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Journal of Virological Methods 173 (2011) 203–212

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

etecting protein–protein interactions in vesicular stomatitis virus using aytoplasmic yeast two hybrid system

egan Moerdyk-Schauwecker, Darla DeStephanis, Eric Hastie, Valery Z. Grdzelishvili ∗

epartment of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA

rticle history:eceived 6 December 2010eceived in revised form 3 February 2011ccepted 7 February 2011vailable online 12 February 2011

eywords:rotein interaction

a b s t r a c t

Protein–protein interactions play an important role in many virus-encoded functions and in virus–hostinteractions. While a “classical” yeast two-hybrid system (Y2H) is one of the most common techniques todetect such interactions, it has a number of limitations, including a requirement for the proteins of interestto be relocated to the nucleus. Modified Y2H, such as the Sos recruitment system (SRS), which detectinteractions occurring in the cytoplasm rather than the nucleus, allow proteins from viruses replicatingin the cytoplasm to be tested in a more natural context. In this study, a SRS was used to detect interactionsinvolving proteins from vesicular stomatitis virus (VSV), a prototypic non-segmented negative strand RNA

ytoplasmic yeast two hybridesicular stomatitis virusegative strand RNA virusost protein

(NNS) virus. All five full-length VSV proteins, as well as several truncated proteins, were screened againsteach other. Using the SRS, most interactions demonstrated previously involving VSV phosphoprotein,nucleocapsid (N) and large polymerase proteins were confirmed independently, while difficulties wereencountered using the membrane associated matrix and glycoproteins. A human cDNA library was alsoscreened against VSV N protein and one cellular protein, SFRS18, was identified which interacted with

tem p

N in this context. The sysviruses.

. Introduction

Protein–protein interactions are essential for many biologicalunctions and are also involved in host–pathogen interplay. Manyteps of virus replication (e.g., virus genome replication and tran-cription, virion assembly) involve protein complexes comprisedf virus-encoded proteins. Furthermore, all viruses depend heav-ly on host cell functions for their replication, while, at the sameime, cellular innate immune components respond to and combathe invading virus. Although direct contacts between viral and hostell components are not necessary for all aspects of these processes,direct, physical interaction between a viral and host protein may

ndicate these proteins are influencing each other in a way thatffects viral replication.

Multiple biochemical and cell based methods have been devel-ped for the detection of protein–protein interactions (reviewed

ecently in Guan and Kiss-Toth, 2008; Lalonde et al., 2008; Mendez-ios and Uetz, 2010; Miernyk and Thelen, 2008). A “classical”east two-hybrid (Y2H) assay (Fields and Song, 1989) using arotein–protein interaction to reconstitute a functional transcrip-

∗ Corresponding author at: Department of Biology, University of North Carolinat Charlotte, 9201 University City Blvd., Charlotte, NC 28223, USA.el.: +1 704 687 7778; fax: +1 704 687 3128.

E-mail address: vzgrdzel@uncc.edu (V.Z. Grdzelishvili).

166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2011.02.006

resented can be redesigned easily for studies in other less tractable NNS

© 2011 Elsevier B.V. All rights reserved.

tion factor has been, and continues to be, one of the mostcommon techniques used to detect protein–protein interactions.The yeast Saccharomyces cerevisiae can be grown quickly, easilyand inexpensively and outcomes can be measured readily throughuse of reporter genes such as �-galactosidase. These featuresmake the Y2H amenable to high-throughput applications suchas cDNA library screens and mapping whole organism interac-tomes (reviewed in Parrish et al., 2006), including those of viruses(Flajolet et al., 2000; Fossum et al., 2009; McCraith et al., 2000;Uetz et al., 2006) and viruses and their host (Calderwood et al.,2007; de Chassey et al., 2008). However, the classical Y2H sys-tem also has a number of limitations, including a large numberof false positives due to cryptic transcription activation domainsand a requirement for the proteins of interest to be relocated tothe nucleus (Causier and Davies, 2002). Y2H variants (reviewedin Bao et al., 2009; Suter et al., 2008) have been developed toaddress many of these limitations including systems designedto analyze protein–protein interactions in a cytoplasmic context.Among these is the Sos recruitment system (SRS) first described byAronheim et al. (1997). SRS utilizes a S. cerevisiae strain cdc25Hwith a point mutation in the CDC25 gene making it tempera-

ture sensitive, and takes advantage of the fact that this defectcan be complemented by its human homolog, son of sevenlesshomolog 1 (Sos1), but only when that protein is recruited to thecellular membrane. In this system, one protein (a “prey”) is fusedto a myristoylation signal (Myr), causing it to be directed to the

204 M. Moerdyk-Schauwecker et al. / Journal of Virological Methods 173 (2011) 203–212

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ig. 1. (A) Overview of the Cytotrap yeast two-hybrid system based on the Sos rcreen viral or HeLa cell baits cloned into the pMyr plasmid (pSos and pMyr diagraomplement a temperature-sensitive mutation, allowing yeast to grow at the non-

nner surface of the cellular membrane where it has the poten-ial to interact with and recruit a second protein (a “bait”) fusedo a fragment of the Sos protein. Thus, if there is an interaction,rowth can occur at the non-permissive temperature (Fig. 1A). Foriruses with a cytoplasmic site of replication, it has the advantagef allowing proteins to be tested in the natural replication environ-ent.The use of the SRS for detecting protein interactions involving

nonsegmented negative-strand RNA (NNS) virus was evalu-ted for the first time using vesicular stomatitis virus (VSV,amily Rhabdoviridae). VSV is a prototypic NNS virus, with itseplication occurring exclusively in the cytoplasm, and it has

small genome, encoding only five proteins (Fig. 1B). In thistudy, all five VSV proteins, including some protein fragments,ere screened against each other using the SRS. Results of

his assay were compared to those achieved previously usingclassical Y2H or other methods of detecting protein–protein

nteraction. Furthermore, potentially functional viral baits weredentified for use in screening a human cDNA library for viral–hostrotein interactions and the results of one such screen are pre-ented.

Since NNS viruses (all belong to the order Mononegavirales)hare a similar genome structure and virus replication schemeLamb and Parks, 2007; Lyles and Rupprecht, 2007), these stud-es on VSV should be relevant to other members of this order.

he system presented can be redesigned easily for studies onrotein–protein interactions in other less tractable NNS virusessuch as Ebola, Nipah and rabies), for which the use of mammalianxperimental systems is more challenging due to obvious biosafetyssues.

ent system. Viral protein baits were cloned into the pSos plasmids and used toe from the Stratagene CytoTrap instruction manual). Protein–protein interactions

ssive temperature. (B) Schematic diagram of the VSV genome.

2. Materials and methods

2.1. Plasmids

The pSos and pMyr vectors, used for expression of Sos1 and Myrfusions, respectively, provided in the CytoTrap Two-Hybrid Systemkit (Stratagene) were amplified by transformation into chemicallycompetent DH5� Escherichia coli cells and subsequent purificationusing a HiSpeed Maxi Prep kit (Qiagen). All other control plasmidscontained in the kit were used as supplied.

Insert sequences were PCR amplified from the plasmidpVSVFL(+)g.1 which contains a complete cDNA copy of the VSV(Indiana strain; IND) antigenome (Lawson et al., 1995). Primers(Table 1) were designed to be complimentary to the target sequenceas well as introduce the desired restriction sites. PCR was conductedusing the Phusion high-fidelity DNA polymerase (Finnzymes) inaccordance with the manufacturer’s instructions. PCR products andvectors were then digested with the indicated restriction enzymes(New England Biolabs), gel purified, ligated utilizing T4 DNA lig-ase (Promega) and transformed into chemically competent JM109E. coli with selection for successful transformants using media con-taining 100 �g/ml ampicillin or 30 �g/ml chloramphenicol for pSosand pMyr constructs, respectively. Transformants were amplifiedand plasmids extracted with commercial kits in accordance withmanufacturer instructions. Plasmid inserts were sequenced using

the primers indicated in Table 1 to confirm a correct sequence andin-frame insertion.

Site directed mutagenesis of the M gene was carried out usingan overlap extension PCR strategy as in Higuchi et al. (1988).Mutagenic primers containing the 5′-aatgctgctatacgatcg-3′ positive

M. Moerdyk-Schauwecker et al. / Journal of Virological Methods 173 (2011) 203–212 205

Table 1List of primers used to perform PCR-based cloning and mutagenesis and plasmid sequencing.

Primera Restriction enzymeb Usec

5′-agtaggatccccatgagttccttaaagaagattc-3′ BamHI pSos-Mwt (+)5′-agtagcggccgctttgaagtggctgatagaatcc-3′ NotI pSos-Mwt (−)5′-agtaccatggccatggataatctcacaaaagttcg-3′ NcoI pSos-Pwt, pSos-PDomainI (+)5′-agtagtcgaccagagaatatttgactctcg-3′ SalI pSos-Pwt (−)5′-agtagtcgacccactgggatttctgctctcc-3′ SalI pSos-PDomainI (−)5′-agtaccatggccatgtctgttacagtcaagaga-3′ NcoI pSos-Nwt, pSos-N�10 (+)5′-agtagtcgactttgtcaaattctgacttagc-3′ SalI pSos-Nwt (−)5′-agtagtcgacaattgtcttctctcttaggcc-3′ SalI pSos-N�10 (−)5′agtaggatccccaagttcaccatagtttttccac-3′ BamHI pSos-Gwt(mature) (+)5′-agtagcggccgcctttccaagtcggttcatctc-3′ NotI pSos-Gwt(mature) (−)5′-agtaacgcgtgatggaagtccacgattttgag-3′ MluI pSos-Lwt, pSos-L1-1338 (+)5′-agtagcggccgcatctctccaagagttttcc-3′ NotI pSos-Lwt (−)5′-agtagcggccgcttgtccccacgaaccttcc-3′ NotI pSos-L1-1338 (−)5′-atctgaaggagatgagtgca-3′ BamHId pSos-L1139-2127 (+)5′-agtaacgcgtatctctccaagagttttcc-3′ MluI pSos-L1139-2127 (−)5′-agtagaattcatgagttccttaaagaagattc-3′ EcoRI pMyr-Mwt (+)5′-agtactcgagtttgaagtggctgatagaatcc-3′ XhoI pMyr-Mwt (−)5′-agtagaattcatggataatctcacaaaagttcg-3′ EcoRI pMyr-Pwt (+)5′-agtactcgagcagagaatatttgactctcg-3′ XhoI pMyr-Pwt (−)5′-agtagaattcatgtctgttacagtcaagag-3′ EcoRI pMyr-Nwt (+)5′-agtactcgagtttgtcaaattctgacttagc-3′ XhoI pMyr-Nwt (−)5′-agtagaattcaagttcaccatagtttttccac-3′ EcoRI pMyr-Gwt(mature) (+)5′-agtactcgagctttccaagtcggttcatctc-3′ XhoI pMyr-Gwt(mature) (−)5′-agtacccgggccatggaagtccacgattttgag-3′ SmaI pMyr-Lwt (+)5′-agtactcgagatctctccaagagttttcc-3′ XhoI pMyr-Lwt (−)5′-aatgctgctatacgatcgctcggtctgaaggggaaaggtaagaaatc-3′ pSos-M2-7 mutagenesis (+)5′-cgatcgtatagcagcattcatggggatcctactataactaattttccttgg-3′ pSos-M2-7 mutagenesis (−)5′-aatgctgctatacgatcgggtaagaaatctaagaaattagggatcgcacc-3′ pSos-M8-13 mutagenesis (+)5′-cgatcgtatagcagcattaatcttctttaaggaactcatggggatcc-3′ pSos-M8-13 mutagenesis (−)5′-gagcaaataccaagtcgccagaag-3′ Overlap PCR (+)5′-ccaagaccaggtaccatg-3′ pSos insert sequencing (+)5′-gccagggttttcccagt-3′ pSos insert sequencing, overlap PCR (−)5′-actactagcagctgtaatac-3′ pMyr insert sequencing (+)5′-cgtgaatgtaagcgtgacat-3′ pMyr insert sequencing (−)5′-ggcctttaaacatagggacg-3′ L insert sequencing (−)5′-ttttgaaatacccgacttac-3′ L insert sequencing (+)5′-cccagtgttccgagttatgg-3′ L insert sequencing (+)5′-atctgaaggagatgagtgca-3′ L insert sequencing (+)5′-gggtctaaaacatctgaatctaca-3′ L insert sequencing (+)5′-cctctatctatacaaggtcgtat-3′ L insert sequencing (+)5′-agatgctagagatgcctcca-3′ L insert sequencing (+)

s(pmpupatc

2

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a Nucleotides complimentary to viral sequence shown in bold.b Restriction site introduced for cloning.c (+), forward primer; (−), reverse primer.d Present naturally within viral gene.

trand DNA sequence encoding the NAAIRS amino acid sequenceArmbruster et al., 2001) are shown in Table 1. This sequence canarticipate in either �-helices or �-sheets (Wilson et al., 1985),inimizing the disruption this substitution causes to the overall

rotein structure. The first round of PCR was conducted as abovesing a mutagenic primer paired with the appropriate flankingrimer. Following gel purification, the PCR products were mixednd used as the template for a second round of PCR in combina-ion with the two flanking primers. These PCR products were thenloned into pSos as above.

.2. Media

Minimal synthetic defined (SD) media using either glucoseFisher Chemical) (SD/Glucose) or a combination of galactoseSigma–Aldrich, contains ≤0.01% glucose) and raffinose (Acros)SD/Galactose) as a carbon source were used for all SRS screens.he latter allowed, while the former repressed expression of theyr fusion proteins that are under the control of an inducible

AL1 promoter. Sos fusions are under the control of a constitutiveDH1 promoter so that expression occurs with both media types.mission of uracil (-U) and/or leucine (-L) from the media allowed

or selection of yeast transformed with pMyr and pSos constructs,espectively.

2.3. Fusion protein expression

pMyr constructs were transformed individually into cdc25H(�)cells using a lithium acetate method in accordance with theCytoTrap kit instructions. Transformants were selected by plat-ing on SD/Glucose(-U) and incubating at room temperature (RT;20–24 ◦C). Individual colonies were then inoculated into liquidSD/Galactose(-U) and incubated at RT for 2–3 days with rotation.Prior to protein extraction, yeast were pelleted at 900 × g for 2 minat RT.

In the same manner, pSos constructions were co-transformedwith pMyr-Sos binding protein (SB). SB interacts directly withSos1, allowing this construct to serve as a positive control forgrowth at the non-permissive temperature when paired with anypSos plasmid. Co-transformed yeast were selected by plating onSD/Glucose(-UL) and incubated at RT. Isolated colonies were thentransferred to SD/Galactose(-UL) plates and incubated at 34 ◦C for7 days.

To extract protein, yeast pellets or colonies were resuspended

in a buffer containing 50 mM Tris (pH 8.0), 10 mM MgCl2, 1 mMEGTA, 2 mM EDTA (pH 8.0), 20% glycerol and 15 mM KCl anddisrupted mechanically using acid washed glass beads. An equalvolume of a second buffer was added to give a final concen-

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06 M. Moerdyk-Schauwecker et al. / Journa

ration of 1% SDS, 45 mM HEPES (pH7.5) and 15 mM DTT andhe samples were heated at 95 ◦C for 10 min. Insoluble mate-ial was pelleted at 16,000 × g for 3 min at 4 ◦C and the resultingupernatant combined with an equal volume of 2× SDS–PAGEample buffer. VSV virions purified as in Kalvodova et al. (2009)ere disrupted in RIPA buffer [25 mM Tris–HCl pH 7.6, 150 mMaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS] andsed as a control where indicated. For western blot analysis,he resulting protein samples were separated by electrophore-is on 10% (Myr fusions) or 7.5% (Sos1 fusions) SDS–PAGE gelsnd electroblotted to polyvinylidene difluoride (PVDF) membranes.embranes were blocked using 5% non-fat powdered milk in TBS-[0.5 M NaCl, 20 mM Tris (pH 7.5), 0.1%Tween20], which was

lso used for antibody dilutions. Membranes were first incubatedith 1:1000 mouse monoclonal anti-Sos antibodies (clone 25;D Biosciences), 1:2000 rabbit polyclonal anti-VSV L antibodiesraised against the N-terminal half of the L protein fused to TrpE;anti-VSV-L1-2”) or 1:5000 rabbit polyclonal anti-VSV antibod-es (raised against VSV virions). Detection was with 1:5000 goatnti-mouse or anti-rabbit horseradish peroxidase-conjugated sec-ndary antibodies (Jackson ImmunoResearch) using the Enhancedhemiluminescence Plus (ECL+) protein detection system (GEealthcare).

.4. Screen of viral–viral protein interactions

Specific plasmid pairings were used to transform cdc25H(�)ells and cotransformants were selected on SD/Glucose(-UL) platesncubated at RT. At 8 days post transformation (dpt), five isolatedolonies from each pairing were resuspended separately in 20 �lterile water in a 96-well plate. These yeast suspensions were thenransferred to SD/Glucose(-UL) and SD/Galactose(-UL) single-welllates in 96-well format using a pin-replicator and incubated at the

ndicated temperatures (RT, 30 ◦C, 34 ◦C and/or 37 ◦C). Plates werevaluated for growth at 3, 7 and 14 days after transfer.

.5. Screen of a HeLa cDNA library using pSos-Nwt

The CytoTrap XR HeLa cDNA Library (Stratagene) was ampli-ed in accordance with the manufacturer’s instructions. Cdc25H(�)east were transformed with 40 �g of library plasmids and 40 �gf pSos-Nwt (wt, wild-type), plated onto 150 mm SD/Glucose(-L) plates and incubated at RT to select for cotransformants. Atdpt, yeast colonies were replica plated onto SD/Galactose(-UL)lates using velvet and incubated at 34 ◦C. Colonies appear-

ng on these plates at 4, 8, 11, 14 and 18 days after replicalating were picked manually and transferred to single-wellD/Glucose(-UL) plates in a 96-well arrangement, to repress Myrusion protein expression, and incubated at RT. These putative-ositives were then further screened following the scheme laidut by Aronheim et al. (1997). Briefly, once good growth wasbserved, colonies were transferred to a SD/Glucose(-UL) and aD/Galactose(-UL) 150 mm plate using a pin-replicator and incu-ated at 34 ◦C. Colonies showing growth on SD/Galactose(-UL)ut not SD/Glucose(-UL) at 34 ◦C were rescreened to confirm thisrowth pattern. Plasmid DNA was isolated from putative-positiveeast colonies using hot phenol extraction as in Leeds et al. (1991)nd transformed into chemically competent JM109 E. coli whichere plated onto LB agar plates with 30 �g/ml chlorampheni-

ol to select for bacteria transformed with the pMyr constructs.

lasmids were extracted using the PureYield plasmid miniprepystem (Promega). These plasmids were then sequenced usinghe same primers as for the viral inserts and transformed intodc25H� along with pSos or pSos-Nwt and screened as in Section.4.

rological Methods 173 (2011) 203–212

3. Results

3.1. VSV fusion constructs

The full-length open reading frames of the VSV matrix (Mwt),phosphoprotein (Pwt), nucleocapsid (Nwt) and large polymerase(Lwt) proteins were cloned into both the pSos and pMyr vec-tors. The sequence encoding the mature form of the glycoprotein[Gwt(mature)], lacking the N-terminal 16 amino acids (aa) whichare cleaved off following insertion of the nascent peptide chaininto the endoplasmic reticulum (Irving et al., 1979), was alsocloned into both vectors. Also cloned into pSos were: aa 1–138of P encompassing Domain I of the P protein (PdomainI) (Chen,Ogino, and Banerjee, 2006); N protein with the C-terminal 10 aadeleted (N�10); overlapping fragments of the L protein encod-ing aa 1–1338 (L1–1338) or aa 1139–2127 (L1139–2127); andM protein with aa 2–7 (M2–7) or aa 8–13 (M8–13) mutated toNAAIRS.

Expression of the VSV fusion proteins was confirmed by westernblot (Fig. 2). Some variation in protein expression was seen, withPwt, PdomainI, Lwt and L1–1138 in particular being detected athigher levels (compare products of interest to non-specific bandsin Fig. 2), indicating there may be variability in expression and/orstability between some of these fusion proteins. However, all viralfusion proteins were detected and many of the proteins detectedat lower levels participated in protein–protein interactions withinthe SRS, suggesting lower expression is not necessarily detrimental.Expression of pMyr constructs transformed individually into yeastwas detected after induction of protein expression by incubation inliquid SD/Galactose(-U) at RT. As expected, all proteins were similarin size to their virion encoded counterparts as Myr adds only 15 aawhile only 16 aa are lost from the mature form of G (Fig. 2B and C).This also suggests P is being phosphorylated, as the absence of phos-phorylation would cause a marked protein mobility shift (Barik andBanerjee, 1991, 1992). Interestingly, expression of Sos-fusion pro-teins, encoded by either viral constructs or kit provided controlplasmids, could not be detected using similar transformations ofpSos plasmids alone and incubation in liquid SD/Glucose(-L) at RTdespite gene expression being under the control of the constitu-tive ADH1 promoter (data not shown). However, when yeast wereco-transformed with pMyr-SB and a pSos construct and incubatedon SD/Galactose(-UL) plates at 34 ◦C, the fusion proteins could bedetected using an antibody against Sos1 (Fig. 2A). As the Sos1 pro-tein fragment used in these fusions is itself quite large (1067 aa),little size variation could be seen between most of the viral fusionproteins although all were significantly larger than the Sos frag-ment expressed alone (Fig. 2A).

3.2. Optimization of screening conditions

Initial experiments using a subset of the viral fusions paired witheach other at the recommended non-permissive temperature of37 ◦C, failed to detect several interactions shown previously. There-fore, using the same subset of pairings, the incubation temperaturewas varied from the recommended non-permissive temperatureof 37 ◦C in an attempt to increase sensitivity. At 30 ◦C, signifi-cant growth was seen for all plasmid pairings (including negativecontrols) on SD/Glucose(-UL) as well as SD/Galactose(-UL) mediamaking this condition unsuitable for screening purposes (data notshown). In contrast, by using 34 ◦C as the non-permissive tem-

perature, an increase in the number of pairings showing growthon SD/Galactose(-UL) media was observed, as well as improvedgrowth for most of the interactions detected previously, without asignificant increase in growth on SD/Glucose(-UL) media. Examplesof this observation are shown in Fig. 3.

M. Moerdyk-Schauwecker et al. / Journal of Virological Methods 173 (2011) 203–212 207

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ig. 2. Verification of fusion protein expression. Total protein was extracted froD/Galactose(-UL) agar at 34 ◦C or (B and C) transformed with the indicated plasmidsrotein separation on 7.5% (A) or 10% (B and C) SDS–PAGE gels using anti-Sos, -VSight of the blots.

.3. Screen for viral–viral protein interactions

Using 34 ◦C as the non-permissive temperature, a full screenf all possible pairing of the viral constructs against each others well as the empty vector (pSos or pMyr) and pMyr-SB con-rols was undertaken. As expected, there was no growth at theon-permissive temperature when the pMyr viral constructs wereaired with the “empty” pSos plasmid while all pSos constructsaired with the pMyr-SB positive control showed good growthn SD/Galactose media. There also was no growth at the non-ermissive temperature for pSos empty vector or pSos viral fusionsaired with pMyr empty vector except for Sos1 fusions with the Mroteins. In these cases, growth at the non-permissive temperatureas seen for all pairings regardless of the pMyr construct and even

ccurred on SD/Glucose media, where the Myr fusion protein is notxpressed (Fig. 4B). As Sos1 must be present at the cellular mem-rane for growth to occur at the non-permissive temperature, this

ndicates that M is trafficking to the membrane, thereby relocat-ng the Sos1 protein it is fused to and allowing activation of the

as signaling cascade in the absence of pMyr protein expression.eplacing aa 2–7 (M2–7) or 8–13 (M8–13) of the M protein with thea NAAIRS, mutations that have been shown previously to reduceembrane affinity in eukaryotic cells while retaining at least some

f the capabilities of M to bind VSV nucleocapsids (Dancho et al.,

st (A) transformed with the indicated plasmid plus pMyr-SB then incubated onincubated in SD/Galactose(-U) broth at RT. Immunoblots were performed followingL antibodies as indicated. The expected position of the proteins is indicated to the

2009), allowed differences in growth to be distinguished betweenthe positive and negative controls (Fig. 4B). However, backgroundremained high enough to make these mutant pSos-M fusionsunsuitable for screening against other viral proteins. Growth onSD/Galactose only at the non-permissive temperature did indicateinteractions between pSos-Pwt and pMyr-Nwt, pSos-PdomainI andpMyr-Nwt, pSos-PdomainI and pMyr-Lwt, pSos-Nwt and pMyr-Pwt and pSos-N�10 and pMyr-Pwt (Fig. 4A). Interactions betweenpSos-Gwt(mature), pSos-Lwt, pSos-L1-1338 and pSos-L1172-2127and any of the pMyr viral constructs were not observed (data notshown).

3.4. Screen of a HeLa cDNA library for interactions with VSV-Nwt

Approximately 2.0 × 106 yeasts cotransformed with pSos-Nwtand a pMyr plasmid from a HeLa cDNA library were obtained andscreened. After replica plating onto SD/Galactose(-UL) media at thenon-permissive temperature, 2449 colonies formed. In additionto well defined, independent colonies, a number of tight clus-

ters of small colonies were observed. In all cases tested, yeastsin these clusters contained the same pMyr plasmid (data notshown) and are treated as a single colony. These colonies werefurther screened to eliminate yeast capable of growth on bothglucose and galactose containing media at the non-permissive

208 M. Moerdyk-Schauwecker et al. / Journal of Virological Methods 173 (2011) 203–212

Fig. 3. Temperature optimization. Yeasts were co-transformed with the indicated plasmids, grown initially on SD/Glucose(-UL) at RT, then replica plated to created matchingplates for comparison of growth at 34 and 37 ◦C. Growth on SD/Galactose(-UL) but not SD/Glucose(-UL) is indicative of a protein–protein interaction. For each plasmid pairing,results for 3 of 5 independent transformants are shown. +, protein–protein interaction; −, no interaction.

Table 2Identity of inserts from pMyr plasmids causing non-specific growth at the non-permissive temperature.

Protein Gene NCBI Entrez Gene ID # of colonies Day(s) picked

Natriuretic peptide receptor A/guanylate cyclase A NPR1 4881 11 4,8,11Son of sevenless homolog 1 SOS1 6654 5 4v-Ha-ras Harvey rat sarcoma viral oncogene homolog HRAS 3265 2 4Related RAS viral (r-ras) oncogene homolog 2 RRAS2 22800 2 8Poly (ADP-ribose) polymerase family, member 14 PARP14 54625 2 8

tfC2idmynirtcceatpnNs

Poly (ADP-ribose) polymerase family, member 10 PARP10No sequence obtained N/A

emperature, meaning that growth was independent of the Myrusion protein expression, most likely due to a reversion in theDC25 mutant gene allowing temperature-independent growth.359 colonies were eliminated in the first screen and another 65

n a repeat of the screen, leaving 25 colonies where growth wasependent on Myr fusion protein expression. When pMyr plas-ids isolated from these colonies were transformed back into

east with either pSos or pSos-Nwt, 24 showed growth at theon-permissive temperature when paired with either plasmid,

ndicated that growth is due to properties of the Myr fusion proteinather than an interaction with the VSV N protein. The identi-ies of these proteins, as determined by plasmid insert sequencingombined with PCR screening, are given in Table 2. One insert asso-iated with non-specific growth was unable to be sequenced usingither primer. The plasmid containing the cDNA for splicing factor,rginine/serine-rich 18 (SFRS18; NCBI Entrez Gene ID: 25957) was

he only plasmid that allowed for growth only when paired withSos-Nwt, indicating a specific association with N. Growth at theon-permissive temperature for colonies transformed with pSos-wt and pMyr-SFRS18 was very weak but reproducible (data not

hown).

84875 1 18N/A 1 11

4. Discussion

In this study, the SRS was used to identify interactions of VSVproteins with each other and with host proteins. SRS has been usedpreviously to test interactions of a specific viral protein with otherviral or host proteins (Chomchan et al., 2003; Frischmuth et al.,2004; Kim et al., 2006; Takemoto and Hibi, 2005; Yamanaka et al.,2000); or in the case of hepatitis B virus X protein (Barak et al.,2001; Shamay et al., 2002), tobacco mosaic virus movement protein(Kragler et al., 2003), Papaya ringspot virus helper component-proteinase (Shen et al., 2010) and influenza A virus protein NS1(Zhao et al., 2009) to screen a host protein library. In this report, adetailed feasibility study was conducted to uncover the strengthsand weaknesses of the SRS for defining whole virus interactomesand finding host protein partners for a viral protein. The SRS hasnever been used before for VSV or any other NNS virus.

Results of the screen of the viral protein pairings were mixedwhen compared to previous studies of protein–protein interactionsamong VSV proteins. Overall, it showed a propensity of the SRS tofalse-negatives rather than false-positives, at least for the proteinstested, as not all interactions shown previously were detected but

M. Moerdyk-Schauwecker et al. / Journal of Virological Methods 173 (2011) 203–212 209

Fig. 4. Screen for viral–viral protein interactions. Yeasts were cotransformed with the indicated plasmids, grown initially on SD/Glucose(-UL) at RT, then replica plated asshown with incubation at the non-permissive temperature of 34 ◦C. In (A), growth on SD/Galactose(-UL) but not SD/Glucose(-UL) is indicative of a protein–protein interaction.In (B), growth was seen on both media types for all pairings, including pMyr empty vector, indicating the Sos fusions are trafficking to the cell membrane. For each plasmidpairing, results for 2 of 5 independent transformants are shown. (C) Summary of results: +, protein–protein interaction; −, no interaction; I, growth independent of pMyrexpression.

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o unexpected interactions were seen. This may be one reason whyelaxing the selective conditions by lowering the non-permissiveemperature 34 ◦C allowed for identification of a greater number ofrotein–protein interactions.

For VSV proteins that are entirely cytoplasmic (N, P and L), aumber of documented interactions were confirmed while someere not. P is capable of binding to both free and RNA-bound N pro-

ein although different regions of the P protein are involved in thosenteractions. Binding RNP has been shown to involve P aa from posi-ions 213–263 (Gill et al., 1986; Green and Luo, 2009). In contrast,sing a mammalian two-hybrid assay, both the C-terminal 10 aa ofand residues within Domain I (aa 1–138), have been shown to be

mportant for binding of P to free N protein, although differences inhe relative importance of these domains were seen between theew Jersey and IND strains of VSV. For both strains, phosphoryla-

ion of P was not required for binding to N. In this same system,emoval of the C-terminal 10 aa of the N protein (equivalent to�10) almost abolished completely the interaction with P (Takacsnd Banerjee, 1995; Takacs et al., 1993). Consistent with theseeports for free N, using the SRS Pwt and Nwt were capable of inter-cting with each other regardless of what vector they were clonedn. Furthermore, Pdomain I was capable of binding Nwt. Bindingetween Pwt and N�10 was also detected, although growth ofhese colonies was reduced compared to the other N–P combina-ions, suggesting this deletion weakens the interaction (Fig. 4A).

ammalian two-hybrid has also shown that the N-terminal regionf P, particularly Domain I, is also important for binding of P to the Lrotein, with phosphorylation of P increasing the efficiency of thatinding (Takacs and Banerjee, 1995). In this study, an interactionetween PdomainI and Lwt but not Pwt and Lwt was detected, pos-ibly due to conformational differences between the PdomainI andwt fusion proteins or to differences in phosphorylation state orultimerization. P also self associates to form dimers. Using a tradi-

ional yeast two-hybrid assay, aa 161–210 within the hinge regionf P has been shown to be important for this interaction (Chen et al.,006) and phosphorylation of P appears to be required (Gao andenard, 1995). Using the SRS, no interaction was shown betweenwt proteins. PdomainI would not be expected to participate in Pimerization.

Transformation with a plasmid encoding the VSV M proteinused to Sos1 resulted in growth at the non-permissive tempera-ure when paired with any pMyr construct, including the emptyector, and even occurred when Myr fusion protein expressionas repressed by growth on SD/Glucose(-UL) media (Fig. 4B). This

ndicates that in this case, Sos1 relocalization from the cytoplasmo the plasma membrane, a requirement for growth at the non-ermissive temperature, is not due to a protein–protein interactionut to M protein trafficking to the yeast membrane. This observa-ion is consistent with the previous studies in mammalian cells,emonstrating that M protein expressed on its own can traffico the cellular membrane and cause budding of vesicles from theell surface (Chong and Rose, 1993; Justice et al., 1995). Mutationsn M shown to reduce membrane trafficking in mammalian cells,educed yeast growth at the non-permissive temperature, confirm-ng the observed phenotype is influenced by properties of the Mrotein and demonstrating that these same mutations also reduceembrane trafficking of M in yeast. While further mutation or dele-

ion of residues involved in membrane association may make Msuitable bait for some applications, these mutations have also

een shown to reduce dramatically the association of M with RNPDancho et al., 2009) and were not made. Due to this property, the

Sos-M plasmids could not be used to screen for protein–protein

nteractions. This precluded testing for the interaction of M withtself shown previously (Ge et al., 2010; Graham et al., 2008). How-ver, pMyr-Mwt could be used to test for interactions with otheriral proteins as this fusion is designed to go to the membrane,

rological Methods 173 (2011) 203–212

but no interactions were detected despite the fact that M has beenshown previously to bind RNPs (Chong and Rose, 1993) and Gtrimers (Lyles et al., 1992) in vitro. In addition to the potentialcauses of false negatives common to all proteins used in a Y2Hassay, M has been shown to have a cooperative mechanism of bind-ing to the nucleocapsid (Flood and Lyles, 1999; Lyles and McKenzie,1998) which may have contributed to the failure to detect anyinteractions with these proteins in this assay.

The signal sequence of the G protein was removed prior tocloning into the expression vectors to prevent possible membraneinsertion. Unlike for M, the Sos1-G fusion, did not cause growth inthe absence of a pMyr construct, indicating it did not traffic to themembrane despite G being an integral membrane protein. How-ever, no interactions were seen between either G fusion and anyof the other viral proteins. In addition to its interaction with M,G has been shown previously to form homotrimers (Doms et al.,1987; Roche et al., 2006; Roche et al., 2007). G undergoes exten-sive post-translational modification some of which are necessaryfor proper protein folding and trimer formation (Machamer et al.,1985; Machamer and Rose, 1988). It is possible that in this system,one or more of those modifications is lacking.

Based on the results from the screen of viral protein interac-tions, pSos-Nwt, -Pwt and -PdomainI were identified as potentialbaits to use in the screening of a HeLa cDNA library. Results of thescreen using pSos-Nwt are presented, while future screens usingthe other two baits are planned. The identification of potential cel-lular proteins interacting with the VSV N protein was somewhatencumbered by the large number of colonies with a reversion inthe CDC25 gene (2424 out of 2449). That number may have beenexacerbated by use of the longest incubation time prior to replicaplating recommended by the manufacturer in an attempt to max-imize detection of interactions. A further difficulty was the largenumbers of cellular cDNAs encoding proteins capable of rescuinggrowth even in the absence of an interaction with a Sos fusionprotein (24 out of 25), although development of primers capableof amplifying some of the most common of these inserts by PCRaided the screening process (data not shown). The identity of theseinserts (Table 2) is largely consistent with the functions of theseproteins and previous findings. Trafficking of Sos1 directly to themembrane by means of a Myr tag avoids the need to recruit it viaa protein–protein interaction, while mammalian members of theRas family of proteins have been shown to circumvent the yeastRas signaling system (Aronheim et al., 1997). Natriuretic peptidereceptor A/guanylate cyclase A (NPR1) may allow the initial acti-vation steps to be bypassed as cGMP has been shown to be aseffective as cAMP in activating S. cerevisiae cAMP-dependent pro-tein kinase (Cytrynska et al., 1999), a downstream effector of theRas signaling pathway (Broach and Deschenes, 1990). Why expres-sion of two members of the poly (ADP-ribose) polymerase familyleads to growth at the non-permissive temperature is less appar-ent. While to the authors’ best knowledge, PARP activity has notbeen demonstrated in yeast, many members of this family, includ-ing PARP10, have been shown to be involved in cell cycle regulationin other eukaryotic systems (Chou et al., 2006; Yu et al., 2005).Colonies containing PARP10 or PARP14 were some of the last to beisolated during the initial screen and showed slower growth duringrescreening compared to most of the other non-specific activators(data not shown), indicating the mechanism responsible for growthis likely inefficient.

The only protein found to interact specifically with Nwt inyeast was SFRS18 (also known as SRrp130). This serine/arginine-

rich (SRr) protein is poorly characterized, but has been shown tocolocalize with pinin in nuclear speckles and may be involved inmRNA splicing (Zimowska et al., 2003). Interestingly, several otherSRr proteins have been shown to shuttle continuously betweenthe nucleus and the cytoplasm, suggesting that at least some

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R proteins may function in cytoplasmic processes (Long andaceres, 2009). Moreover, the Swedish human protein atlas projectwww.proteinatlas.org) (Berglund et al., 2008) indicates cytoplas-

ic localization of SFRS18 in many human tissues, suggesting an-SFRS18 interaction in VSV-infected cells is at least hypotheticallyossible, although this would need to be confirmed experimentally.he failure to detect a larger number of proteins interacting withwt is not necessarily an indictment of the SRS as it is unclear howany cellular proteins interact with the N protein. Furthermore,hen N is expressed in E. coli in the absence of P, it tends to form

ggregates (Das and Banerjee, 1993). If the same happens in yeast,t may hinder detection of interactions involving N.

Using VSV as a representative NNS many, although not all, of thenteractions previously shown to occur between the viral proteins

ere confirmed independently, particularly those involving N, Pnd L, which are strictly cytoplasmic. This trend is likely to hold forther viruses used with the SRS in the future. One cellular proteinhat interacts with the VSV N protein in the context of SRS wasdentified, as well as two additional baits for use in future screensf host proteins.

cknowledgements

The authors are grateful to Dr. Sue Moyer (University of Floridaollege of Medicine) for providing VSV reagents for this project.he authors also thank Joshua Stokell, Robert Brown, Sean Vidulich,ames Wang, and Natascha Moestl for providing technical assis-ance. This work was supported by NIH Grant 5R03AI078122 (to V.. G.).

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