scholarly journals The Strand-biased Transcription of SARS-CoV-2 and Unbalanced Inhibition by Remdesivir

2020 ◽  
Author(s):  
Yan Zhao ◽  
Jing Sun ◽  
Yunfei Li ◽  
Zhengxuan Li ◽  
Yu Xie ◽  
...  
Keyword(s):  
Immune Responses ◽  
Rna Polymerase ◽  
Vaccine Development ◽  
Rna Virus ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Negative Strand ◽  
Sense Strand ◽  
Viral Vaccine ◽  
Polymerase Inhibitor

AbstractSARS-CoV-2, a positive single-stranded RNA virus, caused the COVID-19 pandemic. During the viral replication and transcription, the RNA dependent RNA polymerase (RdRp) “jumps” along the genome template, resulting in discontinuous negative-stranded transcripts. In other coronaviruses, the negative strand RNA was found functionally relevant to the activation of host innate immune responses. Although the sense-mRNA architectures of SARS-CoV-2 were reported, its negative strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand. During negative strand synthesis, apart from canonical sub-genomic ORFs, numerous non-canonical fusion transcripts are formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor Remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the anti-viral vaccine development and drug design.One Sentence SummaryStrand-biased transcription of SARS-CoV-2.

scholarly journals The deubiquitinating activity of Middle East respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model

2019 ◽  
Author(s):  
Robert C. M. Knaap ◽  
Raúl Fernández-Delgado ◽  
Tim J. Dalebout ◽  
Nadia Oreshkova ◽  
Peter J. Bredenbeek ◽  
...  
Keyword(s):  
Immune Response ◽  
Middle East ◽  
Mouse Model ◽  
Immune Responses ◽  
Innate Immune Response ◽  
Vaccine Development ◽  
Innate Immune ◽  
Middle East Respiratory Syndrome ◽  
Mrna Levels ◽  
Innate Immune Responses

AbstractMiddle East respiratory syndrome coronavirus (MERS-CoV) continues to cause zoonotic infections and serious disease, primarily in the Arabian Peninsula, due to repeated spill-over from dromedary camels and subsequent nosocomial transmission. Approved MERS vaccines for use in animals or humans are not currently available. MERS-CoV replication requires the virus-encoded papain-like protease (PLpro) to cleave multiple sites in the viral replicase polyproteins, thereby releasing functional non-structural proteins. Additionally, PLpro is a deubiquitinating enzyme (DUB) that can remove ubiquitin(-like) moieties from substrates, presumably to counteract host antiviral responses. In previous work, we determined the crystal structure of MERS-CoV PLpro in complex with ubiquitin, facilitating the design of PLpro mutations that impair DUB activity without affecting viral polyprotein cleavage. Here, we introduced these DUB-inactivating mutations into the viral genome and examined their impact on MERS-CoV infection both in cell culture and in a lethal mouse model. Although overall replication of DUB-negative and wild-type (wt) recombinant MERS-CoV was comparable in multiple cell lines, infection with DUB-negative virus markedly increased mRNA levels for interferon (IFN)-β and IFN-stimulated genes. Moreover, compared to a wt virus infection, the survival rate was significantly increased when DUB-negative MERS-CoV was used to infect transgenic mice expressing a human MERS-CoV receptor. Interestingly, DUB-negative and wt MERS-CoV replicated to the same titers in lungs of infected mice, but the DUB-negative virus was cleared faster, likely due to the observed accelerated and better-regulated innate immune responses, in contrast to delayed and subsequently excessive responses in wt virus-infected mice. This study provides the first direct evidence that the DUB activity of a coronaviral protease contributes to innate immune evasion and can profoundly enhance virulence in an animal model. Thus, reduction or removal of the innate immune-suppressive DUB activity of PLpros is a promising strategy for coronavirus attenuation in the context of rational vaccine development.Author SummaryAlthough zoonotic coronaviruses such as Middle East respiratory coronavirus (MERS-CoV) have pandemic potential, therapeutics and vaccines that counteract this public health threat are not currently available. Coronaviruses typically employ multiple strategies to evade the host’s innate immune response, which may enhance clinical disease and/or reduce the efficacy of modified live vaccines. The MERS-CoV-encoded papain-like protease (PLpro) is not only crucial for the expression of functional replicase proteins, but has also been postulated to antagonize ubiquitination-dependent steps during the activation of the innate immune response. Here, we report the generation of engineered MERS-CoVs mutants in which PLpro’s deubiquitinating (DUB) activity was specifically disrupted without affecting virus viability. In this manner, we could demonstrate that the DUB activity of PLpro suppresses the interferon response in MERS-CoV-infected cells. Strikingly, in the lungs of mice infected with DUB-negative MERS-CoV, innate immune responses were induced at an earlier stage of infection than in wt virus-infected mice. This group also showed a clearly increased survival, indicating that the DUB activity is an important MERS-CoV virulence factor. This proof-of-concept study establishes that the engineering of DUB-negative coronaviruses, which elicit a more effective immune response in the host, is a viable strategy for vaccine development.

scholarly journals The Nucleolus Is the Site of Borna Disease Virus RNA Transcription and Replication

1998 ◽  
Vol 72 (9) ◽  
pp. 7697-7702 ◽  
Author(s):  
J. M. Pyper ◽  
J. E. Clements ◽  
M. C. Zink
Keyword(s):  
Rna Polymerase ◽  
Disease Virus ◽  
Rna Virus ◽  
Neuroblastoma Cells ◽  
Rna Polymerase I ◽  
Borna Disease Virus ◽  
Negative Strand ◽  
Sense Strand ◽  
Polymerase I ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) is a neurotropic nonsegmented negative-strand RNA virus with limited homology to rhabdoviruses and paramyxoviruses. A distinguishing feature of BDV is that it replicates in the nucleus of infected cells. Strand-specific probes used for in situ hybridization of infected rat brain showed that there was differential localization of positive- and negative-strand RNAs within the nucleus of neurons. Within nuclei, sense-strand RNAs were preferentially localized within nucleolar regions while genomic-sense RNAs were found in both nucleolar and nonnucleolar regions. These results suggested a role for the nucleolus in BDV replication. Nucleoli isolated from persistently infected neuroblastoma cells contained both genomic and antigenomic BDV RNA species as well as an enrichment of the 39/38-kDa and gp18 BDV proteins. Since the nucleolus is the site of rRNA transcription, we examined BDV transcription in the presence of inhibitors of RNA polymerase I. Inhibition of RNA polymerase I did not affect levels of BDV transcription.

scholarly journals Rotavirus NSP1 Requires Casein Kinase II-Mediated Phosphorylation for Hijacking of Cullin-RING Ligases

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Kaitlin A. Davis ◽  
Marco Morelli ◽  
John T. Patton
Keyword(s):  
Immune Responses ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Ubiquitin Ligases ◽  
Innate Immune ◽  
E3 Ubiquitin Ligases ◽  
Innate Immune Responses ◽  
Double Stranded Rna ◽  
Severe Diarrhea ◽  
Ring E3

ABSTRACT The rotavirus nonstructural protein NSP1 repurposes cullin-RING E3 ubiquitin ligases (CRLs) to antagonize innate immune responses. By functioning as substrate adaptors of hijacked CRLs, NSP1 causes ubiquitination and proteasomal degradation of host proteins that are essential for expression of interferon (IFN) and IFN-stimulated gene products. The target of most human and porcine rotaviruses is the β-transducin repeat-containing protein (β-TrCP), a regulator of NF-κB activation. β-TrCP recognizes a phosphorylated degron (DSGΦXS) present in the inhibitor of NF-κB (IκB); phosphorylation of the IκB degron is mediated by IκB kinase (IKK). Because NSP1 contains a C-terminal IκB-like degron (ILD; DSGXS) that recruits β-TrCP, we investigated whether the NSP1 ILD is similarly activated by phosphorylation and whether this modification is required to trigger the incorporation of NSP1 into CRLs. Based on mutagenesis and phosphatase treatment studies, we found that both serine residues of the NSP1 ILD are phosphorylated, a pattern mimicking phosphorylation of IκB. A three-pronged approach using small-molecule inhibitors, small interfering RNAs, and mutagenesis demonstrated that NSP1 phosphorylation is mediated by the constitutively active casein kinase II (CKII), rather than IKK. In coimmunoprecipitation assays, we found that this modification was essential for NSP1 recruitment of β-TrCP and induced changes involving the NSP1 N-terminal RING motif that allowed formation of Cul3-NSP1 complexes. Taken together, our results indicate a highly regulated stepwise process in the formation of NSP1-Cul3 CRLs that is initiated by CKII phosphorylation of NSP1, followed by NSP1 recruitment of β-TrCP and ending with incorporation of the NSP1–β-TrCP complex into the CRL via interactions dependent on the highly conserved NSP1 RING motif. IMPORTANCE Rotavirus is a segmented double-stranded RNA virus that causes severe diarrhea in young children. A primary mechanism used by the virus to inhibit host innate immune responses is to hijack cellular cullin-RING E3 ubiquitin ligases (CRLs) and redirect their targeting activity to the degradation of cellular proteins crucial for interferon expression. This task is accomplished through the rotavirus nonstructural protein NSP1, which incorporates itself into a CRL and serves as a substrate recognition subunit. The substrate recognized by the NSP1 of many human and porcine rotaviruses is β-TrCP, a protein that regulates the transcription factor NF-κB. In this study, we show that formation of NSP1 CRLs is a highly regulated stepwise process initiated by CKII phosphorylation of the β-TrCP recognition motif in NSP1. This modification triggers recruitment of the β-TrCP substrate and induces subsequent changes in a highly conserved NSP1 RING domain that allow anchoring of the NSP1–β-TrCP complex to a cullin scaffold. IMPORTANCE Rotavirus is a segmented double-stranded RNA virus that causes severe diarrhea in young children. A primary mechanism used by the virus to inhibit host innate immune responses is to hijack cellular cullin-RING E3 ubiquitin ligases (CRLs) and redirect their targeting activity to the degradation of cellular proteins crucial for interferon expression. This task is accomplished through the rotavirus nonstructural protein NSP1, which incorporates itself into a CRL and serves as a substrate recognition subunit. The substrate recognized by the NSP1 of many human and porcine rotaviruses is β-TrCP, a protein that regulates the transcription factor NF-κB. In this study, we show that formation of NSP1 CRLs is a highly regulated stepwise process initiated by CKII phosphorylation of the β-TrCP recognition motif in NSP1. This modification triggers recruitment of the β-TrCP substrate and induces subsequent changes in a highly conserved NSP1 RING domain that allow anchoring of the NSP1–β-TrCP complex to a cullin scaffold.

PS2-025. Activation of RIG-I-dependent innate immune responses by incoming RNA virus nucleocapsids containing a 5’-triphosphorylated genome

Cytokine ◽  
2011 ◽  
Vol 56 (1) ◽  
pp. 69
Author(s):  
Friedemann Weber ◽  
Ali Gawanbacht-Ramorrhose ◽  
Michaela Weber ◽  
Matthias Habjan
Keyword(s):  
Immune Responses ◽  
Rna Virus ◽  
Innate Immune ◽  
Innate Immune Responses

scholarly journals Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans

2011 ◽  
Vol 208 (12) ◽  
pp. 2357-2366 ◽  
Author(s):  
Marina Caskey ◽  
François Lefebvre ◽  
Abdelali Filali-Mouhim ◽  
Mark J. Cameron ◽  
Jean-Philippe Goulet ◽  
...  
Keyword(s):  
Immune Responses ◽  
Subcutaneous Administration ◽  
Innate Immune ◽  
Transcriptional Analysis ◽  
Yellow Fever Vaccine ◽  
Type I ◽  
Innate Immune Responses ◽  
Double Stranded Rna ◽  
Viral Vaccine ◽  
Immune Pathways

Adjuvants are critical for the success of vaccines. Agonists of microbial pattern recognition receptors (PRRs) are promising new adjuvant candidates. A mechanism through which adjuvants enhance immune responses is to stimulate innate immunity. We studied the innate immune response in humans to synthetic double-stranded RNA (polyinosinic:polycytidylic acid [poly IC] stabilized with poly-l-lysine [poly ICLC]), an agonist for toll-like receptor (TLR) 3, and the cytosolic RNA helicase MDA-5. Transcriptional analysis of blood samples from eight volunteers, after subcutaneous administration of poly ICLC, showed up-regulation of genes involved in multiple innate immune pathways in all subjects, including interferon (IFN) and inflammasome signaling. Blocking type I IFN receptor ex vivo significantly dampened the response to poly IC. Comparative transcriptional analysis showed that several innate immune pathways were similarly induced in volunteers immunized with the highly efficacious yellow fever vaccine. Therefore, a chemically defined PRR agonist like poly ICLC can be a reliable and authentic microbial mimic for inducing innate immune responses in humans.

The Tissue Factor/Thrombin/Protease-Activated Receptor 1 Pathway Enhances Double-Strand RNA Induced Immune Responses in Macrophages

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4114-4114
Author(s):  
Silvio Antoniak ◽  
Kohei Tatsumi ◽  
Nigel Mackman
Keyword(s):  
Immune Response ◽  
Immune Responses ◽  
Tissue Factor ◽  
Rna Virus ◽  
Innate Immune ◽  
Poly I:C ◽  
Type I ◽  
Innate Immune Responses ◽  
Protease Activated Receptor 1 ◽  
Viral Responses

Abstract Introduction: Co-regulation of the immune response and the coagulation cascade after infection is thought to be an ancient response to limit pathogen spread. Recently, we showed that activation of the thrombin receptor, protease-activated receptor 1 (PAR1), on fibroblasts enhanced the innate immune responses to RNA virus infection. Here, we investigated whether PAR1 activation by the extrinsic coagulation pathway contributes to dsRNA-induced innate immune responses in macrophages. Methods: Activation of the type-I interferon (IFN) pathway in the murine macrophage cell line RAW264.7 and bone-marrow derived macrophages (BMDM) from WT and PAR1-/- was analyzed after dsRNA (poly I:C) and/or PAR-1 stimulation. In addition, innate immune responses in the spleen were analyzed in vivo 4 hours after poly I:C (8mg/kg) injection in mice with reduced tissue factor expression (LowTF) or global PAR1 deletion (PAR1-/-) as well as in WT mice with a thrombin inhibitor (dabigatran etexilate, 10g/kg chow) or PAR-1 inhibitor (SCH79797, 25μg/kg). Lastly, we investigated the innate immune response in the spleen of WT and PAR1-/-mice after infection with the single-stranded RNA virus coxsackievirus B3. Results: RAW264.7 and BMDM exhibited a toll-like receptor 3 dependent induction of IFNβ and CXCL10 after poly I:C stimulation. Activation of PAR-1 with either thrombin or agonist peptide enhanced poly I:C induction of IFNβ and CXCL10. A deficiency of tissue factor levels, thrombin inhibition, PAR-1 inhibition or PAR1 deficiency resulted in reduced expression levels of type-I IFNs and IFN-response genes such as CXCL10 in the spleen and plasma in mice given poly I:C. Last, PAR1-/-mice exhibited impaired IFNβ immune response 4 days after coxsackievirus B3 infection compared to WT mice. Conclusion: Our study indicates that the coagulation dependent activation of PAR1 on macrophages is important for anti-viral responses to dsRNA. We speculate that PAR1 inhibition may interfere with anti-viral responses in humans. Disclosures No relevant conflicts of interest to declare.

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