scholarly journals Structural characterization of Nonstructural protein 1 from SARS-CoV-2

2020 ◽  
Author(s):  
Cameron Semper ◽  
Nobuhiko Watanabe ◽  
Alexei Savchenko
Keyword(s):  
Crystal Structure ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Ribosomal Subunit ◽  
Structural Features ◽  
Globular Domain ◽  
Nonstructural Protein 1 ◽  
C Terminus ◽  
40S Ribosomal Subunit ◽  
Efficient Replication

AbstractSevere acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) is a single-stranded, enveloped RNA virus and the etiological agent of the current COVID-19 pandemic. Efficient replication of the virus relies on the activity of nonstructural protein 1 (Nsp1), a major virulence factor shown to facilitate suppression of host gene expression through promotion of host mRNA degradation and interaction with the 40S ribosomal subunit. Here, we report the crystal structure of the globular domain of SARS-CoV-2 Nsp1, encompassing residues 13 to 127, at a resolution of 1.65 Å. Our structure features a six-stranded, capped β-barrel motif similar to Nsp1from SARS-CoV and reveals how variations in amino acid sequence manifest as distinct structural features. Through comparative analysis of structural homologues, we identified a topological signature associated with this protein fold that facilitated modeling of Nsp1 from MERS-CoV. Combining our high-resolution crystal structure with existing data on the C-terminus of Nsp1 from SARS-CoV-2, we propose a model of the full-length protein. Our results provide unparalleled insight into the molecular structure of a major pathogenic determinant of SARS-CoV-2.

scholarly journals Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. 1249-1255 ◽  
Author(s):  
Matthias Thoms ◽  
Robert Buschauer ◽  
Michael Ameismeier ◽  
Lennart Koepke ◽  
Timo Denk ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Nonstructural Protein ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Structural Basis ◽  
Innate Immune Responses ◽  
Structure Based Drug Design ◽  
Nonstructural Protein 1 ◽  
C Terminus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40S ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo–electron microscopy of in vitro–reconstituted Nsp1-40S and various native Nsp1-40S and -80S complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid–inducible gene I–dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.

scholarly journals SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

2020 ◽  
Author(s):  
Ming Shi ◽  
Longfei Wang ◽  
Pietro Fontana ◽  
Setu Vora ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Ribosomal Subunit ◽  
Direct Interaction ◽  
Mrna Translation ◽  
Protein Translation ◽  
Host Protein ◽  
Terminal Domain ◽  
Nonstructural Protein 1 ◽  
40S Ribosomal Subunit ◽  
Open Question

SUMMARYThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus that underlies the current COVID-19 pandemic. SARS-CoV-2 is thought to disable various features of host immunity and cellular defense. The SARS-CoV-2 nonstructural protein 1 (Nsp1) is known to inhibit host protein translation and could be a target for antiviral therapy against COVID-19. However, how SARS-CoV-2 circumvents this translational blockage for the production of its own proteins is an open question. Here, we report a bipartite mechanism of SARS-CoV-2 Nsp1 which operates by: (1) hijacking the host ribosome via direct interaction of its C-terminal domain (CT) with the 40S ribosomal subunit and (2) specifically lifting this inhibition for SARS-CoV-2 via a direct interaction of its N-terminal domain (NT) with the 5’ untranslated region (5’ UTR) of SARS-CoV-2 mRNA. We show that while Nsp1-CT is sufficient for binding to 40S and inhibition of host protein translation, the 5’ UTR of SARS-CoV-2 mRNA removes this inhibition by binding to Nsp1-NT, suggesting that the Nsp1-NT-UTR interaction is incompatible with the Nsp1-CT-40S interaction. Indeed, lengthening the linker between Nsp1-NT and Nsp1-CT of Nsp1 progressively reduced the ability of SARS-CoV-2 5’ UTR to escape the translational inhibition, supporting that the incompatibility is likely steric in nature. The short SL1 region of the 5’ UTR is required for viral mRNA translation in the presence of Nsp1. Thus, our data provide a comprehensive view on how Nsp1 switches infected cells from host mRNA translation to SARS-CoV-2 mRNA translation, and that Nsp1 and 5’ UTR may be targeted for anti-COVID-19 therapeutics.

scholarly journals Dynamics of Coronavirus Replication-Transcription Complexes

2009 ◽  
Vol 84 (4) ◽  
pp. 2134-2149 ◽  
Author(s):  
Marne C. Hagemeijer ◽  
Monique H. Verheije ◽  
Mustafa Ulasli ◽  
Indra A. Shaltiël ◽  
Lisa A. de Vries ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Nonstructural Protein ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
C Terminus ◽  
Efficient Replication ◽  
Infected Cells ◽  
Biochemical Analyses ◽  
Transcription Complexes ◽  
Cytoplasmic Side

ABSTRACT Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.

scholarly journals Lysine 164 is critical for SARS-CoV-2 Nsp1 inhibition of host gene expression

2020 ◽  
Author(s):  
Zhou Shen ◽  
Guangxu Zhang ◽  
Yilin Yang ◽  
Mengxia Li ◽  
Siqi Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Ribosomal Subunit ◽  
Virus Production ◽  
Host Gene ◽  
Host Protein ◽  
Renilla Luciferase ◽  
Host Gene Expression ◽  
Nonstructural Protein 1

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

scholarly journals Structure of nonstructural protein 1 from SARS-CoV-2.

2020 ◽  
Author(s):  
Lauren K. Clark ◽  
Todd J. Green ◽  
Chad M. Petit
Keyword(s):  
Crystal Structure ◽  
Life Cycle ◽  
High Resolution ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Viral Life Cycle ◽  
Structure And Function ◽  
Future Studies ◽  
Nonstructural Protein 1 ◽  
And Function

The periodic emergence of novel coronaviruses (CoVs) represents an ongoing public health concern with significant health and financial burden worldwide. The most recent occurrence originated in the city of Wuhan, China where a novel coronavirus (SARS-CoV-2) emerged causing severe respiratory illness and pneumonia. The continual emergence of novel coronaviruses underscores the importance of developing effective vaccines as well as novel therapeutic options that target either viral functions or host factors recruited to support coronavirus replication. The CoV nonstructural protein 1 (nsp1) has been shown to promote cellular mRNA degradation, block host cell translation, and inhibit the innate immune response to virus infection. Interestingly, deletion of the nsp1-coding region in infectious clones prevented the virus from productively infecting cultured cells. Because of nsp1’s importance in the CoV life cycle, it has been highlighted as a viable target for both antiviral therapy and vaccine development. However, the fundamental molecular and structural mechanisms that underlie nsp1 function remain poorly understood, despite its critical role in the viral life cycle. Here we report the high-resolution crystal structure of the amino, globular portion of SARS-CoV-2 nsp1 (residues 10 – 127) at 1.77 Å resolution. A comparison of our structure with the SARS-CoV-1 nsp1 structure reveals how mutations alter the conformation of flexible loops, inducing the formation of novel secondary structural elements and new surface features. Paired with the recently published structure of the carboxyl end of nsp1 (residues 148 – 180), our results provide the groundwork for future studies focusing on SARS-CoV-2 nsp1 structure and function during the viral life cycle. IMPORTANCE The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. One protein known to play a critical role in the coronavirus life cycle is nonstructural protein1 (nsp1). As such, it has been highlighted in numerous studies as a target for both the development of antivirals and for the design of live-attenuated vaccines. Here we report the high-resolution crystal structure of nsp1 derived from SARS-CoV-2 at 1.77 Å resolution. This structure will facilitate future studies focusing on understanding the relationship between structure and function for nsp1. In turn, understanding these structure-function relationships will allow nsp1 to be fully exploited as a target for both antiviral development and vaccine design.

scholarly journals Crystal Structure of the Eukaryotic 40S Ribosomal Subunit in Complex with Initiation Factor 1

Science ◽  
2010 ◽  
Vol 331 (6018) ◽  
pp. 730-736 ◽  
Author(s):  
J. Rabl ◽  
M. Leibundgut ◽  
S. F. Ataide ◽  
A. Haag ◽  
N. Ban
Keyword(s):  
Crystal Structure ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
40S Ribosomal Subunit

scholarly journals The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

2015 ◽  
Vol 90 (3) ◽  
pp. 1178-1189 ◽  
Author(s):  
Li-Chen Yen ◽  
Jia-Teh Liao ◽  
Hwei-Jen Lee ◽  
Wei-Yuan Chou ◽  
Chun-Wei Chen ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Virus Replication ◽  
Japanese Encephalitis ◽  
Protective Immunity ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
The Core ◽  
Nonstructural Protein 1 ◽  
C Terminus ◽  
Infected Cells

ABSTRACTNS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using atrans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain.IMPORTANCEThe positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

scholarly journals Structure of nonstructural protein 1 from SARS-CoV-2

2020 ◽  
Author(s):  
Lauren K. Clark ◽  
Todd J. Green ◽  
Chad M. Petit
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Structure And Function ◽  
Health Concern ◽  
Future Studies ◽  
Nonstructural Protein 1 ◽  
And Function ◽  
Viral Lifecycle

ABSTRACTThe periodic emergence of novel coronaviruses (CoVs) represents an ongoing public health concern with significant health and financial burden worldwide. The most recent occurrence originated in the city of Wuhan, China where a novel coronavirus (SARS-CoV-2) emerged causing severe respiratory illness and pneumonia. The continual emergence of novel coronaviruses underscores the importance of developing effective vaccines as well as novel therapeutic options that target either viral functions or host factors recruited to support coronavirus replication. The CoV nonstructural protein 1 (nsp1) has been shown to promote cellular mRNA degradation, block host cell translation, and inhibit the innate immune response to virus infection. Interestingly, deletion of the nsp1-coding region in infectious clones prevented the virus from productively infecting cultured cells. Because of nsp1’s importance in the CoV lifecycle, it has been highlighted as a viable target for both antiviral therapy and vaccine development. However, the fundamental molecular and structural mechanisms that underlie nsp1 function remain poorly understood, despite its critical role in the viral lifecycle. Here we report the high-resolution crystal structure of the amino, globular portion of SARS-CoV-2 nsp1 (residues 10 – 127) at 1.77Å resolution. A comparison of our structure with the SARS-CoV-1 nsp1 structure reveals how mutations alter the conformation of flexible loops, inducing the formation of novel secondary structural elements and new surface features. Paired with the recently published structure of the carboxyl end of nsp1 (residues 148 – 180), our results provide the groundwork for future studies focusing on SARS-CoV-2 nsp1 structure and function during the viral lifecycle.IMPORTANCEThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. One protein known to play a critical role in the coronavirus lifecycle is nonstructural protein1 (nsp1). As such, it has been highlighted in numerous studies as a target for both the development of antivirals and for the design of live-attenuated vaccines. Here we report the high-resolution crystal structure of nsp1 derived from SARS-CoV-2 at 1.77Å resolution. This structure will facilitate future studies focusing on understanding the relationship between structure and function for nsp1. In turn, understanding these structure-function relationships will allow nsp1 to be fully exploited as a target for both antiviral development and vaccine design.

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