A temperature-sensitive mutation in the acidic polymerase gene of an influenza A virus alters the regulation of viral protein synthesis

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.

Download Full-text

RNase L limits host and viral protein synthesis via inhibition of mRNA export

10.1101/2021.04.18.440343 ◽

2021 ◽

Cited By ~ 1

Author(s):

James M. Burke ◽

Alison R. Gilchrist ◽

Sara L. Sawyer ◽

Roy Parker

Keyword(s):

Protein Synthesis ◽

Influenza A Virus ◽

Viral Protein ◽

Influenza A ◽

Cytokine Production ◽

Mrna Export ◽

Virus Protein ◽

Rnase L ◽

Viral Mrna ◽

Viral Protein Synthesis

AbstractRNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Herein, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L-mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. Importantly, the heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L-mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.One Sentence SummaryRNase L-mediated shutoff of nuclear mRNA export limits viral protein synthesis and regulates antiviral cytokine production.

Download Full-text

Inhibition of Ongoing Influenza A Virus Replication Reveals Different Mechanisms of RIG-I Activation

Journal of Virology ◽

10.1128/jvi.02066-18 ◽

2019 ◽

Vol 93 (6) ◽

Cited By ~ 8

Author(s):

GuanQun Liu ◽

Yao Lu ◽

Qiang Liu ◽

Yan Zhou

Keyword(s):

Protein Synthesis ◽

Viral Replication ◽

Influenza A Virus ◽

Viral Protein ◽

Influenza A ◽

Viral Rna ◽

Chain Elongation ◽

Type I ◽

Viral Polymerase ◽

Viral Protein Synthesis

ABSTRACTPattern recognition receptors provide essential nonself immune surveillance within distinct cellular compartments. Retinoic acid-inducible gene I (RIG-I) is one of the primary cytosolic RNA sensors, with an emerging role in the nucleus. It is involved in the spatiotemporal sensing of influenza A virus (IAV) replication, leading to the induction of type I interferons (IFNs). Nonetheless, the physiological viral ligands activating RIG-I during IAV infection remain underexplored. Other than full-length viral genomes, cellular constraints that impede ongoing viral replication likely potentiate an erroneous viral polymerase generating aberrant viral RNA species with RIG-I-activating potential. Here, we investigate the origins of RIG-I-activating viral RNA under two such constraints. Using chemical inhibitors that inhibit continuous viral protein synthesis, we identify the incoming, but notde novo-synthesized, viral defective interfering (DI) genomes contributing to RIG-I activation. In comparison, deprivation of viral nucleoprotein (NP), the key RNA chain elongation factor for the viral polymerase, leads to the production of aberrant viral RNA species activating RIG-I; however, their nature is likely to be distinct from that of DI RNA. Moreover, RIG-I activation in response to NP deprivation is not adversely affected by expression of the nuclear export protein (NEP), which diminishes the generation of a major subset of aberrant viral RNA but facilitates the accumulation of small viral RNA (svRNA). Overall, our results indicate the existence of fundamentally different mechanisms of RIG-I activation under cellular constraints that impede ongoing IAV replication.IMPORTANCEThe induction of an IFN response by IAV is mainly mediated by the RNA sensor RIG-I. The physiological RIG-I ligands produced during IAV infection are not fully elucidated. Cellular constraints leading to the inhibition of ongoing viral replication likely potentiate an erroneous viral polymerase producing aberrant viral RNA species activating RIG-I. Here, we demonstrate that RIG-I activation during chemical inhibition of continuous viral protein synthesis is attributable to the incoming DI genomes. Erroneous viral replication driven by NP deprivation promotes the generation of RIG-I-activating aberrant viral RNA, but their nature is likely to be distinct from that of DI RNA. Our results thus reveal distinct mechanisms of RIG-I activation by IAV under cellular constraints impeding ongoing viral replication. A better understanding of RIG-I sensing of IAV infection provides insight into the development of novel interventions to combat influenza virus infection.

Download Full-text

RNase L limits host and viral protein synthesis via inhibition of mRNA export

Science Advances ◽

10.1126/sciadv.abh2479 ◽

2021 ◽

Vol 7 (23) ◽

pp. eabh2479

Author(s):

James M. Burke ◽

Alison R. Gilchrist ◽

Sara L. Sawyer ◽

Roy Parker

Keyword(s):

Protein Synthesis ◽

Influenza A Virus ◽

Viral Protein ◽

Influenza A ◽

Mrna Export ◽

Virus Protein ◽

Rnase L ◽

Viral Mrna ◽

Viral Protein Synthesis ◽

Translation Arrest

RNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Here, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L–mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. The heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L–mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.

Download Full-text

Synthesis of the NS 2 nonstructural protein messenger RNA of influenza A viruses occurs in the absence of viral protein synthesis

Archives of Virology ◽

10.1007/bf01310483 ◽

1991 ◽

Vol 120 (3-4) ◽

pp. 281-288 ◽

Cited By ~ 2

Author(s):

T. Odagiri ◽

K. Tobita ◽

M. Tashiro

Keyword(s):

Protein Synthesis ◽

Viral Protein ◽

Messenger Rna ◽

Influenza A ◽

Nonstructural Protein ◽

Influenza A Viruses ◽

Viral Protein Synthesis

Download Full-text

The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant

10.1101/438176 ◽

2018 ◽

Cited By ~ 2

Author(s):

Boris Bogdanow ◽

Katrin Eichelbaum ◽

Anne Sadewasser ◽

Xi Wang ◽

Immanuel Husic ◽

...

Keyword(s):

Protein Synthesis ◽

Host Range ◽

Influenza A Virus ◽

Rna Splicing ◽

Influenza A ◽

Matrix Protein ◽

Shotgun Proteomics ◽

Influenza Viruses ◽

Species Barrier ◽

Viral Protein Synthesis

SUMMARYA century ago, influenza A virus (IAV) infection caused the 1918 flu pandemic and killed an estimated 20-40 million people. Pandemic IAV outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular details of this species barrier are incompletely understood. We combined metabolic pulse labeling and quantitative shotgun proteomics to globally monitor protein synthesis upon infection of human cells with a human-and a bird-adapted IAV strain. While production of host proteins was remarkably similar, we observed striking differences in the kinetics of viral protein synthesis over the course of infection. Most importantly, the matrix protein M1 was inefficiently produced by the bird-adapted strain at later stages. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Experiments with reporter constructs and recombinant influenza viruses revealed that strain-specific M segment splicing is controlled by the 3’ splice site and functionally important for permissive infection. Independentin silicoevidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, our data identifies M segment RNA splicing as a viral determinant of host range.

Download Full-text

The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant

Nature Communications ◽

10.1038/s41467-019-13520-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Boris Bogdanow ◽

Xi Wang ◽

Katrin Eichelbaum ◽

Anne Sadewasser ◽

Immanuel Husic ◽

...

Keyword(s):

Protein Synthesis ◽

Host Range ◽

Influenza A Virus ◽

Rna Splicing ◽

Rna Structure ◽

Influenza A ◽

Matrix Protein ◽

Species Barrier ◽

Viral Protein Synthesis ◽

The Matrix

AbstractPandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3′ splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.

Download Full-text

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.

Download Full-text