Interactions

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase–ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2–infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host–virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

scholarly journals SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes

2020 ◽  
Author(s):  
Yize Li ◽  
David M Renner ◽  
Courtney E Comar ◽  
Jillian N Whelan ◽  
Hanako M Reyes ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Immune Responses ◽  
Innate Immune ◽  
Alveolar Type ◽  
Rnase L ◽  
Innate Immune Responses ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Ribonuclease L

SummaryCoronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase–ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.SignificanceSARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase–ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.

Introns encode dsRNAs undetected by RIG-I/MDA5/interferons and sensed via RNase L

2021 ◽  
Vol 118 (46) ◽  
pp. e2102134118
Author(s):  
Alisha Chitrakar ◽  
Kristina Solorio-Kirpichyan ◽  
Eliza Prangley ◽  
Sneha Rath ◽  
Jin Du ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Immune Responses ◽  
Innate Immune ◽  
Rna Decay ◽  
Antiviral Defense ◽  
Rnase L ◽  
Innate Immune Responses ◽  
Double Stranded Rna ◽  
Turn On ◽  
Nuclear Rna

Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNA (endo-dsRNA). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs activate endo-dsRNA responses distinct from antiviral defense programs. IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting actively protect from these innate immune responses to self. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. OAS3 provides a receptor for IIIRs, whereas RNase L cleaves IIIR-carrying introns and intergenic RNAs.

CS08-6 Extracellular 2’-5’ Oligoadenylate Synthetase stimulates RNase L-independent antiviral activity: a novel mechanism of virus-induced innate immunity

Cytokine ◽  
2011 ◽  
Vol 56 (1) ◽  
pp. 55
Author(s):  
H. Kristiansen ◽  
M.T. Madsen ◽  
H.H. Gad ◽  
K. Horan ◽  
S.R. Paludan ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Antiviral Activity ◽  
Rnase L ◽  
Oligoadenylate Synthetase

PS2-72 Extracellular 2′-5′ Oligoadenylate synthetase stimulates RNase L-independent antiviral activity: a novel mechanism of virus-induced innate immunity

Cytokine ◽  
2010 ◽  
Vol 52 (1-2) ◽  
pp. 65
Author(s):  
Helle Kristiansenp ◽  
Susanne Vends ◽  
Karthiga Thavachelvam ◽  
Thomas B. Steffensen ◽  
Søren R. Paludan ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Antiviral Activity ◽  
Rnase L ◽  
Oligoadenylate Synthetase

scholarly journals Double-Stranded RNA Sensors and Modulators in Innate Immunity

2019 ◽  
Vol 37 (1) ◽  
pp. 349-375 ◽  
Author(s):  
Sun Hur
Keyword(s):  
Innate Immunity ◽  
Immune Defense ◽  
Innate Immune ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Adenosine Deaminases ◽  
Binding Domains ◽  
Dsrna Binding ◽  
Rna Sensors ◽  
Antiviral Innate Immunity

Detection of double-stranded RNAs (dsRNAs) is a central mechanism of innate immune defense in many organisms. We here discuss several families of dsRNA-binding proteins involved in mammalian antiviral innate immunity. These include RIG-I-like receptors, protein kinase R, oligoadenylate synthases, adenosine deaminases acting on RNA, RNA interference systems, and other proteins containing dsRNA-binding domains and helicase domains. Studies suggest that their functions are highly interdependent and that their interdependence could offer keys to understanding the complex regulatory mechanisms for cellular dsRNA homeostasis and antiviral immunity. This review aims to highlight their interconnectivity, as well as their commonalities and differences in their dsRNA recognition mechanisms.

scholarly journals Reverse Genetics Reveals a Role of Rotavirus VP3 Phosphodiesterase Activity in Inhibiting RNase L Signaling and Contributing to Intestinal Viral Replication In Vivo

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Yanhua Song ◽  
Ningguo Feng ◽  
Liliana Sanchez-Tacuba ◽  
Linda L. Yasukawa ◽  
Lili Ren ◽  
...  
Keyword(s):  
Small Intestine ◽  
Viral Replication ◽  
Reverse Genetics ◽  
Innate Immune ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Rotavirus Vaccines

ABSTRACT Our understanding of how rotavirus (RV) subverts host innate immune signaling has greatly increased over the past decade. However, the relative contribution of each virus-encoded innate immune antagonist has not been fully studied in the context of RV infection in vivo. Here, we present both in vitro and in vivo evidence that the host interferon (IFN)-inducible 2′-5′-oligoadenylate synthetase (OAS) and RNase L pathway effectively suppresses the replication of heterologous RV strains. VP3 from homologous RVs relies on its 2′-5′-phosphodiesterase (PDE) domain to counteract RNase L-mediated antiviral signaling. Using an RV reverse-genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at low levels in the small intestine and were shed less in the feces of wild-type mice, and such defects were rescued in Rnasel−/− suckling mice. Collectively, these findings highlight an important role of VP3 in promoting viral replication and pathogenesis in vivo in addition to its well-characterized function as the viral RNA-capping enzyme. IMPORTANCE Rotaviruses are significant human pathogens that result in diarrhea, dehydration, and deaths in many children around the world. Rotavirus vaccines have suboptimal efficacy in low- to middle-income countries, where the burden of the diseases is the most severe. With the ultimate goal of improving current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virus-encoded VP3 antagonizes RNase L activity both in vitro and in vivo. These studies highlight an ever-evolving arms race between antiviral factors and viral pathogens and provide a new means of targeted attenuation for next-generation rotavirus vaccine design.

scholarly journals Cnidarian Pattern Recognition Receptor Repertoires Reflect Both Phylogeny and Life History Traits

2021 ◽  
Vol 12 ◽  
Author(s):  
Madison A. Emery ◽  
Bradford A. Dimos ◽  
Laura D. Mydlarz
Keyword(s):  
Pattern Recognition ◽  
Innate Immunity ◽  
Life History ◽  
Life History Traits ◽  
Innate Immune ◽  
Immune Pathways ◽  
Immune Pathway ◽  
Molecular Patterns ◽  
Insight Into ◽  
Recognition Receptor

Pattern recognition receptors (PRRs) are evolutionarily ancient and crucial components of innate immunity, recognizing danger-associated molecular patterns (DAMPs) and activating host defenses. Basal non-bilaterian animals such as cnidarians must rely solely on innate immunity to defend themselves from pathogens. By investigating cnidarian PRR repertoires we can gain insight into the evolution of innate immunity in these basal animals. Here we utilize the increasing amount of available genomic resources within Cnidaria to survey the PRR repertoires and downstream immune pathway completeness within 15 cnidarian species spanning two major cnidarian clades, Anthozoa and Medusozoa. Overall, we find that anthozoans possess prototypical PRRs, while medusozoans appear to lack these immune proteins. Additionally, anthozoans consistently had higher numbers of PRRs across all four classes relative to medusozoans, a trend largely driven by expansions in NOD-like receptors and C-type lectins. Symbiotic, sessile, and colonial cnidarians also have expanded PRR repertoires relative to their non-symbiotic, mobile, and solitary counterparts. Interestingly, cnidarians seem to lack key components of mammalian innate immune pathways, though similar to PRR numbers, anthozoans possess more complete immune pathways than medusozoans. Together, our data indicate that anthozoans have greater immune specificity than medusozoans, which we hypothesize to be due to life history traits common within Anthozoa. Overall, this investigation reveals important insights into the evolution of innate immune proteins within these basal animals.

scholarly journals Structure and Activities of the NS1 Influenza Protein and Progress in the Development of Small-Molecule Drugs

2021 ◽  
Vol 22 (8) ◽  
pp. 4242
Author(s):  
Hyeonjin Kim ◽  
Misuk Jeong ◽  
Sebok Jang
Keyword(s):  
Influenza Virus ◽  
Small Molecule ◽  
Global Scale ◽  
Antigenic Drift ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Viral Protein Synthesis ◽  
Antiviral Responses ◽  
Virus Replication Cycle

The influenza virus causes human disease on a global scale and significant morbidity and mortality. The existing vaccination regime remains vulnerable to antigenic drift, and more seriously, a small number of viral mutations could lead to drug resistance. Therefore, the development of a new additional therapeutic small molecule-based anti-influenza virus is urgently required. The NS1 influenza gene plays a pivotal role in the suppression of host antiviral responses, especially by inhibiting interferon (IFN) production and the activities of antiviral proteins, such as dsRNA-dependent serine/threonine-protein kinase R (PKR) and 2′-5′-oligoadenylate synthetase (OAS)/RNase L. NS1 also modulates important aspects of viral RNA replication, viral protein synthesis, and virus replication cycle. Taken together, small molecules that target NS1 are believed to offer a means of developing new anti-influenza drugs.

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