The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19 and 20) and another within NSP1 (R124) which are required for viral evasion, and thus might present promising drug targets. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of anti-viral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

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Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements

Journal of Virology ◽

10.1128/jvi.00160-09 ◽

2009 ◽

Vol 83 (12) ◽

pp. 6087-6097 ◽

Cited By ~ 26

Author(s):

Kortney M. Gustin ◽

Bo-Jhih Guan ◽

Agnieszka Dziduszko ◽

David A. Brian

Keyword(s):

Rna Binding ◽

Nonstructural Protein ◽

Binding Protein ◽

Rna Binding Protein ◽

Coding Region ◽

Bovine Coronavirus ◽

Stem Loop ◽

Nonstructural Protein 1 ◽

Gel Shift ◽

Rna Levels

ABSTRACT Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5′-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of ∼60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5′ untranslated region (UTR)- and one 3′ UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5′ UTR with ∼2.5 μM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.

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Interaction between NS1 and Cellular MAVS Contributes to NS1 Mitochondria Targeting

Viruses ◽

10.3390/v13101909 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1909

Author(s):

Yeu-Yang Tseng ◽

Chih-Ying Kuan ◽

Masaki Mibayashi ◽

Chi-Jene Chen ◽

Peter Palese ◽

...

Keyword(s):

Influenza A ◽

Early Stage ◽

Nonstructural Protein ◽

Influenza Virus Infection ◽

Type I ◽

Mitochondrial Localization ◽

Independent Manner ◽

Nonstructural Protein 1 ◽

Mitochondrial Antiviral Signaling ◽

Mitochondria Targeting

Influenza A virus nonstructural protein 1 (NS1) plays an important role in evading host innate immunity. NS1 inhibits interferon (IFN) responses via multiple mechanisms, including sequestering dsRNA and suppressing retinoic acid-inducible gene I (RIG-I) signaling by interacting with RIG-I and tripartite motif-containing protein 25 (TRIM25). In the current study, we demonstrated the mitochondrial localization of NS1 at the early stage of influenza virus infection. Since NS1 does not contain mitochondria-targeting signals, we suspected that there is an association between the NS1 and mitochondrial proteins. This hypothesis was tested by demonstrating the interaction of NS1 with mitochondrial antiviral-signaling protein (MAVS) in a RIG-I-independent manner. Importantly, the association with MAVS facilitated the mitochondrial localization of NS1 and thereby significantly impeded MAVS-mediated Type I IFN production.

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Rotavirus Nonstructural Protein NSP3 Is Not Required for Viral Protein Synthesis

Journal of Virology ◽

10.1128/jvi.00437-06 ◽

2006 ◽

Vol 80 (18) ◽

pp. 9031-9038 ◽

Cited By ~ 59

Author(s):

Hilda Montero ◽

Carlos F. Arias ◽

Susana Lopez

Keyword(s):

Protein Synthesis ◽

Viral Protein ◽

Nonstructural Protein ◽

Consensus Sequence ◽

Initiation Factor ◽

Cell Protein ◽

Viral Protein Synthesis ◽

Viral Mrnas ◽

Initiation Of Translation ◽

Cellular Mrnas

ABSTRACT Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5′ end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3′ end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3′ ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.

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Modeling the SARS-CoV-2 nsp1–5’-UTR complex via extended ensemble simulations

10.1101/2021.02.24.432807 ◽

2021 ◽

Author(s):

Shun Sakuraba ◽

Xie Qilin ◽

Kota Kasahara ◽

Junichi Iwakiri ◽

Hidetoshi Kono

Keyword(s):

Nonstructural Protein ◽

Binding Mode ◽

Viral Rna ◽

Dynamics Simulation ◽

Recognition Mechanism ◽

Stem Loop ◽

Intrinsically Disordered ◽

Nonstructural Protein 1 ◽

Selective Translation ◽

Simulation Results

AbstractNonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 180-residue protein that blocks translation of host mRNAs in SARS-CoV-2-infected cells. Although it is known that SARS-CoV-2’s own RNA evades nsp1’s host translation shutoff, the molecular mechanism underlying the evasion was poorly understood. We performed an extended ensemble molecular dynamics simulation to investigate the mechanism of the viral RNA evasion. Simulation results showed that the stem loop structure of the SARS-CoV-2 RNA 5’-untranslated region (SL1) is recognized by both nsp1’s globular region and intrinsically disordered region. The recognition presumably enables selective translation of viral RNAs. Cluster analysis of the binding mode and detailed analysis of the binding poses revealed several residues involved in the SL1 recognition mechanism. The simulation results imply that the nsp1 C-terminal helices are lifted from the 40S ribosome upon the binding of SL1 to nsp1, unblocking translation of the viral RNA.

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Targeting Stem-loop 1 of the SARS-CoV-2 5’UTR to suppress viral translation and Nsp1 evasion

10.1101/2021.09.09.459641 ◽

2021 ◽

Author(s):

Setu M. Vora ◽

Pietro Fontana ◽

Valerie Leger ◽

Ying Zhang ◽

Tian-Min Fu ◽

...

Keyword(s):

Protein Synthesis ◽

Nonstructural Protein ◽

Host Protein ◽

Locked Nucleic Acid ◽

Viral Protein Synthesis ◽

Stem Loop ◽

Viral Translation ◽

Nonstructural Protein 1 ◽

Host Protein Synthesis

SARS-CoV-2 is a highly pathogenic virus that evades anti-viral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the mRNA entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the SL1 region of the SARS-CoV-2 5’ UTR is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides (ASOs) inhibits viral translation and makes SARS-CoV-2 5’ UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus’ own virulence mechanism against itself could force a critical trade off between drug resistance and pathogenicity.

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Mechanisms of Coronavirus Nsp1-Mediated Control of Host and Viral Gene Expression

Cells ◽

10.3390/cells10020300 ◽

2021 ◽

Vol 10 (2) ◽

pp. 300

Author(s):

Keisuke Nakagawa ◽

Shinji Makino

Keyword(s):

Gene Expression ◽

Virulence Factor ◽

Nonstructural Protein ◽

Host Gene ◽

Viral Gene ◽

Host Gene Expression ◽

Viral Mrnas ◽

Nonstructural Protein 1 ◽

Cellular Environment ◽

High Amino Acid

Many viruses disrupt host gene expression by degrading host mRNAs and/or manipulating translation activities to create a cellular environment favorable for viral replication. Often, virus-induced suppression of host gene expression, including those involved in antiviral responses, contributes to viral pathogenicity. Accordingly, clarifying the mechanisms of virus-induced disruption of host gene expression is important for understanding virus–host cell interactions and virus pathogenesis. Three highly pathogenic human coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2, have emerged in the past two decades. All of them encode nonstructural protein 1 (nsp1) in their genomes. Nsp1 of SARS-CoV and MERS-CoV exhibit common biological functions for inducing endonucleolytic cleavage of host mRNAs and inhibition of host translation, while viral mRNAs evade the nsp1-induced mRNA cleavage. SARS-CoV nsp1 is a major pathogenic determinant for this virus, supporting the notion that a viral protein that suppresses host gene expression can be a virulence factor, and further suggesting the possibility that SARS-CoV-2 nsp1, which has high amino acid identity with SARS-CoV nsp1, may serve as a major virulence factor. This review summarizes the gene expression suppression functions of nsp1 of CoVs, with a primary focus on SARS-CoV nsp1 and MERS-CoV nsp1.

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Novel Coronavirus SARS-CoV-2 (COVID-19) and Pregnancy: A hypothetical view

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530320666201023124843 ◽

2020 ◽

Vol 20 ◽

Author(s):

Ahmed RG

Keyword(s):

Immune System ◽

Cellular Therapy ◽

Early Stage ◽

Control Strategies ◽

Vital Role ◽

Healthy Life ◽

Global Pandemic ◽

Maternal Immune System ◽

Perinatal Management ◽

Novel Coronavirus

Background: The complications of the SARS-CoV-2 infection and its COVID-19 disease on mothers and their offspring are less known. Objective: The aim of this review was to determine the transmission, severity, complications of SARS-CoV-2 infection during the pregnancy. This review showed the influence of COVID-19 disease on the neonatal neurogenesis. Owing to no specific vaccines or medicines that were reported for the treatment of COVID-19 disease, this review suggested some control strategies like treatments (medicinal plants, antiviral therapy, cellular therapy, and immunotherapy), nutrition uptake, prevention, and recommendations. Discussion: This overview showed in severely states that SARS-CoV-2 infection during the early stage of pregnancy might increase the risk of stress, panic, and anxiety. This disorder can disturb the maternal immune system, and thus causing a neurodevelopmental disturbance. This hypothesis may be depending on the severity and intensity of the SARS-CoV-2 infection during pregnancy. However, vertical transmission of SARS-CoV-2 from dams to their fetuses is absent until now. Conclusion: During this global pandemic disease, maintaining safety during pregnancy, vaginal delivery, and breastfeeding may play a vital role in a healthy life for the offspring. Thus, international and national corporations should be continuing for perinatal management, particularly during the next pandemic or disaster time.

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Cerebellar hypoplasia and dysplasia in a juvenile raccoon with parvoviral infection

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638720912229 ◽

2020 ◽

Vol 32 (3) ◽

pp. 463-466 ◽

Cited By ~ 1

Author(s):

Arno Wünschmann ◽

Robert Lopez-Astacio ◽

Anibal G. Armien ◽

Colin R. Parrish

Keyword(s):

Granule Cells ◽

Nonstructural Protein ◽

Canine Parvovirus ◽

Cerebellar Hypoplasia ◽

Diagnostic Laboratory ◽

Vp2 Gene ◽

Nonstructural Protein 1 ◽

Cell Processes ◽

Ns1 Gene ◽

Pcr Targeting

A juvenile raccoon ( Procyon lotor) was submitted dead to the Minnesota Veterinary Diagnostic Laboratory for rabies testing without history. The animal had marked hypoplasia of the cerebellum. Histology demonstrated that most folia lacked granule cells and had randomly misplaced Purkinje cells. Immunohistochemistry revealed the presence of parvoviral antigen in a few neurons and cell processes. PCR targeting feline and canine parvovirus yielded a positive signal. Sequencing analyses from a fragment of the nonstructural protein 1 ( NS1) gene and a portion of the viral capsid protein 2 ( VP2) gene confirmed the presence of DNA of a recent canine parvovirus variant (CPV-2a–like virus) in the cerebellum. Our study provides evidence that (canine) parvovirus may be associated with cerebellar hypoplasia and dysplasia in raccoons, similar to the disease that occurs naturally and has been reproduced experimentally by feline parvoviral infection of pregnant cats, with subsequent intrauterine or neonatal infections of the offspring.

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Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽

10.3390/v13071393 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1393

Author(s):

Thanyaporn Dechtawewat ◽

Sittiruk Roytrakul ◽

Yodying Yingchutrakul ◽

Sawanya Charoenlappanit ◽

Bunpote Siridechadilok ◽

...

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Antiviral Drug ◽

Denv Infection ◽

Site Directed Mutagenesis ◽

Phosphorylation Sites ◽

Post Translational Modification ◽

Multifunctional Protein ◽

Nonstructural Protein 1 ◽

Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

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SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Cells ◽

10.3390/cells10030530 ◽

2021 ◽

Vol 10 (3) ◽

pp. 530

Author(s):

Soo Jin Oh ◽

Ok Sarah Shin

Keyword(s):

Nucleocapsid Protein ◽

Nuclear Translocation ◽

Nonstructural Protein ◽

Poly I:C ◽

Interferon Response ◽

Type I ◽

Antiviral Immune Response ◽

N Protein ◽

Nonstructural Protein 1 ◽

Polycytidylic Acid

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

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