Rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection.

ABSTRACT Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments withXenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at482VANTSTQTM↓GPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.

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Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation

Journal of Virology ◽

10.1128/jvi.02023-07 ◽

2007 ◽

Vol 82 (2) ◽

pp. 828-839 ◽

Cited By ~ 36

Author(s):

Maria D. Gainey ◽

Patrick J. Dillon ◽

Kimberly M. Clark ◽

Mary J. Manuse ◽

Griffith D. Parks

Keyword(s):

Protein Synthesis ◽

Hela Cells ◽

Viral Protein ◽

Time Course ◽

Translation Initiation Factor ◽

Host Protein ◽

Initiation Factor ◽

Viral Protein Synthesis ◽

Viral Mrnas ◽

V Protein

ABSTRACT The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI− mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI− mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI− virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2α. WT SV5 was a poor activator of the eIF-2α kinase protein kinase R (PKR). By contrast, the P/V-CPI− mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI− mutant was restored to ∼50% that of control HeLa cells. By contrast, the rates of P/V-CPI− viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI− mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI− mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

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

10.1101/2020.08.09.243451 ◽

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

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Stress granule-inducing eukaryotic translation initiation factor 4A inhibitors block influenza A virus replication

10.1101/194589 ◽

2017 ◽

Cited By ~ 1

Author(s):

Patrick D. Slaine ◽

Mariel Kleer ◽

Nathan Smith ◽

Denys A. Khaperskyy ◽

Craig McCormick

Keyword(s):

Protein Synthesis ◽

Influenza A Virus ◽

Viral Protein ◽

Influenza A ◽

Translation Initiation Factor ◽

Host Protein ◽

Initiation Factor ◽

Viral Protein Synthesis ◽

Infected Cells ◽

Pateamine A

ABSTRACTEukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5’ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection results in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates the feasibility of targeting core host protein synthesis machinery to prevent viral replication.IMPORTANCEInfluenza A virus (IAV) relies on cellular protein synthesis to decode viral messenger RNAs. Pateamine A and silvestrol are natural products that inactivate an essential protein synthesis protein known as eIF4A. Here we show that IAV is sensitive to these eIF4A inhibitor drugs. Treatment of infected cells with pateamine A or silvestrol prevented synthesis of viral proteins, viral genome replication and release of infectious virions. The irreversible eIF4A inhibitor pateamine A sustained long-term blockade of viral replication, whereas viral protein synthesis quickly resumed after silvestrol was removed from infected cells. Prolonged incubation of either infected or uninfected cells with these drugs induced the programmed cell death cascade called apoptosis. Our findings suggest that core components of the host protein synthesis machinery are viable targets for antiviral drug discovery. The most promising drug candidates should selectively block protein synthesis in infected cells without perturbing bystander uninfected cells.

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Multi-Alignment Comparison of Coronavirus Non-Structural Proteins Nsp13-16 with Ribosomal proteins and other DNA/RNA modifying Enzymes Suggested Their Roles in the Regulation of Host Protein Synthesis

10.35543/osf.io/qrxc5 ◽

2020 ◽

Author(s):

ASIT KUMAR CHAKRABORTY

Keyword(s):

Protein Synthesis ◽

Ribosomal Proteins ◽

Dna Methyltransferase ◽

Atp Synthesis ◽

Host Protein ◽

Structural Proteins ◽

23S Rrna ◽

Structural Protein ◽

Viral Protein Synthesis ◽

Host Protein Synthesis

Recently we proposed that Coronavirus Nsp2 protein is a RNA topoisomerase and Nsp16 is a 2'-O-Ribose Uridine Methyltransferase. BLAST search found that Nsp13 non-structural protein was a 2'-O-Ribose Guanosine Capping Methyltransferase although it has been implicated as RNA helicase. Search with 200 RNA/DNA binding-modifying proteins confirmed Nsp13 protein homology to ribosomal L6 and L9 proteins and Nsp2 protein to L1 protein and Nsp15 protein to S1 and S22 ribosomal proteins. Further, Nsp13 has some homology with Cfr 23S rRNA methyltransferase and RNaseT whereas Nsp15 had close relation to RecA recombinase and Dcm DNA methyltransferase. Similarly, Nsp14 had homology with Cfr 23S rRNA methyltransferase and Nsp16 2’-O-Ribose MTase had some similarity to UvrC exinuclease and RNase. These suggested that Nsp2, Nsp13, Nsp14, Nsp15 and nsp16 non-structural proteins may be recruited easily to mitoribosome making chimera ribosome to methylate the rRNA or change its topology favouring viral protein synthesis and inhibiting host protein synthesis. Such change in host protein synthesis in the mitochondria may cause a inhibition in oxidative phosphorylation and ATP synthesis causing quick coma or heart failure as seen in many Corona-infected patients. Thus, targeting those viral proteins with drugs, antisense, ribozyme and CRISPR-Cas6 may cure Corona-infected patients.

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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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Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

Journal of Virology ◽

10.1128/jvi.76.3.1206-1212.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1206-1212 ◽

Cited By ~ 89

Author(s):

Mirella Salvatore ◽

Christopher F. Basler ◽

Jean-Patrick Parisien ◽

Curt M. Horvath ◽

Svetlana Bourmakina ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Expression ◽

Influenza A Virus ◽

Viral Protein ◽

Influenza A ◽

Vero Cells ◽

Cellular Protein ◽

Host Protein ◽

Ns1 Protein ◽

Host Protein Synthesis

ABSTRACT The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-α/β) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein—lacking approximately half of its carboxy-terminal end—showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.

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