Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

Krüger, Martin; Beger, Carmela; Li, Qiang-Xin; Welch, Peter J.; Tritz, Richard; Leavitt, Mark C.; Barber, James David; Wong‐Staal, Flossie

doi:10.1073/pnas.97.15.8566

Cited by 86 publications

(56 citation statements)

References 23 publications

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“…Phenotypic screens using such libraries can reveal new potential therapeutic targets as well as new functions of known genes, as has been done using randomized ribozyme libraries instead of siRNAs (Kruger et al 2000;Li et al 2000;. Any siRNA sequences that may be lethal to the host cell will be eliminated from the library, and sequences toxic enough to retard growth can be eliminated by allowing the cells harboring nontoxic siRNAs to outgrow them.…”

Section: Discussionmentioning

confidence: 99%

Complete, gene-specific siRNA libraries: Production and expression in mammalian cells

Seyhan

Vlassov²,

Ilves³

et al. 2005

RNA

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Short interfering RNAs (siRNAs) are widely used to silence the expression of specific genes. Current practice for designing effective siRNAs is to use algorithms based on sequence-efficacy correlations; however, there are many highly effective sequences that these algorithms do not anticipate. To ensure that the best siRNAs are identified, all possible gene-specific siRNA sequences of appropriate lengths should be screened in cell culture. Synthesizing and testing all such sequences individually is costly. A potentially much easier alternative is to prepare a mixture of all these sequences (a gene-specific library), express them in cells, select cells having the desired phenotype, and identify the siRNA contained within the selected cells. Here we describe two new methods for preparing and expressing such libraries. The first uses cloned Dicer or RNase III to digest gene-specific RNA duplexes to siRNAs, which are then converted to the corresponding DNA sequences by attaching RNA primers and performing reverse transcription-PCR. The second method involves partial DNase I digestion of gene-specific DNA, purification of a 20-30-bp fraction, and amplification by attaching DNA adapters followed by PCR. DNA libraries specific for TNF-␣, DsRed, and part of the hepatitis C virus genome, generated by methods, were inserted into siRNA expression vectors between convergent human U6 and H1 promoters. Randomly selected clones from each library together with vectors expressing the corresponding target genes were cotransfected into 293FT cells and assayed for target gene inhibition. About 10%-20% of siRNAs represented in these libraries show significant inhibition of their target genes. Most of these inhibitory sequences are not predicted by existing algorithms.

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Section: Discussionmentioning

confidence: 99%

Complete, gene-specific siRNA libraries: Production and expression in mammalian cells

Seyhan

Vlassov²,

Ilves³

et al. 2005

RNA

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“…EIF2S3 binds to the 5Ј nontranslated region (5Ј NTR) of HCV and has been reported to modulate HCV translation (35). PTBP1 binds to both 5Ј and 3Ј NTRs, suggesting a possible interaction between the 5Ј and 3Ј termini of HCV RNA (35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

Cellular cofactors affecting hepatitis C virus infection and replication

Randall

Panis²,

Cooper

et al. 2007

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

496

454

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Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.antivirals ͉ miR-122 ͉ RNAi ͉ HCVcc-siRNA H epatitis C virus (HCV) has a notable ability to establish persistent infections in Ϸ70% of cases, resulting in 130 million chronically infected people throughout the world (1). This prevalence has spurred considerable interest in the study of HCV-host interactions, on both cellular and molecular levels. The inability to grow HCV in cell culture led some groups to focus on the identification of cellular proteins that interact with individual HCV proteins or RNA elements, resulting in the accumulation of a large number of putative HCV-host interactions. Unfortunately, the significance of most of these with respect to the HCV life cycle is currently unknown (reviewed in ref.2). Over the past 6 years, cell culture systems have been developed that enable the characterization of HCV replication and entry (3-6). This effort recently culminated in the development of cell culture systems that reproduce the entire viral life cycle (7-9). A number of virus-host interactions have been characterized by using these experimental systems. For example, CD81 has been demonstrated to play a role in HCV entry (10-12).Sequence-specific gene silencing of RNAi is ideal for assessing the genetic phenotypes associated with virus-host interactions. We have previously shown that siRNAs are highly effective at silencing either host or viral RNAs in cells that contain replicating HCV, demonstrating th...

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“…These properties would be expected to render HCV translation relatively resistant to cellular stresses or apoptosis-inducing conditions that compromise the availability or integrity of eIF4E, eIF4B, and eIF4G (Bushell et al , 2001Clemens et al 2000). However HCV IRES-driven protein synthesis may potentially still be sensitive to regulation of eIF2 and/or eIF2B activity (Kruger et al 2000). Previous reports have suggested that the function of several cellular IRESes is resistant to inhibition in stressed or apoptotic cells (Coldwell et al 2000;Holcik et al 2000a;Stoneley et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Vyas

Elia²,

Clemens³

2003

RNA

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Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5 untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA f -binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.

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Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

Abstract: CorrectionsMICROBIOLOGY. For the article ''Identification of eIF2B␥ and eIF2␥ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach''

Cited by 86 publications

References 23 publications

Complete, gene-specific siRNA libraries: Production and expression in mammalian cells

Complete, gene-specific siRNA libraries: Production and expression in mammalian cells

Cellular cofactors affecting hepatitis C virus infection and replication

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

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