Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor

Li, Zhenghe; Pogany, Judit; Panavas, Tadas; Xu, Kai; Esposito, Anthony M.; Kinzy, Terri Goss; Nagy, Peter D.

doi:10.1016/j.virol.2008.11.041

Cited by 122 publications

(187 citation statements)

References 85 publications

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“…Given a tight association between RT and IN (22), it is possible that an IN and eEF1A interaction resulted in coprecipitation of the RT p51 subunit. eEF1A has been implicated as a host cofactor for other RNA viruses (12,14,(23)(24)(25). However, recently, the plant and yeast homologs of eEF1A and eEF1G (called eEF1Bγ) were found to be important for RNA replication of tomato bushy stunt virus (TBSV) through interactions with the viral RNA-dependent RNA polymerase (RdRp) and RNA stem-loop structures in the viral genome (15).…”

Section: Discussionmentioning

confidence: 99%

“…S2A). However, eEF1A has many noncanonical roles in the cell (11) and in virus replication (12)(13)(14)(15). In addition, eEF1A was reported to interact with HIV-1 Gag (16) and the cytoskeletal networks (17) that are thought to play crucial role in the completion of HIV-1 reverse transcription (18).…”

Section: Purification and Identification Of Cellular Proteins In Fracmentioning

confidence: 99%

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Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Warren

Wei

et al. 2012

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

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Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus. purification | mass spectrometry | cellular factor | siRNA knock down | virus replication T he HIV type-1 (HIV-1) reverse transcriptase (RT) enzyme carries out reverse transcription through DNA polymerase and RNase H activities, which mediate a sequential series of steps that include the initiation of DNA synthesis producing negative strand strong stop DNA (−ssDNA), full-length negative-strand DNA, positive-strand DNA, and intact preintegrative doublestrand DNA (reviewed elsewhere 1). The ability of HIV-1 to efficiently produce early reverse transcription products in vitro has been described (2). However, under in vitro conditions, the production of late reverse transcription products is less efficient than that observed in HIV-1-infected cells (3, 4), suggesting that host cellular factors are required. After entry into the host cell cytoplasm, the HIV-1 core, containing the viral genomic RNA, RT, and integrase (IN), reorganizes to form the reverse transcription complex (RTC). The RTC requires both IN and RT to be active (5), and they are believed to recruit cellular factors to facilitate DNA synthesis (6). Although two-hybrid library and genomewide RNAi screening methods have implicated more than 50 cellular proteins as important for reverse transcription (6, 7), whether any of these cellular proteins are RTC components, or whether they direct the RTC in other ways, is unclear.Attempts to purify and to identify cellular RTC cofactors by more conventional methods ...

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Section: Discussionmentioning

confidence: 99%

Section: Purification and Identification Of Cellular Proteins In Fracmentioning

confidence: 99%

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Warren

Wei

et al. 2012

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

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“…To induce the expression of p33 or p92, 50 M CuSO 4 was added to the yeast cultures for 30 min at 29°C, followed by the addition of cycloheximide to a final concentration of 100 g/ml to inhibit protein synthesis. Equal amounts of yeast cells were collected at given time points as indicated in the figure legends after cycloheximide treatment (26), and cell lysates were prepared by the NaOH method as described previously (37 total protein samples were analyzed by SDS-PAGE and Western blotting with anti-His antibody using ECL (Amersham) as described previously (15). In vitro translation assay.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants

Sharma

Sasvari

Nagy

2010

J Virol

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The replication of plus-strand RNA viruses depends on subcellular membranes. Recent genome-wide screens have revealed that the sterol biosynthesis genes ERG25 and ERG4 affected the replication of Tomato bushy stunt virus (TBSV) in a yeast model host. To further our understanding of the role of sterols in TBSV replication, we demonstrate that the downregulation of ERG25 or the inhibition of the activity of Erg25p with an inhibitor (6-amino-2-n-pentylthiobenzothiazole; APB) leads to a 3-to 5-fold reduction in TBSV replication in yeast. In addition, the sterol biosynthesis inhibitor lovastatin reduced TBSV replication by 4-fold, confirming the importance of sterols in viral replication. We also show reduced stability for the p92 pol viral replication protein as well as a decrease in the in vitro activity of the tombusvirus replicase when isolated from APB-treated yeast. Moreover, APB treatment inhibits TBSV RNA accumulation in plant protoplasts and in Nicotiana benthamiana leaves. The inhibitory effect of APB on TBSV replication can be complemented by exogenous stigmasterol, the main plant sterol, suggesting that sterols are required for TBSV replication. The silencing of SMO1 and SMO2 genes, which are orthologs of ERG25, in N. benthamiana reduced TBSV RNA accumulation but had a lesser inhibitory effect on the unrelated Tobacco mosaic virus, suggesting that various viruses show different levels of dependence on sterol biosynthesis for their replication.Plus-stranded RNA [(ϩ)RNA] viruses usurp various intracellular/organellar membranes for their replication. These cellular membranes are thought to facilitate the building of viral factories, promote a high concentration of membrane-bound viral proteins, and provide protection against cellular nucleases and proteases (1,12,35,44). The membrane lipids and proteins may serve as scaffolds for targeting the viral replication proteins or for the assembly of the viral replicase complex. The subcellular membrane also may provide critical lipid or protein cofactors to activate/modulate the function of the viral replicase. Indeed, the formation of spherules, consisting of lipid membranes bended inward and viral replication proteins as well as recruited host proteins, has been demonstrated for several (ϩ)RNA viruses (20,30,48). These virus-induced spherules serve as sites of viral replication. Importantly, (ϩ)RNA viruses also induce membrane proliferation that requires new lipid biosynthesis. Therefore, it is not surprising that several genome-wide screens for the identification of host factors affecting (ϩ)RNA virus replication unraveled lipid biosynthesis/metabolism genes (8,23,38,50). However, in spite of these intensive efforts, understanding the roles of various lipids and lipid biosynthesis enzymes and pathways in (ϩ)RNA virus replication is limited.Tomato bushy stunt virus (TBSV) is among the most advanced model systems regarding the identification of host factors affecting (ϩ)RNA virus replication (32). Among the five proteins encoded by the TBSV genome, only the p33 re...

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“…This factor is also known to participate in processes including export of tRNAs from the nucleus and the targeting of damaged and misfolded proteins to the proteasome (Sasikumar et al, 2012). EF1A is also reported to have interactions with the tombusvirus replication complex and the 3' tRNA-like structure of turnip yellow mosaic virus (Matsuda et al, 2004;Li et al, 2009). …”

Section: The Actors In Elongation Eef1amentioning

confidence: 99%

Mechanism of Cytoplasmic mRNA Translation

Browning

Bailey‐Serres

2015

The Arabidopsis Book

186

220

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Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings.

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Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor

Cited by 122 publications

References 85 publications

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants

Mechanism of Cytoplasmic mRNA Translation

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