research papers
Structural genomics of the Epstein–Barr virus
aEMBL-Grenoble Outstation, BP 181, F-38042 Grenoble CEDEX 9, France, bInstitut de Virologie Moléculaire et Structurale, FRE 2854 CNRS–UJF, BP 181, F-38042 Grenoble CEDEX 9, France, cLaboratoire de Virologie, CHU Michallon, BP 217, F-38043 Grenoble CEDEX 9, France, dWellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, England, and eInstitut Universitaire de France, 103 Boulevard Saint-Michel, F-75005 Paris, France
*Correspondence e-mail: wpb@embl-grenoble.fr
Epstein–Barr virus is a herpesvirus that causes infectious mononucleosis, carcinomas and immunoproliferative disease. Its genome encodes 86 proteins, which provided targets for a structural genomics project. After updating the annotation of the genome, 23 open reading frames were chosen for expression in Escherichia coli, initially selecting for those with known and then supplementing this set based on a series of predicted properties, in particular secondary structure. The major obstacle turned out to be poor expression and low solubility. Surprisingly, this could not be overcome by modifications of the constructs, changes of expression temperature or strain or renaturation. Of the eight soluble proteins, five were crystallized using robotic nanolitre-drop crystallization trials, which led to four solved structures. Although these results depended on individual treatment rather than standardized protocols, a high-throughput miniaturized crystallization screening protocol was a key component of success with these difficult proteins.
Keywords: structural genomics; EBV; Epstein–Barr virus; genome annotation; protein expression.
1. Introduction
Human herpesviruses comprise three subfamilies: (i) α-herpesviruses [herpes simplex viruses (HSV) 1 and 2 and varicella zoster virus (VZV)], (ii) β-herpesviruses [cytomegalovirus (CMV) and human herpesvirus (HHV) 6 and 7] and (iii) γ-herpesviruses, comprising the Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) and Epstein–Barr virus (EBV or HHV4). The last infects the vast majority of the world's human population, establishing and maintaining a lifelong persistence in the infected host.
Primary infection typically occurs in childhood and is frequently asymptomatic. In contrast, a delayed primary infection in adolescents or young adults results in infectious mononucleosis (IM) in approximately half of cases, with symptoms including fever, pharyngitis, lymphadenopathy and splenomegaly. IM is a self-limiting lymphoproliferative disorder characterized by an expansion of EBV-infected B-lymphocytes associated with viral lytic replication in the oropharynx, controlled by a vigorous CD8+ cytotoxic T-cell The majority of cases of acute IM recover, but serious complications can occasionally lead to death. EBV is associated with a number of cancers in the immunocompetent host (Rickinson & Kieff, 1996), in particular Burkitt's lymphoma and nasopharyngeal carcinoma, which are endemic in African and Asian populations (Raab-Traub, 2005). Furthermore, EBV can lead to immunoproliferative disease in immunosuppressed patients, notably those infected with HIV (Rickinson & Kieff, 1996). Currently licensed anti-herpesvirus drugs (acyclovir and related compounds) directed against viral DNA synthesis (Coen & Schaffer, 2003) show little effect against EBV.
EBV is composed of an inner capsid that contains the viral double-stranded DNA genome, surrounded by a membrane carrying various surface ), meaning that EBV has one of the largest genomes of human viruses. The principal viral functions are receptor binding and cell entry, maintenance of latency, nucleotide metabolism, DNA replication and packaging and capsid assembly (Fig. 1a, Table 1). EBV also codes for a number of immune-modulators. Some little-studied proteins shuttle viral particles from the nucleus, the site of viral replication, to the extracellular space and a number of proteins still have no assigned function. With the aim of obtaining insight into the protein functions and in order to identify new drug targets, SPINE (Structural Genomics In Europe) included the structural proteomics of herpesviruses in workpackage 9 (human pathogen targets; see Fogg et al., 2006) and here we report our contribution to this, namely the analysis of a cohort of 23 EBV proteins.
Tegument fills the space between the capsid and the membrane. During the latent stage of infection in B-lymphocytes a very limited set of proteins is expressed. The viral DNA forms a circular episome which is associated with the cellular chromosomes and is replicated by the cellular machinery during cell division. After activation, the infection can switch to the lytic cycle, leading to the expression of the full set of viral proteins and production of viral particles. This complex lifestyle utilizes about 86 predicted proteins (Table 1
‡Expressed. §Structure solved. ¶Purified protein. ††Crystals. ‡‡Soluble protein. |
2. Project design, methods and results
2.1. Target annotation
The project included a major continued effort in protein annotation since the information available in databases [principally SWISS-PROT (Boeckmann et al., 2003) and VIDA (Alba, 2002)] was rather incomplete, in particular for spliced reading frames, or no longer up to date. Our annotation is given in Table 1 with the results on the SPINE targets, together with as much bibliographic information as possible. We identified 86 proteins encoded by the EBV genome. The existence of a few of these remains questionable, owing to alternative splicing. The function of 15 proteins is unknown and could not be inferred from sequence homology or bibliographic information (Table 1, Fig. 1a). In general, little is known about the role of the tegument proteins, even though they have been recently localized unambiguously in the virus particle (Johannsen et al., 2004).
2.2. Target selection
As one aim of the project was to obtain structures of potential new drug targets, we first targeted proteins with known enzymatic activity (11 ORFs; Table 1). Next, proteins were ranked according to several predicted properties. Firstly, they were given priority if they had a high predicted secondary structure by the NSP@ server (Deleage et al., 1997), small size and a high stability index according to the ExPASy ProtParam tool (Gasteiger et al., 2005). Known membrane proteins, surface and proteins involved in the packaging mechanism were omitted in order to avoid redundancy with other teams of the SPINE project. Furthermore, we selected against components of known multi-protein assemblies and eliminated proteins containing transmembrane domains using the DAS software (Cserzo et al., 1997) and the TMHMM server (Krogh et al., 2001) available from the ExPASy web site.
2.3. Cloning and protein production
We opted for a small-scale parallel approach using simple restriction-based cloning into a vector containing a tobacco etch virus protease (TEV) cleavable N-terminal His6 tag, allowing the targets to be closely followed through purification and crystallization.
2.3.1. Cloning and expression tests
The selected genes were cloned by PCR amplification of EBV DNA extracted from the B95-8 cell line using primers introducing restriction sites at the 5′ and 3′ ends of the gene and ligated into the pPROEX-HTb plasmid (Invitrogen) using standard methods. The PCR products were cloned between NcoI or BamHI sites as a first choice, EcoRI as a second choice and HindIII or XhoI sites. The ligated products were directly transformed into Escherichia coli BL21(DE3) GOLD cells (Invitrogen), which were used for both DNA preparation for sequencing and small-scale expression tests. DNA preparation was performed either manually or automatically on the RoBioMol platform at the IBS (Grenoble). Small-scale expression tests used 1 ml LB media inoculated with single colonies. Protein production was induced with 0.5 mM isopropyl β-D-thiogalactoside and continued for 3–5 h at 310 and 303 K and overnight at 296 and 289 K. Cells were lysed with BugBuster (Novagen). Protein solubility was checked on SDS–PAGE by loading both the cell extract and the soluble fraction after centrifugation at 18 000g for 20 min. If soluble protein was not detected, the E. coli strains Rosetta, Origami, BL21 (DE3) STAR (Invitrogen), C41 and C43 (Avidis) were tested with overnight induction at 289 K.
2.3.2. Protein expression and purification
Proteins were produced using either classical LB or an auto-inducible medium (Studier, 2005). Cells were lysed by sonication and cell debris was removed by centrifugation at 30 000g for 30 min. The supernatant was loaded onto an Ni–NTA (Qiagen) column equilibrated with 20 mM Tris–HCl pH 7.5, 100 mM NaCl and 20 mM imidazole, washed using the same buffer containing 50 mM imidazole and eluted at an imidazole concentration of 500 mM. After buffer exchange back to the loading buffer, the protein was incubated overnight at room temperature with a ratio of 1/100 of recombinant His-tagged TEV protease. This was loaded again on an Ni–NTA column and the of this column was concentrated by ultrafiltration and loaded onto a Superdex S75 or S200 gel-filtration column (GE/Amersham), depending on the protein size.
2.3.3. Refolding
When good expression levels of insoluble protein were obtained, refolding was attempted. Following large-scale production with induction at 310 K for 4–5 h, the protein was purified from inclusion bodies using buffers supplemented with 8 M urea. After purification and concentration to 5 mg ml−1, a 20-fold dilution in refolding buffers was followed by 24 h incubation at 277 K. Refolding buffers varied in salt concentration (0 or 500 mM NaCl), pH (Bis-Tris–HCl pH 5, Tris–HCl pH 7 or Tris–HCl pH 9) or divalent cation contents (10 mM EDTA or 5 mM CaCl2/5 mM MgCl2), leading to 12 different basic conditions. Samples were centrifuged for 15 min at 16 000g and supernatants were assayed for soluble protein either by ammonium sulfate precipitation and SDS–PAGE or by concentration followed by gel filtration.
2.4. Crystallization
Proteins were analyzed by dynamic
(Protein Solutions) prior to crystallization. Crystallization screening was carried out at the High Throughput Crystallization Laboratory of the EMBL Grenoble Outstation (HTX Lab). Typically, 576 conditions were tested per sample using a PixSys4200 robot (Cartesian) and the vapour-diffusion method in CrystalQuick (Greiner Bio-One) 96-well sitting-drop crystallization plates with square wells. Drops contained 100 nl protein solution and 100 nl buffer solution. Crystal Screen, Crystal Screen II, PEG/Ion Screen, Crystal Screen Lite, Natrix, Membfac, Grid Screens and Index Screen (Hampton Research) were used as well as Clear Strategy Screens (Molecular Dimensions). Crystallization plates were stored and automatically imaged by a CrystalMation robot (RoboDesign) including a RoboIncubator and a Minstrel III module. Successful crystallizations were reproduced and refined manually using 1 + 1 µl hanging drops.3. Discussion
A significant bottleneck in the structure-determination pipeline for EBV proteins was obtaining levels of protein expression (16/23) and soluble protein sufficient for crystallization (7/23; Fig. 1b, Table 1), although the success rate at crystallization was unexpectedly high (5/7). Surprisingly, changing the bacterial strain or expression temperature did not increase soluble expression levels compared with our standard protocol using BL21 cells at 303 K. A bioinformatics analysis using secondary-structure prediction (Deleage et al., 1997) and ClustalW-based alignments (Thompson et al., 1994) only rarely suggested obvious truncations. Perhaps as a consequence of this, modification of the constructs by N-terminal and C-terminal truncations, although attempted for the majority of the studied reading frames (Table 1), was successful in only one case, uracil-DNA glycosylase (UNG), where deletion of the N-terminal 24 residues increased expression levels and led to diffraction-grade crystals. The deleted residues may contain a nuclear localization signal based on sequence identity with human UNG2 (Otterlei et al., 1998). Seven soluble proteins were expressed in E. coli: the EBV protease domain, dUTPase, uracil-DNA glycosidase, BHRF1, BLRF2, BDLF1 and a fragment of BMLF1 (EB2), but the last three proteins were unstable after purification. In the case of the dUTPase, the low solubility of the protein necessitated intensive optimization of purification and crystallization conditions (Tarbouriech et al., 2005). Work on the EBV protease domain predated the SPINE project (Buisson et al., 2002). Structural determination of BHRF1 was abandoned despite the existence of small crystals when an NMR structure was reported (Huang et al., 2003). BARF1 was obtained through an external collaboration and expressed in eukaryotic cells (de Turenne-Tessier et al., 2005) before entering our structure-determination pipeline. Protein purification using an N-terminal His6 tag together with a TEV protease cleavage site, sometimes including reliably produced pure protein for crystallization. In line with other unpublished results in SPINE, refolding from inclusion bodies failed to produce soluble protein from any of the 12 cases. However, we subsequently tested expression in insect cells using baculovirus and obtained three soluble proteins from six ORFs. Overall in SPINE the experience has been that viral proteins tend to be more difficult to express in bacterial systems than prokaryotic proteins (e.g. 27% of viral proteins were expressed in E. coli compared with 33–77% of some bacterial proteins; Fogg et al., 2006). It is clear that eukaryotic expression is a real alternative for difficult viral proteins. Crystallization screening used 200 nl sitting drops dispensed robotically and achieved a very high success rate; however, for proteins except BARF1 this required the addition of enzyme inhibitors (Table 2). Crystallographic details for each EBV structure are given in Table 2 and further details on the structure determinations and have been or will be published elsewhere.
‡Method used for MR, SAD, single-wavelength on a heavy-atom derivative. §Buisson et al. (2002). ¶Tarbouriech et al. (2005). ††Tarbouriech et al. (2006). ‡‡Unpublished. |
The study described here highlights the particular problems associated with the application of pipeline technologies to difficult proteins. In this case, EBV proteins were poorly suited to bacterial expression systems and success was dependent on a much more individual approach to protein production. Although a simple pipeline approach with standard protocols is unlikely to be universally applicable for structural determination, pipeline components can be extremely effective, exemplified here by the high-throughput nanolitre crystallization platform. This major breakthrough in crystallization screening undoubtedly contributed to the high crystallization rates observed with the soluble EBV proteins.
Acknowledgements
This work was undertaken as part of the European Union Framework Programme `Quality of Life and Management of Living Resources', Integrated Project SPINE (Structural Proteomics In Europe), contract No. QLG2-CT-2002-00988. We thank Jean-Marie Seigneurin for providing DNA from the B95.8 cell line and support for the project, Florine Dupeux, Benoit Gallet, José-Antonio Marquez, Martin Rower and Thierry Vernet for the operation of high-throughput facilities at the Grenoble Partnership for Structural Biology (PSB), and Lucy Freeman and Lucie Rivail for work on individual proteins. We are grateful to Henri Gruffat and Tadamasa Ooka for help with the genome annotation.
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