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

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.

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

2021 ◽  
Author(s):  
Lucija Bujanic ◽  
Olga Shevchuk ◽  
Nicolai von Kuegelgen ◽  
Katarzyna Ludwik ◽  
David Koppstein ◽  
...  
Keyword(s):  
Drug Targets ◽  
Early Stage ◽  
Nonstructural Protein ◽  
Viral Escape ◽  
Cellular Mechanisms ◽  
Viral Mrnas ◽  
Stem Loop ◽  
Nonstructural Protein 1 ◽  
Global Pandemic ◽  
Cellular Mrnas

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.

scholarly journals Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses

2020 ◽  
Vol 117 (12) ◽  
pp. 6550-6558 ◽  
Author(s):  
Jae-Hyun Cho ◽  
Baoyu Zhao ◽  
Jie Shi ◽  
Nowlan Savage ◽  
Qingliang Shen ◽  
...  
Keyword(s):  
Molecular Recognition ◽  
Influenza A ◽  
Nonstructural Protein ◽  
Conformational Dynamics ◽  
Relaxation Dispersion ◽  
Binding Kinetics ◽  
Host Protein ◽  
Dynamics Simulation ◽  
Nonstructural Protein 1 ◽  
Strain Dependent

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85β subunit. Here we present the mechanism underlying the molecular recognition of the p85β subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85β of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85β. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1EDexists in a dynamic equilibrium between p85β-binding–competent and –incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1EDproteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85β. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1EDcan result in the faster hijacking of p85β compared to Ud NS1ED, although the former has a lower affinity to p85β than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

scholarly journals A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle

2019 ◽  
Vol 15 (5) ◽  
pp. e1007736 ◽  
Author(s):  
Anna Płaszczyca ◽  
Pietro Scaturro ◽  
Christopher John Neufeldt ◽  
Mirko Cortese ◽  
Berati Cerikan ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Nonstructural Protein 1

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

2009 ◽  
Vol 83 (12) ◽  
pp. 6087-6097 ◽  
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.

scholarly journals Nonstructural protein 1 of SARS-CoV-2 is a potent pathogenicity factor redirecting host protein synthesis machinery toward viral RNA

2020 ◽  
Author(s):  
Shuai Yuan ◽  
Lei Peng ◽  
Jonathan J. Park ◽  
Yingxia Hu ◽  
Swapnil C. Devarkar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Nonstructural Protein ◽  
Differential Expression Analysis ◽  
Human Cells ◽  
Viral Rna ◽  
Host Protein ◽  
Viral Protein Synthesis ◽  
Pathogenicity Factor ◽  
Nonstructural Protein 1

SummaryThe COVID-19 pandemic affects millions of people worldwide with a rising death toll. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), uses its nonstructural protein 1 (Nsp1) to redirect host translation machinery to the viral RNA by binding to the ribosome and suppressing cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the transcriptome in human cells. The changes include repression of major gene clusters in ribosomal RNA processing, translation, mitochondria function, cell cycle and antigen presentation; and induction of factors in transcriptional regulation. We further gained a mechanistic understanding of the Nsp1 function by determining the cryo-EM structure of the Nsp1-40S ribosomal subunit complex, which shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the cryo-EM structure of the 48S preinitiation complex (PIC) formed by Nsp1, 40S, and the cricket paralysis virus (CrPV) internal ribosome entry site (IRES) RNA, which shows that this 48S PIC is nonfunctional due to the incorrect position of the 3’ region of the mRNA. Results presented here elucidate the mechanism of host translation inhibition by SARS-CoV-2, provide insight into viral protein synthesis, and furnish a comprehensive understanding of the impacts from one of the most potent pathogenicity factors of SARS-CoV-2.HighlightsORF screen identified Nsp1 as a major cellular pathogenicity factor of SARS-CoV-2Nsp1 broadly alters the gene expression programs in human cellsNsp1 inhibits translation by blocking mRNA entry channelNsp1 prevents physiological conformation of the 48S PIC

scholarly journals A crystal structure of alphavirus nonstructural protein 4 (nsP4) reveals an intrinsically dynamic RNA-dependent RNA polymerase

2021 ◽  
Author(s):  
Yaw Bia Tan ◽  
Laura Sandra Lello ◽  
Xin Liu ◽  
Yee-Song Law ◽  
Congbao Kang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
3D Structure ◽  
Rna Replication ◽  
Viral Rna ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Intrinsically Disordered ◽  
Rdrp Domain

Alphaviruses such as Ross River virus (RRV), chikungunya virus and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report a crystal structure of the RdRp domain of nsP4 from RRV determined at a resolution of 2.6 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses and picornaviruses. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, we showed that in-solution nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were able to catalyze in vitro RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

scholarly journals Distinct RNA Elements Confer Specificity to Flavivirus RNA Cap Methylation Events

2007 ◽  
Vol 81 (9) ◽  
pp. 4412-4421 ◽  
Author(s):  
Hongping Dong ◽  
Debashish Ray ◽  
Suping Ren ◽  
Bo Zhang ◽  
Francesc Puig-Basagoiti ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Nonstructural Protein ◽  
Viral Rna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Rna Elements ◽  
Novel Target ◽  
Antisense Oligomer

ABSTRACT The 5′ end of the flavivirus plus-sense RNA genome contains a type 1 cap (m7GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5′ terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2′-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5′ stem-loop structure; in contrast, 2′-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5′ viral RNA of 20 nucleotides. The cap analogues GpppA and m7GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m7Gppp-terminated RNAs suggest that the 5′ termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.

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