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.

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 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.

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

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

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

2019 ◽  
Vol 93 (6) ◽  
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.

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 Formation of the Poliovirus Replication Complex Requires Coupled Viral Translation, Vesicle Production, and Viral RNA Synthesis

2000 ◽  
Vol 74 (14) ◽  
pp. 6570-6580 ◽  
Author(s):  
Denise Egger ◽  
Natalya Teterina ◽  
Ellie Ehrenfeld ◽  
Kurt Bienz
Keyword(s):  
Viral Protein ◽  
Rna Synthesis ◽  
Binding Proteins ◽  
Membrane Binding ◽  
Rna Replication ◽  
Viral Rna ◽  
Viral Protein Synthesis ◽  
Viral Translation ◽  
Replication Complex ◽  
Viral Membrane

ABSTRACT Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes in HeLa cells were modified by the expression of different cellular or viral membrane-binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting PV multiplication. Our data suggest that a functional replication complex is formed in cis, in a coupled process involving viral translation, membrane modification and vesicle budding, and viral RNA synthesis.

scholarly journals Composition of Herpesvirus Ribonucleoprotein Complexes

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 119
Author(s):  
Eric S. Pringle ◽  
Craig McCormick
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Viral Protein ◽  
Lytic Replication ◽  
Viral Mrna ◽  
Viral Protein Synthesis ◽  
Initiation Factors ◽  
Ribonucleoprotein Complexes ◽  
Virion Production ◽  
Translation Initiation Factors

Herpesvirus genomes are decoded by host RNA polymerase enzymes, generating messenger ribonucleotides (mRNA) that are post-transcriptionally modified and exported to the cytoplasm through the combined work of host and viral factors. These viral mRNA bear 5′-m7GTP caps and poly(A) tails that should permit the assembly of canonical host eIF4F cap-binding complexes to initiate protein synthesis. However, the precise mechanisms of translation initiation remain to be investigated for Kaposi’s sarcoma-associated herpesvirus (KSHV) and other herpesviruses. During KSHV lytic replication in lymphoid cells, the activation of caspases leads to the cleavage of eIF4G and depletion of eIF4F. Translating mRNPs depleted of eIF4F retain viral mRNA, suggesting that non-eIF4F translation initiation is sufficient to support viral protein synthesis. To identify proteins required to support viral protein synthesis, we isolated and characterized actively translating messenger ribonucleoprotein (mRNP) complexes by ultracentrifugation and sucrose-gradient fractionation followed by quantitative mass spectrometry. The abundance of host translation initiation factors available to initiate viral protein synthesis were comparable between cells undergoing KSHV lytic or latent replication. The translation initiation factors eIF4E2, NCBP1, eIF4G2, and eIF3d were detected in association with actively translating mRNP complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production. By contrast, the N6-methyladenosine methyltransferase METTL3 was required for optimal late gene expression and virion production, but was dispensable for genome replication. Furthermore, we detected several KSHV proteins in actively translating mRNP complexes that had not previously been shown to play roles in viral protein synthesis. We conclude that KSHV usurps distinct host translation initiation systems during latent and lytic phases of infection.

Relationship between viral RNA and viral protein synthesis

Virology ◽  
1962 ◽  
Vol 17 (1) ◽  
pp. 110-117 ◽  
Author(s):  
Eberhard Wecker ◽  
Klaus Hummeler ◽  
Otmar Goetz
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Viral Rna ◽  
Viral Protein Synthesis

scholarly journals Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12008-12022 ◽  
Author(s):  
Brandon L. Walter ◽  
Todd B. Parsley ◽  
Ellie Ehrenfeld ◽  
Bert L. Semler
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

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