Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection

Aumayr, Martina; Schrempf, Anna; Üzülmez, Öykü; Olek, Karin M.; Skern, Tim

doi:10.1016/j.virol.2017.08.020

Cited by 16 publications

(14 citation statements)

References 43 publications

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“…Approximately 1 hour PI, RVs express 2A pro , a protease that degrades eIF4G and polyA binding protein, to dismantle cellular cap-dependent translation that predominates for host mRNA. Viral translation continues through internal ribosome entry sites, which is a viral RNA-specific sequence that allows the recruitment of ribosomes in a cap-independent manner 77 . Furthermore, 2A pro cleaves Phe/Gly-containing nucleoporin proteins within nuclear pore complexes, which impairs nuclear export 78 .…”

Section: Discussionmentioning

confidence: 99%

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication

Real-Hohn

Provance

Gonçalves

et al. 2017

Sci Rep

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Together, the three human rhinovirus (RV) species are the most frequent cause of the common cold. Because of their high similarity with other viral species of the genus Enterovirus, within the large family Picornaviridae, studies on RV infectious activities often offer a less pathogenic model for more aggressive enteroviruses, e.g. poliovirus or EV71. Picornaviruses enter via receptor mediated endocytosis and replicate in the cytosol. Most of them depend on functional F-actin, Rab proteins, and probably motor proteins. To assess the latter, we evaluated the role of myosin light chain kinase (MLCK) and two myosin V isoforms (Va and Vb) in RV-B14 infection. We report that ML-9, a very specific MLCK inhibitor, dramatically reduced RV-B14 entry. We also demonstrate that RV-B14 infection in cells expressing dominant-negative forms of myosin Va and Vb was impaired after virus entry. Using immunofluorescent localization and immunoprecipitation, we show that myosin Va co-localized with RV-B14 exclusively after viral entry (15 min post infection) and that myosin Vb was present in the clusters of newly synthesized RNA in infected cells. These clusters, observed at 180 min post infection, are reminiscent of replication sites. Taken together, these results identify myosin light chain kinase, myosin Va and myosin Vb as new players in RV-B14 infection that participate directly or indirectly in different stages of the viral cycle.

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Section: Discussionmentioning

confidence: 99%

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication

Real-Hohn

Provance

Gonçalves

et al. 2017

Sci Rep

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“…Poliovirus 2A proteinase cleaves eIF4G, separating the eIF4E-binding domain from the eIF3-binding region (Gradi et al 1998), whereas 3C proteinases cleave the poly(A)-binding protein (PABP) (Rivera and Lloyd 2008;Kobayashi et al 2012a). Cleavage of eIF4G by poliovirus or group A rhinovirus 2A protease is stimulated by eIF4E (Aumayr et al 2017;Avanzino et al 2017). During encephalomyocarditis virus (EMCV) infection, hypophosphorylated 4E-BP1 repressor accumulates, inhibiting eIF4E binding to eIF4G and limiting eIF4F assembly (Gingras et al 1996).…”

Section: Remodeling Host Mrna Translation In Infected Cellsmentioning

confidence: 99%

Translational Control in Virus-Infected Cells

Stern-Ginossar

Thompson

Mathews

et al. 2018

Cold Spring Harb Perspect Biol

137

164

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As obligate intracellular parasites, virus reproduction requires host cell functions. Despite variations in genome size and configuration, nucleic acid composition, and their repertoire of encoded functions, all viruses remain unconditionally dependent on the protein synthesis machinery resident within their cellular hosts to translate viral messenger RNAs (mRNAs). A complex signaling network responsive to physiological stress, including infection, regulates host translation factors and ribosome availability. Furthermore, access to the translation apparatus is patrolled by powerful host immune defenses programmed to restrict viral invaders. Here, we review the tactics and mechanisms used by viruses to appropriate control over host ribosomes, subvert host defenses, and dominate the infected cell translational landscape. These not only define aspects of infection biology paramount for virus reproduction, but continue to drive fundamental discoveries into how cellular protein synthesis is controlled in health and disease.

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“…In contrast, 2A needs only to cleave 32 itself away from the viral polyprotein at its amino terminal boundary. Possibly as a 33 consequence of this, the protease has a less well-defined recognition motif, with 34 cleavage of some host substrates depending not just on active site recognition but also 35 exosite interactions as well [11,12]. Host proteins targeted by 2A include eIF4GI [13], 36 eIF4GII [14], Gemin3 [15] and several nucleoporins (Nup62, 98 and 153) [16,17].…”

Section: Introduction 14mentioning

confidence: 99%

Rhinovirus 2A is the predominant protease responsible for instigating the early block to gene expression encountered in infected cells

Smart

Filippi

Smalley

et al. 2019

Preprint

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1Human rhinoviruses express 2 cysteine proteases, 2A and 3C, that are responsible for 2 viral polyprotein processing. Both proteases also suppress host gene expression by 3 inhibiting mRNA transcription, nuclear export and cap-dependent translation. 4However, the relative contribution that each makes in achieving this goal remains 5 Methods 83Cell lines and reagents 84 293T cells were maintained at 37°C in a 5% CO2 incubator with DMEM (Invitrogen, 85Fisher Scientific UK Ltd, Loughborough, UK) supplemented with 10% foetal bovine 86 serum, 100 U/ml penicillin, 100 µg/ml streptomycin. Experimental treatment of cells 87 included incubation in medium supplemented with 200 µg/ml cycloheximide (Sigma, 88 Gillingham, UK) and/or 5 µg/ml actinomycin D (Cayman Chemical Cambridge 89Bioscience Ltd, Cambridge, UK). 90 91 DNA constructs 92Generation of some constructs relied on gene synthesis (Invitrogen, Fisher Scientific 93 UK Ltd, Loughborough, UK). In instances where this was the case, sequences ordered 94 were codon optimized for mammalian expression and then manually adjusted to 95 minimize the existence of unwanted splice donor and splice acceptor sites using the 96 online programmes HSF3[25] and the NetGene2 server [26]. The initial plasmid 97 expressing GFP, VSV-tagged 2A and HA-tagged 3C from HRV16 was generated by 98 cloning a single synthetic DNA fragment into an in-house dual promoter plasmid via 99EcoRI and SalI restriction sites to generate pCIPEP-A16 TAG (+/+) (see Fig. S1 for 100 sequence). Synthesized DNAs encoding for HRV-B4 and HRV-C2 proteases (Table 101 S1) were exchanged with their respective counterparts in pCIPEP-A16 TAG (+/+) using 102ClaI and SbfI (2A) and BsiWI and SalI (3C) restriction sites. Inactivation of the 103 proteases involved a two-step PCR approach, which introduced a Cys>Ala mutation 104 in the active site of both 2A and 3C. A similar PCR-based strategy was used to 105 generate constructs expressing tag-free proteases and a 3C protease with a c-Myc 106 nuclear localization signal (PAAKRVKLD) fused directly to its C-terminus. Primer 107 sequences are available on request. 108The synthetic DNA encoding for the Thioredoxin-Nanoluciferase(Nluc)PEST 109 fusion protein linked by a peptide derived from translating the hepatitis delta virus 110 (HdV) ribozyme sequence (HdV WT Nluc; Fig. S2) was initially cloned into pCDNA3.

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Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection

Cited by 16 publications

References 43 publications

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication

Translational Control in Virus-Infected Cells

Rhinovirus 2A is the predominant protease responsible for instigating the early block to gene expression encountered in infected cells

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