scholarly journals Cleavage of Poly(A)-Binding Protein by Coxsackievirus 2A Protease In Vitro and In Vivo: Another Mechanism for Host Protein Synthesis Shutoff?

1999 ◽  
Vol 73 (1) ◽  
pp. 709-717 ◽  
Author(s):  
Vaishali Kerekatte ◽  
Brett D. Keiper ◽  
Cornel Badorff ◽  
Aili Cai ◽  
Kirk U. Knowlton ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Viral Protein ◽  
Binding Protein ◽  
Host Protein ◽  
Viral Protein Synthesis ◽  
2A Protease ◽  
Host Protein Synthesis

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.

scholarly journals Vesicular Stomatitis Virus Infection Alters the eIF4F Translation Initiation Complex and Causes Dephosphorylation of the eIF4E Binding Protein 4E-BP1

2002 ◽  
Vol 76 (20) ◽  
pp. 10177-10187 ◽  
Author(s):  
John H. Connor ◽  
Douglas S. Lyles
Keyword(s):  
Protein Synthesis ◽  
Vesicular Stomatitis Virus ◽  
Translation Initiation ◽  
Time Course ◽  
Binding Protein ◽  
Protein Translation ◽  
Host Protein ◽  
Eif4f Complex ◽  
Vesicular Stomatitis ◽  
Host Protein Synthesis

ABSTRACT Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5′-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2α is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.

scholarly journals Targeting Stem-loop 1 of the SARS-CoV-2 5’UTR to suppress viral translation and Nsp1 evasion

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.

scholarly journals KSHV lytic mRNA is efficiently translated in the absence of eIF4F

2018 ◽  
Author(s):  
Eric S. Pringle ◽  
Carolyn-Ann Robinson ◽  
Nicolas Crapoulet ◽  
Andrea L-A. Monjo ◽  
Katrina Bouzanis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Host Cell ◽  
Translation Initiation ◽  
Viral Protein ◽  
Viral Proteins ◽  
Lytic Replication ◽  
Cell Protein ◽  
Viral Protein Synthesis ◽  
Viral Mrnas

ABSTRACTHerpesvirus genomes are decoded by host RNA polymerase II, generating messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5 ′ -m7GTP caps and poly(A) tails that should permit assembly of canonical eIF4F cap-binding complexes to initiate protein synthesis. However, we have shown that chemical disruption of eIF4F does not impede KSHV lytic replication, suggesting that alternative translation initiation mechanisms support viral protein synthesis. Here, using polysome profiling analysis, we confirmed that eIF4F disassembly did not affect the efficient translation of viral mRNAs during lytic replication, whereas a large fraction of host mRNAs remained eIF4F-dependent. Lytic replication altered multiple host translation initiation factors (TIFs), causing caspase-dependent cleavage of eIF2α and eIF4G1 and decreasing levels of eIF4G2 and eIF4G3. Non-eIF4F TIFs NCBP1, eIF4E2 and eIF4G2 associated with actively translating messenger ribonucleoprotein (mRNP) complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production, suggesting that the virus does not exclusively rely on one of these alternative TIFs for efficient viral protein synthesis. METTL3, an N6-methyladenosine (m6A) methyltransferase that modifies mRNAs and influences translational efficiency, was dispensable for early viral gene expression and genome replication but required for late gene expression and virion production. METTL3 was also subject to caspase-dependent degradation during lytic replication, suggesting that its positive effect on KSHV late gene expression may be indirect. Taken together, our findings reveal extensive remodelling of TIFs during lytic replication, which may help sustain efficient viral protein synthesis in the context of host shutoff.IMPORTANCEViruses use host cell protein synthesis machinery to create viral proteins. Herpesviruses have evolved a variety of ways to gain control over this host machinery to ensure priority synthesis of viral proteins and diminished synthesis of host proteins with antiviral properties. We have shown that a herpesvirus called KSHV disrupts normal cellular control of protein synthesis. A host cell protein complex called eIF4F starts translation of most cellular mRNAs, but we observed it is dispensable for efficient synthesis of viral proteins. Several proteins involved in alternative modes of translation initiation were likewise dispensable. However, an enzyme called METTL3 that modifies mRNAs is required for efficient synthesis of certain late KSHV proteins and productive infection. We observed caspase-dependent degradation of several host cell translation initiation proteins during infection, suggesting that the virus alters pools of available factors to favour efficient viral protein synthesis at the expense of host protein synthesis.

Emodin inhibits coxsackievirus B3 replication via multiple signalling cascades leading to suppression of translation

2016 ◽  
Vol 473 (4) ◽  
pp. 473-485 ◽  
Author(s):  
Huifang M. Zhang ◽  
Fengping Wang ◽  
Ye Qiu ◽  
Xin Ye ◽  
Paul Hanson ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Viral Protein ◽  
Natural Compound ◽  
Coxsackievirus B3 ◽  
Signalling Pathways ◽  
Viral Protein Synthesis ◽  
Signalling Cascades

We identified emodin, a natural compound, as an anti-coxsackieviral agent and revealed further an unrecognized mechanism by which emodin inhibits coxsackievirus replication by activating multiple signalling pathways targeting either translation initiation or elongation, leading to suppression of viral protein synthesis.

scholarly journals Specific Inhibition of Coxsackievirus B3 Translation and Replication by Phosphorothioate Antisense Oligodeoxynucleotides

2001 ◽  
Vol 45 (4) ◽  
pp. 1043-1052 ◽  
Author(s):  
Aikun Wang ◽  
Paul K. M. Cheung ◽  
Huifang Zhang ◽  
Christopher M. Carthy ◽  
Lubos Bohunek ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Viral Protein ◽  
Plaque Assay ◽  
Rna Replication ◽  
Coxsackievirus B3 ◽  
Viral Rna ◽  
Antisense Oligodeoxynucleotides ◽  
Viral Protein Synthesis ◽  
Rna Translation

ABSTRACT The 5′ and 3′ untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA form highly ordered secondary structures that have been confirmed to play important regulatory roles in viral cap-independent internal translation initiation and RNA replication. We previously demonstrated that deletions in different regions of the 5′ UTR significantly reduced viral RNA translation and infectivity. Such observations suggested strongly that viral RNA translation and replication could be blocked if highly specific antisense oligodeoxynucleotides (AS-ODNs) were applied to target crucial sites within the 5′ and 3′ UTRs. In this study, seven phosphorothioate AS-ODNs were synthesized, and the antiviral activity was evaluated by Lipofectin transfection of HeLa cells with AS-ODNs followed by infection of CVB3. Analysis by Western blotting, reverse transcription-PCR, and viral plaque assay demonstrated that viral protein synthesis, genome replication, and infectivity of CVB3 were strongly inhibited by the AS-ODNs complementary to different regions of the 5′ and 3′ UTRs. The most effective sites are located at the proximate terminus of the 5′ UTR (AS-1), the proximate terminus of the 3′ UTR (AS-7), the core sequence of the internal ribosome entry site (AS-2), and the translation initiation codon region (AS-4). These AS-ODNs showed highly sequence-specific and dose-dependent inhibitory effects on both viral protein synthesis and RNA replication. It is noteworthy that the highest inhibitory activities were obtained with AS-1 and AS-7 targeting the termini of the 5′ and 3′ UTRs. The percent inhibition values of AS-1 and AS-7 for CVB3 protein VP1 synthesis and RNA replication were 70.6 and 79.6 for AS-1 and 73.7 and 79.7 for AS-7, respectively. These data suggest that CVB3 infectivity can be inhibited effectively by AS-ODNs.

scholarly journals The Autophagy Protein ATG16L1 Is Required for Sindbis Virus-Induced eIF2α Phosphorylation and Stress Granule Formation

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 39
Author(s):  
Matthew Jefferson ◽  
Benjamin Bone ◽  
Jasmine L. Buck ◽  
Penny P. Powell
Keyword(s):  
Immune Response ◽  
Protein Synthesis ◽  
Innate Immune Response ◽  
Viral Protein ◽  
Sindbis Virus ◽  
Binding Protein ◽  
Innate Immune ◽  
Stress Granule ◽  
Viral Protein Synthesis ◽  
Eif2α Phosphorylation

Sindbis virus (SINV) infection induces eIF2α phosphorylation, which leads to stress granule (SG) assembly. SINV infection also stimulates autophagy, which has an important role in controlling the innate immune response. The importance of autophagy to virus-induced translation arrest is not well understood. In this study, we show that the autophagy protein ATG16L1 not only regulates eIF2α phosphorylation and the translation of viral and antiviral proteins, but also controls SG assembly. Early in infection (2hpi), capsids were recruited by host factors Cytotoxic Granule-Associated RNA Binding Protein (TIA1), Y-box binding protein 1 (YBX1), and vasolin-containing protein 1 (VCP), to a single perinuclear body, which co-localized with the viral pattern recognition sensors, double stranded RNA-activated protein-kinase R (PKR) and RIG-I. By 6hpi, there was increased eIF2α phosphorylation and viral protein synthesis. However, in cells lacking the autophagy protein ATG16L1, SG assembly was inhibited and capsid remained in numerous small foci in the cytoplasm containing YBX1, TIA1 with RIG-I, and these persisted for over 8hpi. In the absence of ATG16L1, there was little phosphorylation of eIF2α and low levels of viral protein synthesis. Compared to wild type cells, there was potentiated interferon protein and interferon-stimulated gene (ISG) mRNA expression. These results show that ATG16L1 is required for maximum eIF2α phosphorylation, proper SG assembly into a single perinuclear focus, and for attenuating the innate immune response. Therefore, this study shows that, in the case of SINV, ATG16L1 is pro-viral, required for SG assembly and virus replication.

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