- Open Access
Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1
© Milev et al; licensee BioMed Central Ltd. 2010
- Received: 22 November 2009
- Accepted: 10 May 2010
- Published: 10 May 2010
Human immunodeficiency virus type 1 (HIV-1) uses cellular proteins and machinery to ensure transmission to uninfected cells. Although the host proteins involved in the transport of viral components toward the plasma membrane have been investigated, the dynamics of this process remain incompletely described. Previously we showed that the double-stranded (ds)RNA-binding protein, Staufen1 is found in the HIV-1 ribonucleoprotein (RNP) that contains the HIV-1 genomic RNA (vRNA), Gag and other host RNA-binding proteins in HIV-1-producing cells. Staufen1 interacts with the nucleocapsid domain (NC) domain of Gag and regulates Gag multimerization on membranes thereby modulating HIV-1 assembly. The formation of the HIV-1 RNP is dynamic and likely central to the fate of the vRNA during the late phase of the HIV-1 replication cycle.
Detailed molecular imaging of both the intracellular trafficking of virus components and of virus-host protein complexes is critical to enhance our understanding of factors that contribute to HIV-1 pathogenesis. In this work, we visualized the interactions between Gag and host proteins using bimolecular and trimolecular fluorescence complementation (BiFC and TriFC) analyses. These methods allow for the direct visualization of the localization of protein-protein and protein-protein-RNA interactions in live cells. We identified where the virus-host interactions between Gag and Staufen1 and Gag and IMP1 (also known as VICKZ1, IGF2BP1 and ZBP1) occur in cells. These virus-host interactions were not only detected in the cytoplasm, but were also found at cholesterol-enriched GM1-containing lipid raft plasma membrane domains. Importantly, Gag specifically recruited Staufen1 to the detergent insoluble membranes supporting a key function for this host factor during virus assembly. Notably, the TriFC experiments showed that Gag and Staufen1 actively recruited protein partners when tethered to mRNA.
The present work characterizes the interaction sites of key components of the HIV-1 RNP (Gag, Staufen1 and IMP1), thereby bringing to light where HIV-1 recruits and co-opts RNA-binding proteins during virus assembly.
- Lipid Raft
- Plasma Membrane Domain
- Bioluminescence Resonance Energy Transfer
- mRNA Reporter
- Lipid Raft Microdomains
HIV-1 replication is characterized by multiple virus-host interactions that represent fundamental events enabling viral propagation. While Gag is central to assembly, numerous host proteins are also required for the generation of infectious HIV-1 particles . The vRNA can both be translated to produce Gag and Gag-Pol or packaged into virions . Gag selects the HIV-1 RNA genome (vRNA) for packaging in the cytoplasm. These events involve the regulated assembly of viral ribonucleoprotein (RNP) complexes. This is a prerequisite for successful retroviral vRNA trafficking from the nucleus into the cytoplasm, through the cytoplasm, and then into progeny virions at sites of assembly [3, 4]. Importantly, recent studies show how vRNA transport mechanisms dictate to what extent both the vRNA is translated and to what efficiency Gag is assembled [5, 6]. Studies also suggest that the host factors that interact with viral Gag and RNA might dictate intracellular trafficking events during viral egress (reviewed in ).
Initially Gag is synthesized as a precursor molecule, but is then cleaved to give rise to matrix (MA), capsid (CA), nucleocapsid (NC), a late domain (p6) plus two spacer peptides SP1 and SP2 during and following virus budding. The protein domains of Gag play distinct roles in the HIV-1 replication cycle (reviewed in ). During the assembly process MA targets Gag to membranes via its myristoylated highly basic N-terminus. Both the CA and the NC domain function in Gag-Gag multimerization [9–11]. Gag drives virion assembly and is sufficient for the organization, budding and release of virus-like particles (VLPs) from cells . The association of Gag to membranes is essential for efficient viral replication. In fact, during viral egress, Gag rapidly associates to membranes that target to assembly sites [13, 14] with the concerted activities of motor  and adaptor proteins [16–18]. Despite numerous studies, the contributions by cellular factors to the transport of Gag towards viral assembly platforms remain poorly understood. Recently, it was demonstrated that Gag preferentially mediates viral assembly at membrane lipid rafts. These are specific detergent-resistant microdomains implicated in multiple cellular processes (reviewed in ). HIV-1, like several other pathogens, also relies on membrane lipid rafts to complete its replication cycle (reviewed in ).
Previously, we demonstrated that Staufen1 interacts with Gag via the NC domain and influences Gag multimerization . Staufen1's presence in the HIV-1 RNP that selectively contains the precursor Gag (pr55Gag) and the vRNA and not any other HIV-1 RNA species [22, 23] and its eventual virion incorporation  promote the idea that Staufen1 has a regulatory role in HIV-1 assembly.
In the present study, we use BiFC analysis  to further characterize and visualize the interactions between Gag and Staufen1. Our results demonstrate that Staufen1 and Gag interact at both intracellular and plasma membrane compartments. In addition, we show that Staufen1 is recruited by Gag to the plasma membrane at lipid raft domains. TriFC analysis also showed that Staufen1 and Gag were able to recruit each other while bound to mRNA. Furthermore, when we depleted cells of Staufen1, multimerized Gag molecules were inefficiently localized to the plasma membrane, indicating that Staufen1 modulates the localization of the assembling Gag. This work provides new information on how HIV-1 co-opts cellular factors to ensure proper viral assembly.
Bimolecular fluorescence complementation (BiFC) to visualize Gag-Staufen1 interactions in live mammalian cells
We then characterized the interaction between Gag and Staufen1. These proteins are known to interact in a RNA-independent manner  and are in close proximity (≈10 nm) as determined by bioluminescence resonance energy transfer experiments [21–23], thus we expected to observe BiFC; but in addition, we wanted to identify the interaction sites for this virus-host pair. We detected small and large robust BiFC signals in the cytoplasm. Furthermore, a close examination of cells revealed that the Staufen1 and Gag BiFC signals coincided with the plasma membrane periphery (Figure 1B, top panels), similar to what was found for Gag. This was observed in over 90% of cells (n > 300) exhibiting BiFC.
Association of Gag and cellular factors at GM1-containing lipid rafts on the plasma membrane
Our earlier reports indicated that Staufen1 associates with vRNA and Gag in both cells and virus [22, 24]. Our recent data suggest that this host protein modulates Gag multimerization on membranes . Gag preferentially mediates viral assembly at specific sites on the plasma membrane called lipid raft microdomains [37, 38] that are composed of cholesterol and sphingolipids, and contain several other components such as ganglioside GM1, glycophosphatidylinositol-anchored (GPI-anchored) proteins, tyrosine kinases of the Src family and others. Because the Gag and Staufen1 interaction occurs also on well defined plasma membrane structures (Figure 1B), we next determined the nature of these interaction domains using BiFC concomitant with live cell lipid raft staining. We transfected cells with Gag-VN and Gag-, Staufen1- or IMP1-VC BiFC constructs, and at 24 hr stained lipid rafts using AlexaFluor 594-labeled cholera toxin subunit B (CT-B) as described in Materials and Methods. As a reference condition for the association and multimerization of Gag on the plasma membrane, we again used Gag-VN and Gag-VC in BiFC (Figure 2A). We observed an almost complete co-localization of CT-B label and oligomerizing Gag, which is in accordance with previously published work [39–41]. Furthermore, BiFC between Gag-VN and Staufen1-VC followed by GM1 labeling revealed that a substantial part of these interactions also occurred at lipid rafts (Figure 2B). Likewise, a proportion of the Gag-IMP1 BiFC signals coincided with lipid raft domains, although as reported above, the plasma membrane localization was not as marked (Figure 2C).
Staufen1's abundance at the plasma membrane was puzzling since it is normally distributed in the cytoplasm co-localizing with the endoplasmic reticulum [26, 42]. Therefore, to determine if Staufen1 is recruited by Gag to lipid raft domains, we co-transfected HeLa cells with the BiFC plasmid pair Gag-VN and Gag-VC, and at 24 hr post-transfection, we fixed and then stained the cells for endogenous Staufen1 (Figure 2D). The BiFC signals between Gag-VN/Gag-VC were observed at the plasma membrane and were preserved following fixation. Notably, abundant staining for endogenous Staufen1 coincided with the majority of the Gag BiFC signals in whole cells (Figure 2D, left panel) and in the expanded region on the right (Figure 2D, Gag-Gag positive cell) whereas in Gag-Gag negative cells, Staufen1 was dispersed in the cytoplasm. Thus, Staufen1 is recruited presumably by Gag to plasma membrane lipid rafts. Finally, we performed a similar analysis for endogenous Staufen1 in Jurkat T cells. Upon expression of Gag-VC and Gag-VN, endogenous Staufen1 coincided with Gag BiFC signals at cell-to-cell contact sites in Gag-expressing Jurkat T cells (Figure 2E).
We also observed Staufen1-Gag BiFC signals at intracellular domains marked by CT-B. These sites appeared to be vesicular in nature and were observed in ~75% of all cells examined (in >200 cells in 6 experiments; Figure 2B, white arrow). These sites of interaction with CT-B staining represent either rapidly internalized raft membrane domains or sites of raft biogenesis/synthesis [43, 44]. To characterize them further, we performed time lapse imaging of live cells. The structures were mostly immobile, but several were dynamic showing characteristics of membranes that were capable of fusion, fission, detachment and subsequent trafficking towards the plasma membrane (Additional file 2: Figure S2). This result suggests that Gag passes through intracellular lipid raft membrane domains on its way to the plasma membrane.
Biochemical fractionation of lipid rafts
Depletion of membrane cholesterol by hydroxy-propyl-β-cyclodextrin (HβCD) reduces Gag-Gag and Gag-Staufen1 membrane BiFC
Effects of modulating Staufen1 levels on the distribution of Gag-Gag BiFC complexes
Trimolecular fluorescence complementation (TriFC) to visualize specific Gag- RNA interactions in living mammalian cells
List of plasmids used in this study.
Source or reference
Addgene plasmid 14437, 
Addgene plasmid 12661, 
Addgene plasmid 12677, 
Addgene plasmid 14434, 
Addgene plasmid 1817, 
TriFC visualization of protein-protein recruitment and interactions on mRNA template
When we tethered MS2-VN and MS2-Staufen1 to the mRNA via MS2BS and expressed Gag-VC, TriFC was readily detected, indicating that Staufen1, when bound to mRNA, recruits Gag (Figure 8D). These results demonstrate that Gag potentially recruits cellular factors while bound to an mRNA; and likewise, RNA-binding host factors can recruit Gag to mRNA. The results also support the notion that the HIV-1 RNP, containing at its core the precursor Gag and the genomic RNA, will selectively engage cellular factors such as Staufen1 and IMP1 during assembly.
Gag-Staufen1 interactions in living cells
Staufen1 was previously found as a component of HIV-1 particles [24, 55]. We have uncovered additional roles for this host factor including one in promoting Gag multimerization and assembly and another in the selection of vRNA for encapsidation [24, 49]. In the present study, we extend our understanding of Staufen1's role in HIV-1 replication by characterizing its interactions with Gag using two powerful, live cell fluorescence complementation techniques. We demonstrate that Staufen1 interacts with Gag, that Staufen1 recruits Gag when it is bound to mRNA and likewise, Gag recruits Staufen1 when bound to the cognate psi packaging signal. The results highlight Staufen1's involvement in the HIV-1 RNP in the assembly of HIV-1.
Recruitment of Staufen1 by Gag to lipid rafts and to mRNA
Our earlier work demonstrated that when we modulated Staufen1 levels in cells, Gag-Gag interactions increased, and these accumulated in detergent-resistant complexes . Here, we used BiFC to evaluate where Staufen1 and Gag interact. This approach, which employs native Gag sequences, has become increasingly popular to visualize Gag in cells . While Staufen1 and Gag are shown to associate in cytoplasmic compartments, our results also reveal that these interactions occur on membranes and at the plasma membrane where Gag multimerizes during assembly [6, 21]. Whereas Staufen1 is usually found to localize on reticulotubular structures and the endoplasmic reticulum , we show that Staufen1 is recruited from the cytoplasm to lipid rafts at the plasma membrane where it interacts with Gag. Consistently, Staufen1 co-localizes with Gag and vRNA at this location , and the distribution of the Staufen1-interacting partner, UPF1, also shuttles to the plasma membrane domain in HIV-1 expressing cells . Here, Gag expression alone appears to be the driving force behind Staufen1 recruitment to GM1-positive plasma membrane domains that serve as the main platforms for viral assembly . Gag's ability to recruit host factors and bind psi RNA concomitantly, via the same protein domain (NC), reveals a rather multifaceted property of Gag and further strengthens the implication of Staufen1 in HIV-1 assembly . Furthermore, time-lapse confocal imaging showed that Gag-Staufen1 foci are mobile and dynamic and are able to merge with similar structures and separate over time into smaller particles that traffic to and anchor at the plasma membrane (Additional file 2: Figure S2). Consistently, distinct populations of Gag and Staufen1 (and the vRNA) traffic on endosomal membranes in cells [13, 14]. Moreover, the Gag-vRNA interactions as well intermediary Gag assembly domains have been found in juxtanuclear domains [15, 58–60]. Thus, we propose that Staufen1 is hijacked by Gag shortly after its synthesis in order to assist in trafficking and assembly.
There are potentially two caveats with respect to the TriFc method used here. First is the use of codon-optimized hGag, that when expressed, could result in deviations in transport and assembly. Nevertheless, several recent studies have utilized Rev-independent hGag expressors to uncover new information on intracellular Gag trafficking and Gag interacting partners [6, 29, 56]. Furthermore, we show that by employing a Rev-dependent Gag expressor in a modified TriFC analyses nearly identical TriFC signals were obtained, revealing that codon-optimization of hGag did not have a significant bearing on the results. The second caveat is the possibility for an alternative interpretation that includes the order in which the protein-protein and protein-RNA interactions occur. We are claiming that either Gag or Staufen1 recruits the other partner while bound to mRNA, and this is supported by BiFC and endogenous Staufen1 staining (Figure 2). However, the bimolecular interaction between Gag and Gag and that found between Staufen1 and Gag could represent the initial event after which the bimolecular complex co-traffics to the mRNA substrate. Even though the KD for MS2-MS2BS interaction is in the low nanomolar range, further work will be necessary in order to confidently differentiate between some of these possibilities.
Involvement of Staufen1 in the anterograde trafficking of Gag
We recently showed that a population of Staufen1 associates along with Gag and vRNA on endosomal membranes . Likewise, RNPs translocate within cells by making use of machineries that direct traffic of cellular membranes and vesicles (reviewed in ). In this study we wanted to characterize the possible roles of Staufen1 in the transport and distribution of multimerizing Gag-Gag that we can readily visualize in live cells. To resolve this, we modulated Staufen1 expression levels in cells that expressed Gag-VN/Gag-VC. When we depleted Staufen1, Gag-Gag BiFC signals were found at the plasma membrane but also in the cytoplasm at juxtanuclear regions on what appeared to be endosomal membrane vesicles. We suspect that the Gag-Gag BiFC signals are due to coalescing HIV-1 RNPs that tether to endosomal membrane populations that may not traffic properly. These were identified in earlier work  and were found to form in an HIV-1-dependent manner containing Staufen1, Gag (and Gag multimers) and vRNA reactivity later named Staufen1 HIV-1-dependent ribonucleoproteins (SHRNPs; ). The data shown here are consistent with the enhanced Gag-Gag multimerization in Staufen1-depleted cells that we found using another biophysical technique (bioluminescence resonance energy transfer) , but also reveal some of the principal locations of these events (at juxtanuclear and raft domains) [22, 49]. It is possible that, during viral egress, Gag mediates the formation of its own mixed type of membranes bearing several endosomal marker proteins similar to those that other viruses engineer . Of interest are data from recently published work in which the accumulation of HIV-1 Gag and cholesterol-enriched membranes was observed in juxtanuclear late endosomal compartments in Niemann-Pick type C1-deficient cells . Furthermore, the targeted depletion of the suppressor of cytokine signalling 1 reduced the transport of Gag to the plasma membrane, leading to accumulations of it near the nucleus . In addition, strong co-localization between another late endosomal marker, CD63, and Gag was observed as a result of Rab9 depletion . These domains appear to be important for steps in assembly including Gag-Gag and Gag-vRNA associations [13, 58, 59], vRNA encapsidation  and Gag trafficking [60, 66] and in one case, for Gag degradation . Importantly, we showed that the cytoplasmic accumulations of Gag did not form because of endocytosed Gag or viral particles derived from the plasma membrane (Additional file 4: Figure S4). These results suggest that the Staufen1 plays roles in the anterograde transport of Gag, but is also a host factor involved in the formation of the HIV-1 RNP during viral egress and assembly.
In the present study, we demonstrate that the intermolecular associations between the host mRNA-binding protein Staufen1 and HIV-1 Gag occur in the cytoplasm and at the plasma membrane in HeLa cells and T lymphocytes. Furthermore, we demonstrate that Staufen1 is recruited by Gag to lipid raft microdomains and present results that indicate Staufen1 has potentially novel functions in the intracellular trafficking of HIV-1 Gag during viral egress.
Cell culture and transfections
HeLa cells and Jurkat T cells were maintained under standard conditions at 37°C with in Dulbecco's modified Eagle's medium or RPMI, respectively, supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 mg/ml streptomycin (Invitrogen). Lipofectamine 2000 (Invitrogen) was used for the DNA transfections of Jurkat T cells or HeLa cells according to the manufacturer's protocol. 1 day before transfection 1.5-1.8 × 105 or 4-5 × 105 HeLa cells were seeded in 6-well plates or 60-mm dishes, respectively to have ~60% confluency when transfected. 106 Jurkat T cells were transfected in 25 ml tissue culture flasks.
BiFC and TriFC analysis
For the BiFC and TriFC experiments, HeLa cells were plated on poly-D-lysine-coated 18 mm micro-glass coverslips (VWR) or 40 mm Bioptechs cover glasses 20-24 hr prior to transfection. Cells were transfected with 1.5 μg to 3 μg plasmid DNA per 18 mm and 3 μg to 5 μg per 40 mm depending on the design of the experiment. Jurkat T cells were transfected in suspension. At 24 hr post-transfection coverslips (HeLa cells) were mounted on a Bioptechs FCS3 imaging perfusion chamber (Bioptechs, Inc.), continuously perfused with fresh medium and warmed using a heating element (Bioptechs, Inc.). For Jurkat T cells, cell adherence to coverslips was promoted using poly-D-lysine (Sigma-Aldrich, Inc). Live cell imaging was performed at 37°C using either Zeiss Pascal LSM5 confocal microscope (Carl-Zeiss, Inc.) with 63×1.3 oil immersion objective or inverted Leica fluorescence microscope. Time-lapse images were captured using 63×1.3 oil immersion objective mounted on a motorized Leica microscope equipped with a PerkinElmer ERS spinning disk confocal system with heated stage and chamber to maintain the cells at 37°C and CO2. Images were collected at the times shown or as stated otherwise, for the indicated period. Two-dimensional data sets were deconvoluted using AutoDeblur (MediaCybernetics, Inc.) and compiled using Imaris 6.3.1 software (Bitplane, Inc.). In some experiments, BiFC and TriFC signals were measured in two-dimensional single confocal planes using Imaris software.
Immunofluorescence and Fluorescence in situhybridization (IF/FISH) analyses
Laser scanning confocal microscopy was performed using a Leica microscope equipped with a PerkinElmer ERS spinning disk or a Carl-Zeiss LSM5 Pascal confocal microscope . Combined 2- and 3-colour IF/FISH co-analyses were performed exactly as described . Images were captured at 512×512 or at 1024×1024 pixels resolution.
SDS-PAGE, Western blot analysis and antibodies
SDS-PAGE and western blotting were performed as described earlier . Antibodies used included: rabbit anti-Caveolin-1 (Santa Cruz); rabbit anti-Tuberin (TSC1, Abcam); rabbit anti-p24 (Intracell); mouse anti-p24 (NIH); rabbit anti-IMP1 (gift from Finn Nielsen, Rigshospitalet, Copenhagen, Denmark); mouse or rabbit anti-Staufen1 (gifts from Luc DesGroseillers, Université de Montréal, Canada and Graciela Boccaccio, University LeLoir, Argentina); rabbit anti-ABCE1 (a gift from Jais Lingappa, University of Washington); mouse anti-GFP (Roche) and mouse anti-Luciferase (Sigma-Aldrich).
Lipid rafts staining
To visualize the distribution of insoluble membrane microdomains we used Vybrant Lipid Raft Labeling Kit (Invitrogen) according to the manufacturer's protocol. The method relies on the binding of a red-fluorescent AlexaFluor 594 conjugate of cholera toxin subunit B (CT-B) to the pentasaccharide chain of plasma membrane ganglioside GM1 (a lipid raft marker). An antibody that recognizes CT-B is then used to cross-link the CT-B-labeled lipid rafts into distinct patches on the plasma membrane, which we visualized by microscopy. The coverslips with stained live cells were mounted on Bioptechs FCS3 live cell perfusion chamber and were visualized by laser scanning confocal microscopy.
Isolation and analysis of detergent-free lipid rafts
We used a simplified method for the fractionation of lipid rafts that does not require the use of detergent . All procedures were carried out with RNAse-free equipment and materials and on ice. Briefly, for each of the cases, two 175 cm2 of HeLa cells were transfected with corresponding plasmids. 24 hr later they were washed 3 times and scraped into buffer B1 (20 mM Tris-HCl pH7.8, 250 mM sucrose, 1 mM CaCl2, 1 mM MgCl2 and RNAse out (Invitrogen) 1 μl/5 ml), centrifuged for 5 min at 1500 rpm and resuspended again in 1 ml of buffer B1 containing complete protease inhibitor cocktail (Roche). The cells were homogenized in RNAse free 1.5 ml Eppendorf tubes with plastic pestles, centrifuged at 1000 rpm for 10 min and the supernatant (S1) was collected. The pellet was lysed in 1 ml of B1 and homogenized again. Following centrifugation at 500 × g for 10 min, the second supernatant (S2) was collected and mixed with S1 (total ~2 ml). 2 ml of 50% OptiPrep (diluted in B1 without Ca2+ and Mg2+ to reach 50%) was added to S1 and S2. The resulting 4 ml of 25% OptiPrep mixture was first poured in 12 ml centrifugation tubes (Beckman Coulter). A step gradient 0-20% was then created using 1.6 ml of 20%, 15%, 10%, 5% and 0% OptiPrep mixtures. The samples were centrifuged in a Beckman ultracentrifuge for 90 min at 52 000 × g with rotor SW41. After centrifugation 0.66 ml fractions (18 in total) starting from the top of the tube were collected and 150 μl were used for western blot analysis. Optical densities of resultant bands were quantified using ImageJ software (freeware from the NIH) as described .
For the complete depletion of cellular cholesterol in live cells, cells were first stained with Vybrant Lipid Raft Labeling Kit (Invitrogen). Coverslips were then mounted in Bioptechs environmental chamber and by perfusion DMEM medium was exchanged with DMEM containing 30 mM 2-hydroxypropyl-β-cyclodextrin (HβCD) . Pictures were taken at t = 0 min before addition HβCD and at different time points after addition as indicated in the figures.
Plasmid expression constructs and siRNAs
The plasmids pMS2-Venus, pMS2-VN, pStaufen1-VC, pIMP1-VC, pFMRP-VC, pGL3-basic/site and pGL3-βActin zipcode have been described previously . To generate pMS2-Staufen1, pMS2-humanized(h)Gag and pMS2-Gag(C36S) vectors Staufen1, hGag and Gag(C36S) sequences were amplified by PCR from pcDNA-Staufen1-TAP , pCMV55M1-10  and pNL4.3(C36S)  and replaced the VN sequence from pMS2-VN between XhoI and NotI. To generate phGag-VenusC and pGag(C36S)-VenusC, hGag and Gag(C36S) (amplified from pNL4.3(C36S) ) PCR fragments replaced IMP1 sequence from pIMP1-VenusC between NheI and XhoI. The Rev-responsive Gag expressor, pSVGagRRE-R was generously supplied by David Rekosh (University of Virginia, U.S.A.; ).
For the generation of mRNA reporter expressing plasmids pGL3MS2site-psi or pGL3MS2site-Delta-psi the fragments containing vRNA packaging signal psi (52 bp) and delta psi (33 bp) were amplified from plasmids HxBRU and plasmid pHXBAP1  and cloned in pGL3MS2site/basic vector between NheI and XhoI. All plasmids were purified using the Sigma GeneElute Maxi-prep kit. pCMV-Rev was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. The complete list of plasmids used in this work is given in Table 1.
We thank Graciela Boccaccio, Luc DesGroseillers, Ronald Montelaro, Jing Jin, Jaisri Lingappa, David Rekosh and Finn Nielsen for gifts of plasmids and antibodies; Oliver Rackham for contributions to the generation of plasmids; Laurent Chatel-Chaix for helpful discussions; Bashar Ghoujal for constructive comments on the manuscript and Lara Ajamian for scientific input. A.J.M. is the recipient of a Canadian Institutes of Health Research (CIHR) New Investigator and Fonds de la recherche en santé du Québec Chercheur-boursier Senior career awards. This work was supported by grants from the New Zealand Health Research Council (grant #05/195) to Warren Tate, Elizabeth Poole and C.M.B., the Canadian Foundation for Innovation (project #6848) and the CIHR (#MOP-38111 and #MOP-56974) to A.J.M.
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