Inhibition of Transcription and Translation in Sindbis Virus-Infected Cells

Gorchakov, Rodion; Frolova, Elena I.; Frolov, Ilya

doi:10.1128/jvi.79.15.9397-9409.2005

Cited by 127 publications

(237 citation statements)

References 59 publications

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“…The protein-antibody complex was isolated by protein G/A-agarose beads (Oncogene Inc.). Protein-antibody-protein G/A-agarose complexes were washed with radioimmune precipitation assay buffer (13). The complexes in gel loading buffer (Invitrogen) were heated at 70 -75°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

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The Essential Vertebrate ABCE1 Protein Interacts with Eukaryotic Initiation Factors

Chen

Dong

Ishimura

et al. 2006

Journal of Biological Chemistry

134

118

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The ABCE1 gene is a member of the ATP-binding cassette (ABC) multigene family and is composed of two nucleotide binding domains and an N-terminal Fe-S binding domain. The ABCE1 gene encodes a protein originally identified for its inhibition of ribonuclease L, a nuclease induced by interferon in mammalian cells. The protein is also required for the assembly of the HIV and SIV gag polypeptides. However, ABCE1 is one of the most highly conserved proteins and is found in one or two copies in all characterized eukaryotes and archaea. Yeast ABCE1/RLI1 is essential to cell division and interacts with translation initiation factors in the assembly of the pre-initiation complex. We show here that the human ABCE1 protein is essential for in vitro and in vivo translation of mRNA and that it binds to eIF2␣ and eIF5. Inhibition of the Xenopus ABCE1 arrests growth at the gastrula stage of development, consistent with a block in translation. The human ABCE1 gene contains 16 introns, and the extremely high degree of amino acid identity allows the evolution of its introns to be examined throughout eukaryotes. The demonstration that ABCE1 plays a role in vertebrate translation initiation extends the known functions of this highly conserved protein. Translation is a highly regulated process important to development and pathologies such as cancer, making ABCE1 a potential target for therapeutics. The evolutionary analysis supports a model in which an ancestral eukaryote had large number of introns and that many of these introns were lost in non-vertebrate lineages. The induction of ribobuclease L (RNase L)2 represents an important viral defense mechanism of mammalian cells against RNA viruses (1, 2). RNase L is present in the cell in an inactive form and can be activated by interferon. Interferon causes the activation of oligoadenylate synthases producing 2Ј-5Ј-oligoadenylate. Bisbal et al. (3) described the isolation of a 68-kDa protein that binds to and inhibits RNase L and cloning of the gene. Although originally termed RNase L inhibitor (RLI) the gene is part of the ABC multigene family and its gene symbol is ABCE1. ABCE1 is induced during infection of cells with HIV-1 and an antisense construct directed against ABCE1 resulted in a reduction of viral load (4). In cell-free extracts HIV-1 gag protein can assemble into viral capsids (5). This process is ATP-dependent and was shown to require a 68-kDa protein (HP68) identified as ABCE1/RLI. This same protein also functions in the assembly of HIV-2 and SIVmac (6).Most of the ABC family genes encode large transport proteins that contain 6 -17 transmembrane domains (7). ABCE1 is one of four human ABC genes that contain only nucleotide binding domains and are therefore not likely to be transporters. Of the 48 human ABC proteins, ABCE1 is the most conserved with a single copy of the gene in every characterized eukaryote, except for Arabidopsis, which has two ABCE1-like genes. In addition, there is an ABCE1-related gene in all characterized archae but not in prokaryotes, demonstrating t...

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Section: Methodsmentioning

confidence: 99%

“…Total 35 S activity, 2.3 mCi/181 l) in DMEM lacking methionine/cysteine as described by Gorchakov et al (13). After 30-min labeling, the cells were lysed by mammalian protein extraction buffer as described above.…”

Section: Methodsmentioning

confidence: 99%

The Essential Vertebrate ABCE1 Protein Interacts with Eukaryotic Initiation Factors

Chen

Dong

Ishimura

et al. 2006

Journal of Biological Chemistry

134

118

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“…Vero cells were co-transfected with a plasmid containing the promoter region and 59-untranslated region (UTR) of one of these target genes upstream of a firefly luciferase (Fluc) gene and a plasmid constitutively expressing Renilla luciferase (Rluc). Given that CHIKV infection induces host shut-off and reduces RNA polymerase IIdriven transcription (Akhrymuk et al, 2012;Gorchakov et al, 2005), we studied the induction of UPR reporter genes using a luciferase-based assay in which all Fluc values were normalized for constitutive Rluc expression. One day after transfection, cells were infected with CHIKV at an m.o.i.…”

Section: Effect Of Chikv Infection On Upr Activationmentioning

confidence: 99%

Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response

Fros

Major

Scholte

et al. 2015

Journal of General Virology

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The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1a become activated. PERK phosphorylates eIF2a leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1a splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2a and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV nonstructural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.

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“…Second, the placement of a DLP structure at the 5′ end of the EGFP coding sequence restored translation to a level comparable to that of translation in viral 26S mRNA. Third, the use of a viral promotor that drives the synthesis of reporter mRNA allowed direct measurement of the effect of virus replication on translation, obviating the perturbations that Sindbis and other viruses exert on cellular transcription (3,(31)(32)(33)49). In fact, infection with alphavirus also results in a halt of host transcription that can be separated from the translation shut-off phenomenon (49).…”

Section: Discussionmentioning

confidence: 99%

“…Third, the use of a viral promotor that drives the synthesis of reporter mRNA allowed direct measurement of the effect of virus replication on translation, obviating the perturbations that Sindbis and other viruses exert on cellular transcription (3,(31)(32)(33)49). In fact, infection with alphavirus also results in a halt of host transcription that can be separated from the translation shut-off phenomenon (49). Despite this, we found that the steady-state levels of abundant host RNA, such as ribosomal or β-actin mRNA, did not significantly decrease at 6 h postinfection, when translation of host mRNA was completely inhibited (Fig.1).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of host translation by virus infection in vivo

Toribio

Ventoso

2010

Proc. Natl. Acad. Sci. U.S.A.

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Infection of cultured cells with lytic animal viruses often results in the selective inhibition of host protein synthesis, whereas viral mRNA is efficiently translated under these circumstances. This phenomenon, known as "shut off," has been well described at the molecular level for some viruses, but there is not yet any direct or indirect evidence supporting the idea that it also should operate in animals infected with viruses. To address this issue, we constructed recombinant Sindbis virus (SV)-expressing reporter mRNA, the translation of which is sensitive or resistant to virus-induced shut off. As found in cultured cells, replication of SV in mouse brain was associated with a strong phosphorylation of eukaryotic initiation factor (eIF2) that prevented translation of reporter mRNA (luciferase and EGFP). Translation of these reporters was restored in vitro, in vivo, and ex vivo when a viral RNA structure, termed downstream hairpin loop, present in viral 26S mRNA, was placed at the 5′ end of reporter mRNAs. By comparing the expression of shut off-sensitive and -resistant reporters, we unequivocally concluded that replication of SV in animal tissues is associated with a profound inhibition of nonviral mRNA translation. A strategy as simple as that followed here might be applicable to other viruses to evaluate their interference on host translation in infected animals.eukaryotic initiation factor-2 phosphorylation | dsRNA-dependent protein kinase | Sindbis

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Inhibition of Transcription and Translation in Sindbis Virus-Infected Cells

Cited by 127 publications

References 59 publications

The Essential Vertebrate ABCE1 Protein Interacts with Eukaryotic Initiation Factors

The Essential Vertebrate ABCE1 Protein Interacts with Eukaryotic Initiation Factors

Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response

Inhibition of host translation by virus infection in vivo

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