Department of Veterinary and Biomedical Sciences (202 VBS)1 and USDA, ARS, Department of Plant Pathology2, University of Nebraska-Lincoln, Fair Street and East Campus Loop, Lincoln, NE 68583-0905, USA
Case Western Reserve University, School of Medicine, Department of Biochemistry, Cleveland, OH 44106, USA3
Author for correspondence: Ruben Donis. Fax +1 402 472 9690. e-mail rdonis{at}unlnotes.unl.edu
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Abstract |
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Introduction |
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BVDV initiates infection by attaching to the plasma membrane, after which endocytosis and pH-dependent fusion of the envelope to the endosomal membrane occur, resulting in the delivery of the BVDV genome into the cytosol of the target cell. The genomic RNA is then translated by recruitment of translation initiation factors mediated by the IRES, which is present within the 385 nt 5' untranslated region (UTR). Newly synthesized non-structural proteins are thought to assemble into functional replicase complexes and carry out the first step of genome replication, negative-strand (antigenome) RNA synthesis. The replicase must then complete the synthesis of progeny positive-stranded RNA using the antigenomic RNA as the template. Little is known about either the molecular aspects of this process or the cis- and trans-acting factors involved. However, NS5B bears the glycineaspartateaspartate motif characteristic of RNA-dependent RNA polymerases and the purified protein displays polymerase activity when supplied with suitable substrates and template (Lai et al., 1999 ; Zhong et al., 1998
). NS2-3 is thought to contribute its helicase activity to RNA replicase functions (Gu et al., 2000
). In support of this hypothesis, it has been reported that expression of NS3 lacking the NS2 region is correlated with increased levels of viral RNA accumulation (Vassilev & Donis, 2000
). It is not known, however, if other viral or cellular proteins are involved in the activity and regulation of BVDV replicase.
Interactions among viral proteins play a central role in the assembly and regulation of the functional complexes responsible for viral RNA replication (Andino et al., 1993 ; Lai, 1998
). These replication complexes often include transient or long-lived interactions with host proteins for structural purposes or recruit regulatory and catalytic functions (Lai, 1998
). It is now well established that coupling among the different sequential steps of virus replication is central to the overall infectious cycle of many RNA viruses of bacteria, plants and animals (Eigen et al., 1991
; Gamarnik & Andino, 1998
; Janda & Ahlquist, 1998
; Nguyen et al., 1996
; Novak & Kirkegaard, 1994
; Nugent et al., 1999
). Identification of proteinprotein interactions between viral and cellular proteins may lead to a more complete understanding of the dynamics of RNA replication, virus-mediated cellular modulation and host-range restriction.
In this report, we present the results of a yeast two-hybrid screen that describe the identification of a cellular protein that interacts with BVDV NS5A. 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.
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Methods |
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Strain EGY48 harbouring pLexANS5A and pSH18-34 (a reporter plasmid encoding -galactosidase with a Gal1 promoter transcriptionally controlled by upstream LexA-binding sequences) was transformed with a pJG4.5MDBK cDNA library using the lithiumacetate method. Proceeding transformation, yeast were selected for histidine, uracil and tryptophan prototrophy (his+, ura+ and trp+), thus confirming the presence of all three plasmids within the transformants. Cells were then cultivated, induced with galactoseraffinose media and selected for leucine prototrophy, which is characteristic of a proteinprotein interaction phenotype. Approximately 1x107 primary yeast transformants were selected for the leu+ growth phenotype on plates containing uracil, histidine, tryptophan, leucine and galactose. In order to discard false-positive colonies with the leu+ phenotype, independent of LexANS5A expression, colonies were also analysed for
-galactosidase activity on nitrocellulose filters using the X-Gal substrate. Plasmid DNA was extracted from leucine prototrophic library transformants expressing
-galactosidase only when growing in galactoseraffinose plates. These plasmids were introduced into E. coli strain KC8 cells by electroporation. Library plasmids were selected for tryptophan prototrophy on minimal media M9 agar plates that lacked tryptophan and contained 50 µg/ml kanamycin. Yeast strain EGY48 was transformed with each candidate prey plasmid DNA from E. coli strain KC8 and subsequently mated with yeast strain RFY206 bearing pSH18-34 and one of the various lexA gene fusion baits to assess the specificity of the interaction by
-galactosidase assay (Table 1
). Colonies that remained white after 3 h incubation with X-Gal were scored negative. Library plasmids that mediated transactivation, as demonstrated by leucine prototrophy and
-galactosidase activity in the presence of pLexANS5A, but not in the presence of specificity controls, were saved for further analysis. Sequencing using the dideoxynucleotide chain termination method revealed the identity of the cDNA of each library plasmid encoding a candidate NS5A-interacting protein.
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Expression of myc- and haemagglutinin (HA)-tagged proteins.
pmycNS5A was obtained by subcloning NS5A as a SmaIXhoI fragment from pLexANS5A into pcDNAmyc (D. R. Perez, unpublished data). pmycNS5A-890, -CV24 Oregon, -CP7 and -NCP7 were generated by subcloning an EcoRI fragment encoding NS5A from pCRII-Topo into pcDNAmyc. pmycNS4B was generated by subcloning an EcoRIXhoI fragment from pLexANS4B into pcDNAmyc. pHAeEF1A was generated by subcloning eEF1A as an EcoRIXhoI fragment from pJG4.5eEF1A into pcDNAHA (D. R. Perez, unpublished data). To produce mycNS5A and HAeEF1A fusion proteins in mammalian cells, 5x105 African green monkey kidney cells (CV-1) in 6-well plates were infected with recombinant vaccinia virus vTF7-3 for 45 min at an m.o.i. of 5. After washing twice with minimal essential medium (MEM), cells were subsequently transfected for 4 h with either pmycNS5A or pHAeEF1A using a mixture containing 2 µg of plasmid DNA, 6 µl lipofectamine (Gibco BRL) and 1 ml MEM. At the end of the transfection period, the transfection mixture was removed and cells were maintained in 2 ml MEM supplemented with 10% foetal bovine serum for approximately 12 h. Cells were harvested and lysed by sonication at 4 °C for 45 s in 700 µl of lysis/binding buffer (20 mM TrisHCl pH 7·6, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 2 mM Na4P2O7, 30 mM NaF, 0·05% Triton X-100 and a protease inhibitor cocktail, as described previously). Lysates were clarified by centrifugation at 10000 g and the resulting supernatants were used for in vitro binding assays. In vitro expression of HAeEF1A was performed using a Coupled Wheat Germ T7 Transcription/Translation system (Promega). In vitro-translated HAeEF1A was diluted in lysis/binding buffer and used during in vitro binding assays, as explained below.
In vitro binding assays.
GST fusion proteins were subjected to 10% SDSPAGE, fixed and stained with Coomassie blue to estimate the amount of protein needed for use during in vitro binding assays. In all cases, an equal concentration of GST fusion protein was used by adjusting the amount with additional glutathioneagarose beads to keep the volume constant. A volume of 20 µl of bead slurry was incubated with 200 µl of CV-1 protein lysates for 2 h at room temperature with rotation. Unbound proteins were removed by washing the beads six times with lysis/binding buffer in a cycle consisting of ten 1 min washes and 5 s centrifugation for bead collection. Finally, beads were resuspended in 1x SDS sample buffer and boiled for 2 min (Ausubel et al., 1989 ). To assay the binding of pure eEF1A to GSTNS5A, 5 µg eEF1A, purified as described previously, was diluted in 200 µl lysis/binding buffer and treated as explained above (Carvalho et al., 1984
; Cavallius et al., 1997
).
Western blot assays.
Cell lysates or protein samples dissolved in sample buffer were separated by 10% SDSPAGE and electrotransferred onto Hybond-C nitrocellulose filters (Amersham) using a semi-dry electroblotter (Bio-Rad). Prestained molecular mass standards for electrophoresis were purchased from Sigma (# SDS7B). LexA fusion proteins were detected using an anti-LexA monoclonal antibody (MAb) at a concentration of 20 ng/ml (Clontech, # 5397-1). To detect HA epitope-tagged proteins, anti-HA MAb 12C5 was used in a 1:50 dilution in PBS supplemented with 0·05% Tween 20. Detection of eEF1A was performed with an anti-eEF1A MAb at a concentration of 1 µg/ml (Upstate Biotechnology). Myc epitope-tagged proteins were detected using a 1:400 dilution of an anti-c-myc MAb (Chemicon). Incubation with the primary antibody at room temperature (2224 °C) for 1 h was followed by three washes. Samples were then incubated under the same conditions with a secondary goat anti-mouse IgG MAb conjugated to horseradish peroxidase at a dilution of 1:500 (Sigma, # A5278). Blots were subjected to enhanced chemiluminescence (ECL, Amersham), according to the manufacturers instructions.
Sequence identity, translation and alignments.
Amino acid sequence alignments were produced using the PileUp program within the Wisconsin Package, version 9.1 (Altschul et al., 1990 ). Electronic translation of the nucleotide sequence of bovine eEF1A was accomplished using the Translate program within the same software package. Initial sequences were compared with the NCBI database using the BLAST program and the similarity among the proteins was analysed as described previously (Feng & Doolittle, 1996
).
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Results |
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NS5A binds to eEF1A in vitro
Yeast two-hybrid screens are excellent tools to identify interacting proteins in vivo. However, by virtue of the complexity of the nuclear environment of live yeast, it is not possible to equate reporter gene expression unequivocally with biologically relevant interactions. One approach to determine the significance of the interaction between NS5A and eEF1A revealed in the two-hybrid assay is to study the interaction in vitro. To this end, we performed pull-down assays with GST fusion proteins and epitope-tagged interaction partners. NS5A and bovine eEF1A were subcloned under the control of a T7 promoter and in-frame with N-terminal myc and HA epitope tags, respectively, yielding mycNS5A and HAeEF1A fusion proteins. Expression of these proteins from plasmids transfected into CV-1 cells was achieved by infection with the recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). Expression of mycNS5A and HAeEF1A in CV-1 cell lysates was demonstrated by probing immunoblots with specific anti-HA and anti-c-myc MAbs (Fig. 2A). NS5A and eEF1A were also expressed in E. coli as GST fusion proteins (GSTNS5A and GSTeIEF1A) and purified using glutathioneagarose beads (data not shown). Mammalian cell lysates containing HAeEF1A were incubated with GSTNS5A or GST alone, produced in the prokaryotic system, and bound to agarose beads. Likewise, lysates containing mycNS5A were incubated in the presence of either GSTeEF1A or GST alone. As shown in Fig. 2(B)
, Western blot analysis revealed that HAeEF1A (
54 kDa) was retained by binding to GSTNS5A on the agarose beads. The reverse was also true, as GSTeEF1A interacted with the
60 kDa mycNS5A protein expressed in CV-1 cells (Fig. 2B
). These interactions were specific, as GST alone was incapable of binding to either HAeEF1A or mycNS5A. We also noted that GSTNS5A bound the endogenous
54 kDa eEF1A present in CV-1 lysates (Fig. 2 C
); this is expected given the absolute amino acid sequence identity between bovine and primate eEF1A.
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eEF1A binds to NS5A from divergent BVDV strains
NS5A is the most variable non-structural protein among divergent BVDV isolates (Deng & Brock, 1992 ). Consequently, it was important to establish whether binding to eEF1A is conserved among different strains of BVDV. The ability of eEF1A to bind NS5A from several strains of BVDV, including non-cytopathic and cytopathic viruses from genotypes II and I, was analysed in the two-hybrid assay. We chose BVDV strains NADL, NCP7, CP7, CV24 Oregon and 890 to amplify NS5A-coding regions by RTPCR and cloned the resulting amplicons as LexA chimeras. Subsequently, expression of polypeptides of the expected size by yeast transformed with plasmids encoding each of the LexANS5A chimeras was demonstrated by Western blot analysis (Fig. 3
). To assess their capacity to interact with eEF1A, two-hybrid assays were performed by mating EGY48 and RFY206 yeast strains to yield diploid progeny expressing B42eEF1A and LexANS5A chimeras. All the BVDV NS5A proteins mediated transactivation of
-galactosidase activity and leucine prototrophy, indicating the ability of these NS5A fusions to interact with eEF1A. To extend the significance of the in vivo interactions of the NS5A proteins from divergent isolates with eEF1A, we performed GST pull-down assays. For this purpose, we expressed the viral proteins in CV-1 cells as N-terminal myc-tagged proteins and examined their retention by the GSTeEF1A chimera or by GST expressed alone. No major differences in the levels of expression of the BVDV NS5A proteins were observed, although we noted significant differences in the electrophoretic mobility of all NS5A proteins expressed (Figs 3
and 4A
). Interestingly, we found that all the NS5A proteins bound eEF1A as efficiently as the prototype NS5A from the NADL strain (Fig. 4
B). LexANS5A and mycNS5A proteins from each isolate displayed electrophoretic mobility shifts relative to the prototype NS5A from the NADL strain. Because these proteins are only between 67 and 89% identical, we postulate that amino acid composition and/or post-translational modifications are probably responsible for this effect. NS5A is a phosphoprotein and sequence divergence can result in different patterns of phosphorylation, which could, at least in part, explain the altered migration among these proteins. Nevertheless, our results suggest that, whatever the reasons for the mobility shifts in NS5A, they do not alter binding to eEF1A significantly.
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Discussion |
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The conserved genomic localization of NS5A and the generation of similar processing intermediates among both hepaciviruses and pestiviruses suggest a common and essential role of NS5A in the virus life cycle. However, we were unable to detect an interaction between hepatitis C virus NS5A and bovine eEF1A, whose amino acid sequence is identical to primate eEF1A. Nevertheless, the interaction described herein is conserved among divergent cytopathic and non-cytopathic BVDV isolates as well as in isolates belonging to genotypes I and II. Conservation of the eEF1ANS5A interaction among divergent BVDV strains does not constitute evidence of the essential nature of the binding interface, but it suggests strongly that this character may at least confer some selective advantage to BVDV.
An overwhelming body of data demonstrates the potential roles for the components of the host translation machinery in virus life cycles. Such viralhost interactions in the context of translation factors were demonstrated first within the bacteriophage Q RNA-dependent RNA polymerase or replicase. The active enzyme was found to exist as a heterotetramer consisting of a virus-encoded subunit plus three host proteins: ribosomal protein S1 and elongation factors Tu and Ts (Blumenthal et al., 1972
). More recently, it has been shown that two of these subunits, S1 and EF-Tu, provide the differential template recognition of positive- and negative-strand RNA present during virus replication (Brown & Gold, 1996
). This model demonstrates the direct role of host proteins in the mechanics of virus replication.
Binding of eEF1, -
and -
components to the viral RNA-dependent RNA polymerase of vesicular stomatitis virus is required for its replicase activity in vitro (Das et al., 1998
). Binding of eEF1A to viral RNA, directly or in association with viral proteins, has long been observed among bacterial, plant and animal viruses. These interactions have been demonstrated with poliovirus (Harris et al., 1994
), West Nile virus (Blackwell & Brinton, 1997
), brome mosaic virus (Bastin & Hall, 1976
), furoviruses (Goodwin & Dreher, 1998
) and bacteriophage Q
with EF-Tu, as mentioned previously. Viral RNAeEF1A interactions generally occur within the UTRs of viral genomes at domains containing conserved secondary structures.
Interestingly, the NS5A proteins of two highly divergent strains of BVDV analysed, 890 (genotype II) and NADL (genotype I), are only 77% similar, yet the ability to bind eEF1A is conserved. NS5A is hydrophilic, relatively stable within infected cells and phosphorylated at serine and threonine residues. Phosphorylation is conserved among all NS5A and NS5 proteins within members of the family Flaviviridae, suggesting its importance in the flavivirus life cycle (Reed et al., 1997 , 1998
). The electrophoretic mobility variability of the LexANS5A fusion proteins represented in Figs 3
and 4(A)
may reflect the different phosphorylation states of each polypeptide. However, differential processing events by an exopeptidase, for example, cannot be ruled out. Interestingly, electrophoretic mobility shifts were observed when comparing NS5A expressed in yeast and mammalian cells. This is probably a reflection of different post-translational modification events occurring in these cells. There was no consistent trend towards increased or decreased mobility that could be correlated with the type of host cell. Collectively, changes mediating gel mobility alterations did not abrogate NS5A binding to eEF1A, although they may well modulate binding affinities.
Nucleotide sequence analysis revealed the conserved identity of all the clones obtained in the MDBK cDNA screen as the highly conserved eEF1A. Comparison of the bovine eEF1A amino acid sequence revealed 100% identity to all known mammalian counterparts, with 81% identity to yeast eEF1A and 79% identity to barley eEF1A. eEF1A shows 56% amino acid similarity and conserved function to prokaryotic EF-Tu (Negrutskii & Elskaya, 1998 ; Sprinzl, 1994
). eEF1A constitutes 14% of all soluble proteins within active cells, being second only to actin with regard to protein abundance (Slobin, 1980
). eEF1A is essential for cell viability by virtue of its role in the formation of every peptide bond during protein translation. In addition to this primary role, eEF1A has secondary functions, including the binding and bundling of actin (Condeelis, 1995
; Murray et al., 1996
; Yang et al., 1990
), microtubule severing (Shiina et al., 1994
), protein degradation mediated through ubiquitin-dependent pathways (Gonen et al., 1994
) and association with ribonucleoprotein complexes (Kruse et al., 1998
). Taken together, these roles implicate eEF1A in the global regulation of mRNA translation, stability of expressed proteins and cytoskeletal organization. The astounding versatility of this highly abundant and conserved cellular protein renders it attractive for recruitment by the virus replication machinery. Further insight into the role of this interaction in virus replication may be provided by its manipulation in cell-free BVDV replication systems. Ultimately, functional evidence of the significance of the interaction would be provided by mapping the critical residues of NS5A for the interaction with eEF1A, followed by phenotypic analyses of viruses bearing mutations in these residues obtained through reverse genetics.
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Acknowledgments |
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Footnotes |
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c Present address: Department of Virology and Molecular Biology, St Jude Childrens Research Hospital, 332 North Lauderdale St, Memphis, TN 38105, USA.
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Received 31 May 2001;
accepted 23 August 2001.