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

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

Journal of General Virology ◽

10.1099/jgv.0.001513 ◽

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.

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SARS coronavirus protein nsp1 disrupts localization of Nup93 from the nuclear pore complex

Biochemistry and Cell Biology ◽

10.1139/bcb-2018-0394 ◽

2019 ◽

Vol 97 (6) ◽

pp. 758-766 ◽

Cited By ~ 9

Author(s):

Garret N. Gomez ◽

Fareeha Abrar ◽

Maya P. Dodhia ◽

Fabiola G. Gonzalez ◽

Anita Nag

Keyword(s):

Gene Expression ◽

Nuclear Pore Complex ◽

Rna Binding ◽

Nonstructural Protein ◽

Nuclear Lamina ◽

Host Gene ◽

Nuclear Pore ◽

Host Gene Expression ◽

Nonstructural Protein 1 ◽

Cytoplasmic Transport

Severe acute respiratory syndrome coronavirus nonstructural protein 1 (nsp1) is a key factor in virus-induced down-regulation of host gene expression. In infected cells, nsp1 engages in a multipronged mechanism to inhibit host gene expression by binding to the 40S ribosome to block the assembly of translationally competent ribosome, and then inducing endonucleolytic cleavage and the degradation of host mRNAs. Here, we report a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts the nuclear–cytoplasmic transport of biomolecules. We identified members of the nuclear pore complex from the nsp1-associated protein assembly and found that the expression of nsp1 in HEK cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation, while the nuclear lamina remains unperturbed. Consistent with its role in host shutoff, nsp1 alters the nuclear–cytoplasmic distribution of an RNA binding protein, nucleolin. Our results suggest that nsp1, alone, can regulate multiple steps of gene expression including nuclear–cytoplasmic transport.

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Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic Origin

Journal of Virology ◽

10.1128/jvi.01352-15 ◽

2015 ◽

Vol 89 (21) ◽

pp. 10970-10981 ◽

Cited By ~ 61

Author(s):

Kumari G. Lokugamage ◽

Krishna Narayanan ◽

Keisuke Nakagawa ◽

Kaori Terasaki ◽

Sydney I. Ramirez ◽

...

Keyword(s):

Gene Expression ◽

Middle East ◽

Nonstructural Protein ◽

Mrna Translation ◽

Mrna Degradation ◽

Middle East Respiratory Syndrome ◽

Host Gene ◽

Host Gene Expression ◽

Highly Pathogenic ◽

Nonstructural Protein 1

ABSTRACTThe newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome CoV (SARS-CoV) represent highly pathogenic human CoVs that share a property to inhibit host gene expression at the posttranscriptional level. Similar to the nonstructural protein 1 (nsp1) of SARS-CoV that inhibits host gene expression at the translational level, we report that MERS-CoV nsp1 also exhibits a conserved function to negatively regulate host gene expression by inhibiting host mRNA translation and inducing the degradation of host mRNAs. Furthermore, like SARS-CoV nsp1, the mRNA degradation activity of MERS-CoV nsp1, most probably triggered by its ability to induce an endonucleolytic RNA cleavage, was separable from its translation inhibitory function. Despite these functional similarities, MERS-CoV nsp1 used a strikingly different strategy that selectively targeted translationally competent host mRNAs for inhibition. While SARS-CoV nsp1 is localized exclusively in the cytoplasm and binds to the 40S ribosomal subunit to gain access to translating mRNAs, MERS-CoV nsp1 was distributed in both the nucleus and the cytoplasm and did not bind stably to the 40S subunit, suggesting a distinctly different mode of targeting translating mRNAs. Interestingly, consistent with this notion, MERS-CoV nsp1 selectively targeted mRNAs, which are transcribed in the nucleus and transported to the cytoplasm, for translation inhibition and mRNA degradation but spared exogenous mRNAs introduced directly into the cytoplasm or virus-like mRNAs that originate in the cytoplasm. Collectively, these data point toward a novel viral strategy wherein the cytoplasmic origin of MERS-CoV mRNAs facilitates their escape from the inhibitory effects of MERS-CoV nsp1.IMPORTANCEMiddle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human CoV that emerged in Saudi Arabia in 2012. MERS-CoV has a zoonotic origin and poses a major threat to public health. However, little is known about the viral factors contributing to the high virulence of MERS-CoV. Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors. The nonstructural protein 1 (nsp1) of CoVs is one such protein that inhibits host gene expression and is a major virulence factor. This study presents evidence for a strategy used by MERS-CoV nsp1 to inhibit host gene expression that has not been described previously for any viral protein. The present study represents a meaningful step toward a better understanding of the factors and molecular mechanisms governing the virulence and pathogenesis of MERS-CoV.

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A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.009713 ◽

2019 ◽

Vol 294 (37) ◽

pp. 13606-13618 ◽

Cited By ~ 18

Author(s):

Zhou Shen ◽

Gang Wang ◽

Yiling Yang ◽

Jiale Shi ◽

Liurong Fang ◽

...

Keyword(s):

Gene Expression ◽

Nonstructural Protein ◽

Host Gene ◽

Host Gene Expression ◽

Nonstructural Protein 1 ◽

Viral Virulence ◽

Conserved Region

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Coronavirus nonstructural protein 1: Common and distinct functions in the regulation of host and viral gene expression

Virus Research ◽

10.1016/j.virusres.2014.11.019 ◽

2015 ◽

Vol 202 ◽

pp. 89-100 ◽

Cited By ~ 73

Author(s):

Krishna Narayanan ◽

Sydney I. Ramirez ◽

Kumari G. Lokugamage ◽

Shinji Makino

Keyword(s):

Gene Expression ◽

Nonstructural Protein ◽

Viral Gene Expression ◽

Viral Gene ◽

Nonstructural Protein 1

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The N-terminal and central domains of CoV-2 nsp1 play key functional roles in suppression of cellular gene expression and preservation of viral gene expression

10.1101/2021.05.28.446204 ◽

2021 ◽

Author(s):

Aaron Stephen Mendez ◽

Michael Ly ◽

Angélica M. González-Sánchez ◽

Ella Hartenian ◽

Nicholas Ingolia ◽

...

Keyword(s):

Gene Expression ◽

Mutational Analysis ◽

Nonstructural Protein ◽

Ribosomal Subunit ◽

Viral Gene ◽

Viral Mrna ◽

Cellular Gene ◽

Cellular Gene Expression ◽

Nonstructural Protein 1

Nonstructural protein 1 (nsp1) is the first viral protein synthesized during coronavirus (CoV) infection and is a key virulence factor that dampens the innate immune response. It restricts cellular gene expression through a combination of inhibiting translation by blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We performed a detailed structure-guided mutational analysis of CoV-2 nsp1 coupled with in vitro and cell-based functional assays, revealing insight into how it coordinates these activities against host but not viral mRNA. We found that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, thereby enhancing its restriction of host gene expression. These residues are also critical for the ability of mRNA containing the CoV-2 leader sequence to escape translational repression. Notably, we identify CoV-2 nsp1 mutants that gain the ability to repress translation of viral leader-containing transcripts. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in CoV-2 nsp1 could attenuate the virus.

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Functional Evolution of the 2009 Pandemic H1N1 Influenza Virus NS1 and PA in Humans

Journal of Virology ◽

10.1128/jvi.01206-18 ◽

2018 ◽

Vol 92 (19) ◽

Cited By ~ 9

Author(s):

Aitor Nogales ◽

Luis Martinez-Sobrido ◽

Kevin Chiem ◽

David J. Topham ◽

Marta L. DeDiego

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Amino Acid ◽

H1n1 Influenza ◽

Innate Immune ◽

Host Gene ◽

Pandemic H1n1 ◽

Host Gene Expression ◽

Pandemic H1n1 Influenza ◽

Nonstructural Protein 1

ABSTRACT In 2009, a pandemic H1N1 influenza A virus (IAV) (pH1N1) emerged in the human population from swine causing a pandemic. Importantly, this virus is still circulating in humans seasonally. To analyze the evolution of pH1N1 in humans, we sequenced viral genes encoding proteins inhibiting general gene expression (nonstructural protein 1 [NS1] and PA-X) from circulating seasonal viruses and compared them to the viruses isolated at the origin of the pandemic. Recent pH1N1 viruses contain amino acid changes in the NS1 protein (E55K, L90I, I123V, E125D, K131E, and N205S), as previously described (A. M. Clark, A. Nogales, L. Martinez-Sobrido, D. J. Topham, and M. L. DeDiego, J Virol 91:e00721-17, 2017, https://doi.org/10.1128/JVI.00721-17), and amino acid changes in the PA-X protein (V100I, N204S, R221Q, and L229S). These amino acid differences between early and more recent pH1N1 isolates are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes. In addition, currently circulating pH1N1 viruses have acquired amino acid changes in the PA protein (V100I, P224S, N321K, I330V, and R362K). A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from currently circulating viruses is fitter in replication in cultured cells and in mice and is slightly more pathogenic than the original ancestor pH1N1 virus. These results demonstrate the need to monitor the evolution of pH1N1 in humans for mutations in the viral genome that could result in enhanced virulence. Importantly, these results further support our previous findings suggesting that inhibition of global gene expression mediated by NS1 and PA-X proteins is subject to a balance which can determine virus pathogenesis and fitness. IMPORTANCE IAVs emerge in humans from animal reservoirs, causing unpredictable pandemics. One of these pandemics was caused by an H1N1 virus in 2009, and this virus is still circulating seasonally. To analyze host-virus adaptations likely affecting influenza virus pathogenesis, protein amino acid sequences from viruses circulating at the beginning of the pandemic and those circulating currently were compared. Currently circulating viruses have incorporated amino acid changes in two viral proteins (NS1 and PA-X), affecting innate immune responses, and in the PA gene. These amino acid differences led to increased NS1-mediated and decreased PA-X-mediated inhibition of host gene expression. A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from recently circulating viruses is fitter in replication in tissue culture cells and in mice, and the virus is more pathogenic in vivo. Importantly, these results suggest that a balance in the abilities of NS1 and PA-X to induce host shutoff is beneficial for IAVs.

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Rhinovirus reduces the severity of subsequent respiratory viral infections, which is associated with dampened inflammatory responses

10.1101/2020.11.06.371005 ◽

2020 ◽

Author(s):

James T. Van Leuven ◽

Andres J. Gonzalez ◽

Alexander Q. Wixom ◽

John L. Clary ◽

Benjamin J. Ridenhour ◽

...

Keyword(s):

Gene Expression ◽

Viral Infection ◽

Disease Severity ◽

Respiratory Virus ◽

Inflammatory Responses ◽

Lethal Dose ◽

Host Gene ◽

Viral Gene ◽

Human Populations ◽

Host Gene Expression

AbstractCoinfection by unrelated viruses in the respiratory tract is common and can result in changes in disease severity compared to infection by individual virus strains. We have previously shown that inoculation of mice with rhinovirus (RV) two days prior to inoculation with a lethal dose of influenza A virus (PR8), provides complete protection against mortality. In this study, we extend that finding to a second lethal respiratory virus, pneumonia virus of mice (PVM) and characterize the differences in inflammatory responses and host gene expression in single virus infected vs. coinfected mice. RV prevented mortality and weight loss associated with PVM infection, suggesting that RV-mediated protection is more effective against PVM than PR8. Major changes in host gene expression upon PVM infection were delayed compared to PR8, which likely provides a larger time frame for RV-induced gene expression to alter the course of disease. Overall, RV induced earlier recruitment of inflammatory cells, while these populations were reduced at later times in coinfected mice. Findings common to both coinfection conditions included upregulated expression of mucin-associated genes in RV/PR8 and RV/PVM compared to mock/PR8 and mock/PVM infected mice and dampening of inflammation-related genes late during coinfection. These findings, combined with differences in virus replication levels and disease severity, suggest that the suppression of inflammation in RV/PVM coinfected mice may be due to early suppression of viral replication, while in RV/PR8 coinfected mice may be due to a direct suppression of inflammation. Thus, a mild upper respiratory viral infection can reduce the severity of a subsequent severe viral infection in the lungs through virus-dependent mechanisms.Author SummaryRespiratory viruses from diverse families co-circulate in human populations and are frequently detected within the same host. Though clinical studies suggest that coinfection by more than one unrelated respiratory virus may alter disease severity, animal models in which we can control the doses, timing, and strains of coinfecting viruses are critical to understand how coinfection affects disease severity. In this study, we compared gene expression and immune cell recruitment between two pairs of coinfecting viruses (RV/PR8 and RV/PVM) that both result in reduced severity compared to infection by PR8 or PVM alone in mice. Reduced disease severity was associated with suppression of inflammatory responses in the lungs. However, differences in disease kinetics and host and viral gene expression suggest that protection by coinfection with RV may be due to distinct molecular mechanisms.

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