The NS5A protein of bovine viral diarrhoea virus interacts with the α subunit of translation elongation factor-1

Johnson, Craig; Pérez, Daniel R.; French, Ron; Merrick, William C.; Donis, Ruben O.

doi:10.1099/0022-1317-82-12-2935

Cited by 49 publications

(43 citation statements)

References 47 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%

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%

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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“…GST-NS5A bound specifically to both in vitro translated and mammalian cell expressed eEF1A. This interaction was suggested to play a role in the replication of BVDV (Johnson et al 2001). In addition, Y2H screening identified bovine NIK-and IKK -binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-κB) signalling in cells (Zahoor et al 2010).…”

Section: Virus-host Interacting Partnersmentioning

confidence: 99%

“…NS3-SphK1 binding enhance BVDV replication and BVDVinduced apoptosis (Yamane et al 2009a). Using Y2H screening, the subunit of bovine translation elongation factor 1A (eEF1A) was shown to interact with the NS5A polypeptide of BVDV (Johnson et al 2001). This interaction was further analysed in a cell-free translation system and was found to be conserved among BVDV isolates of both genotypes and biotypes.…”

Section: Virus-host Interacting Partnersmentioning

confidence: 99%

Understanding the Pathogenesis of Cytopathic and Noncytopathic Bovine Viral Diarrhea Virus Infection Using Proteomics

Ammari¹,

McCarthy²,

Nanduri³

et al. 2012

Proteomic Applications in Biology

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“…Several other RNA viruses are proposed to use EF-1␣ for replication (24)(25)(26)(27)(28)(29), which might play a role comparable to that of EF-1␥ for the above viruses.…”

Section: Identification Of 96 Yeast Genes Affecting Accumulation Of Tbsvmentioning

confidence: 99%

Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

Panavas

Servienė

Brasher

et al. 2005

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

201

270

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host factors ͉ plus-stranded RNA virus ͉ tomato bushy stunt tombusvirus ͉ virus replication ͉ yeast-knockout strains T he success of viruses as pathogens of humans, animals, and plants depends on the viruses' ability to reprogram the host-cell metabolism to support the infection. The virus-host interaction is more complex than the term ''reprogramming'' suggests because host cells have antiviral defense mechanisms. Identifying host genes that can affect virus replication and the infection process is central to understanding the complex role of the host in viral infections. The largest group of viruses, the positive-strand RNA viruses, which include the severe acute respiratory syndrome (SARS) coronavirus and hepatitis C and West Nile viruses, has virion RNAs that are directly translated in the infected cell. Synthesized viral proteins and recruited host proteins mediate processes that lead to efficient multiplication of the viral RNA (1, 2). RNA viruses are important not only as infectious agents but also as tools in biotechnology and gene therapy for expressing selected proteins in cells (3)(4)(5).Yeast is a model eukaryotic cell that has been used extensively to study the roles of individual genes in cellular processes based on genome-wide screens (6-9). Many screens have been based on the yeast single-gene-knockout (YKO) library, because the role of each nonessential yeast gene can be tested for selected functions (10). We and others have developed systems for inducing yeast cells to support the replication of certain positive-strand RNA viruses or their surrogates (11)(12)(13)(14). Here, we apply our previously developed system for robust replication of a small RNA replicon of the tomato bushy stunt virus (TBSV) in yeast (13,15) to screen the entire YKO library for genes influencing the efficiency of viral replication. A total of 96 YKO strains were identified. The identified host genes are either involved in many cellular processes, including nucleic acid, protein, and lipid metabolism, protein targeting͞transport, and general and stress metabolism or have unknown functions. Our results show that the replication of positive-strand RNA viruses of different supergroups is influenced by distinct groups of genes and that TBSV replication is associated with sets of genes that also have been associated with certain human disease states. Materials and MethodsYeast Strains and Expression Plasmids. Yeast strain 4741 and the YKO deletion series (10) were obtained from Open Biosystems (Huntsville, AL). Yeast transformation was modified from the standard lithium acetate (LiOAc)͞polyethylene glycol (PEG) protocol (16) to facilitate a 96-well plate format. Briefly, yeast strains were grown overnight in yeast extract͞peptone͞dextrose medium supplemented with 200 mg͞liter geneticin G418. Cultures were then diluted to Ϸ0.3 OD 600 in fresh medium (0.25 ml per well) and cultured for an additional 4 h at 30°C. The cells were pelleted, washed with sterile water, and resuspended in 0.1 M LiOAc. This procedure was followed by rep...

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The NS5A protein of bovine viral diarrhoea virus interacts with the α subunit of translation elongation factor-1

Cited by 49 publications

References 47 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

Understanding the Pathogenesis of Cytopathic and Noncytopathic Bovine Viral Diarrhea Virus Infection Using Proteomics

Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

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