research papers\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

Journal logoBIOLOGICAL
CRYSTALLOGRAPHY
ISSN: 1399-0047

Structural genomics of the Epstein–Barr virus

CROSSMARK_Color_square_no_text.svg

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

(Received 15 November 2005; accepted 1 August 2006)

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 enzyme activity 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.

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 immune response. 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[Rickinson, A. & Kieff, E. (1996). Fields Virology, edited by B. N. Fields, Vol. 2, pp. 2397-2446. Philadelphia: Lippincott-Raven.]), in particular Burkitt's lymphoma and nasopharyngeal carcinoma, which are endemic in African and Asian populations (Raab-Traub, 2005[Raab-Traub, N. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 71-92. Norwich: Caister Academic Press.]). Furthermore, EBV can lead to immunoproliferative disease in immunosuppressed patients, notably those infected with HIV (Rickinson & Kieff, 1996[Rickinson, A. & Kieff, E. (1996). Fields Virology, edited by B. N. Fields, Vol. 2, pp. 2397-2446. Philadelphia: Lippincott-Raven.]). Currently licensed anti-herpesvirus drugs (acyclovir and related compounds) directed against viral DNA synthesis (Coen & Schaffer, 2003[Coen, D. M. & Schaffer, P. A. (2003). Nature Rev. Drug Discov. 2, 278-288.]) 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 glycoproteins. 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[link]), 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. 1[link]a, Table 1[link]). 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[Fogg, M. J. et al. (2006). Acta Cryst. D62, 1196-1207.]) and here we report our contribution to this, namely the analysis of a cohort of 23 EBV proteins.

Table 1
Proteins of EBV

Accession, SwissProt, TrEMBL or PIR (Protein Information Resource) accession number. NCBI, GI numbers assigned by NCBI. Function, information on name, synonyms and function of the protein. ORF EBV, name of the EBV open reading frame. F, classification based on the function into C, capsid; M, membrane (glyco)protein; N, nucleotide metabolism; L, latency; P, packaging; R, replication; S, transcription factors, transactivators, signalling; T, tegument. S, an × means translated from spliced messenger RNA. Occ., occurrence in herpesvirus subfamilies, no entry for proteins present only in EBV and very closely related viruses such as rhesus lymphocryptovirus (LCV). Homologue, name of the homologue in HSV, if existing, otherwise of human CMV. N, number of constructs used in the project. St, current status. Proteins with enzymatic activity are shown in bold. For space reasons, only a limited number of references to original work are given; otherwise, review articles are cited. The annotation extensively used the BLAST program at NCBI (Altschul et al., 1997[Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Nucleic Acids Res. 25, 3389-3402.]).

Accession NCBI Function ORF EBV F S Occ. Homologue Reference N St Comment
P03229 gi:140202 Bcl-2 homologue, negative regulator of anti-apoptosis protein BHRF1 BALF1 S   γ   Marshall et al. (1999[Marshall, W. L., Yim, C., Gustafson, E., Graf, T., Sage, D. R., Hanify, K., Williams, L., Fingeroth, J. & Finberg, R. W. (1999). J. Virol. 73, 5181-5185.]), Bellows et al. (2002[Bellows, D. S., Howell, M., Pearson, C., Hazlewood, S. A. & Hardwick, J. M. (2002). J. Virol. 76, 2469-2479.]), Cabras et al. (2005[Cabras, G., Decaussin, G., Zeng, Y., Djennaoui, D., Melouli, H., Broully, P., Bouguermouh, A. M. & Ooka, T. (2005). J. Clin. Virol. 34, 26-34.]) 1 Toxic
P03227 gi:118744 Single-stranded DNA-binding protein, part of replication fork/machinery BALF2 R   αβγ UL29 Decaussin et al. (1995[Decaussin, G., Leclerc, V. & Ooka, T. (1995). J. Virol. 69, 7309-7314.]), Robertson et al. (1996[Robertson, E. S., Ooka, T. & Kieff, E. D. (1996). Proc. Natl Acad. Sci. USA, 93, 11334-11340.])      
P25939 gi:124087 Terminase large subunit/ATPase BALF3 P   αβγ UL28 Alba (2002[Alba, M. M. (2002). VIDA Database. https://www.biochem.ucl.ac.uk/bsm/virus_database/ .]), Hwang & Bogner (2002[Hwang, J. S. & Bogner, E. (2002). J. Biol. Chem. 277, 6943-6948.]), Savva et al. (2004[Savva, C. G., Holzenburg, A. & Bogner, E. (2004). FEBS Lett. 563, 135-140.])      
P03188 gi:138191 Membrane glycoprotein B (gB, gp110) precursor, fusion and co-receptor binding BALF4 M   αβγ UL27 Gong et al. (1987[Gong, M., Ooka, T., Matsuo, T. & Kieff, E. (1987). J. Virol. 61, 499-­508.]), Spear & Longnecker (2003[Spear, P. G. & Longnecker, R. (2003). J. Virol. 77, 10179-10185.])      
P03198 gi:118858 DNA polymerase BALF5 R   αβγ UL30 Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]) 12 Insoluble
Q8AZJ4 gi:23893662 BARF0, nuclear/perinuclear in epithelial cells, start at amino acid 298 of SWISS-PROT, homologue in rhesus LCV BARF0 U ×     Hitt et al. (1989[Hitt, M. M., Allday, M. J., Hara, T., Karran, L., Jones, M. D., Busson, P., Tursz, T., Ernberg, I. & Griffin, B. E. (1989). EMBO J. 8, 2639-2651.]), Gilligan et al. (1991[Gilligan, K. J., Rajadurai, P., Lin, J. C., Busson, P., Abdel-Hamid, M., Prasad, U., Tursz, T. & Raab-Traub, N. (1991). J. Virol. 65, 6252-6259.]), Fries et al. (1997[Fries, K. L., Sculley, T. B., Webster-Cyriaque, J., Rajadurai, P., Sadler, R. H. & Raab-Traub, N. (1997). J. Virol. 71, 2765-2771.])      
P03175 gi:132620 Ribonucleoside reductase, small 38 kDa subunit BaRF1 N   αγ UL40 Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]) 1 No expression
P03228 gi:115128 CD80 homologue (p33), oncogene, soluble glycoprotein, 2 Ig domains, binds CSF-1 BARF1 S       Wei & Ooka (1989[Wei, M. X. & Ooka, T. (1989). EMBO J. 8, 2897-2903.]), Strockbine et al. (1998[Strockbine, L. D., Cohen, J. I., Farrah, T., Lyman, S. D., Wagener, F., DuBose, R. F., Armitage, R. J. & Spriggs, M. K. (1998). J. Virol. 72, 4015-4021.]), Tarbouriech et al. (2006[Tarbouriech, N., Ruggiero, F., de Turenne-Tessier, M., Ooka, T. & Burmeister, W. P. (2006). J. Mol. Biol. 359, 667-678.]) 1 § PDB 2ch8
P03216 gi:136813 Myristoylated phosphoprotein in tegument (MyrP) BBLF1 T   γ   Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
Not in DB Not in DB Primase-associated factor, spliced, full sequence not in DB, part of helicase–primase complex BBLF2/3 R × αβγ UL8 Yokoyama et al. (1999[Yokoyama, N., Fujii, K., Hirata, M., Tamai, K., Kiyono, T., Kuzushima, K., Nishiyama, Y., Fujita, M. & Tsurumi, T. (1999). J. Gen. Virol. 80, 2879-2887.])      
P03214 gi:122807 Helicase, part of helicase–primase complex BBLF4 R   αβγ UL5 Yokoyama et al. (1999[Yokoyama, N., Fujii, K., Hirata, M., Tamai, K., Kiyono, T., Kuzushima, K., Nishiyama, Y., Fujita, M. & Tsurumi, T. (1999). J. Gen. Virol. 80, 2879-2887.]) 1 No expression
P03213 gi:136792 Portal protein UL6 homologue, function by homology BBRF1 P   αβγ UL6 Newcomb et al. (2001[Newcomb, W. W., Juhas, R. M., Thomsen, D. R., Homa, F. L., Burch, A. D., Weller, S. K. & Brown, J. C. (2001). J. Virol. 75, 10923-10932.])      
P29882 gi:267198 Unknown function BBRF2 U   αβγ UL7 Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03215 gi:140278 Glycoprotein M, part of gN–gM complex involved in envelope–tegument interaction BBRF3 M   αβγ   Lake & Hutt-Fletcher (2000[Lake, C. M. & Hutt-Fletcher, L. M. (2000). J. Virol. 74, 11162-11172.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03226 gi:137569 Major capsid protein, MCP, VP5, 155K BcLF1 C   αβγ   Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.])      
P03180 gi:114886 Viral interleukin-10 homologue precursor, vIL-10 BCRF1 S   βγ   Hsu et al. (1990[Hsu, D. H., de Waal Malefyt, R., Fiorentino, D. F., Dang, M. N., Vieira, P., de Vries, J., Spits, H., Mosmann, T. R. & Moore, K. W. (1990). Science, 250, 830-832.])     PDB 1vlk , 1y6m
P25215 gi:136975 Unknown function BcRF1 U   βγ hcmvUL84 Alba (2002[Alba, M. M. (2002). VIDA Database. https://www.biochem.ucl.ac.uk/bsm/virus_database/ .])      
Q8AZK7 gi:7441833 EBNA-Lp (EBNA-5) nuclear phosphoprotein, highly spliced, 12 exons, proline-rich, enhances EBNA-2 transactivation BWRF1 L × γ   Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]), Waltzer et al. (1996[Waltzer, L., Perricaudet, M., Sergeant, A. & Manet, E. (1996). J. Virol. 70, 5909-5915.])      
P25214 gi:139189 Minor capsid protein (mCP), triplex protein HSV-1 VP23 homologue BDLF1 C   αβγ UL18 Trus et al. (2001[Trus, B. L., Heymann, J. B., Nealon, K., Cheng, N., Newcomb, W. W., Brown, J. C., Kedes, D. H. & Steven, A. C. (2001). J. Virol. 75, 2879-2890.]) 1 Purified
P03225 gi:136827 Tegument protein, transmembrane protein? BDLF2 T   γ   Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.]) 5 Insoluble
P03224 gi:138369 Envelope glycoprotein gp150 (gp117) BDLF3 M   γ   Borza & Hutt-Fletcher (1998[Borza, C. M. & Hutt-Fletcher, L. M. (1998). J. Virol. 72, 7577-7582.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03223 gi:136983 Protein (gp115) BDLF4 U   βγ hcmvUL92 Alba (2002[Alba, M. M. (2002). VIDA Database. https://www.biochem.ucl.ac.uk/bsm/virus_database/ .]), Boeckmann et al. (2003[Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Gasteiger, E., Martin, M. J., Michoud, K., O'Donovan, C., Phan, I., Pilbout, S. & Schneider, M. (2003). Nucleic Acids Res. 31, 365-370.])      
Q66541 gi:1334910 Assemblin without protease domain coded by 2nd reading frame BdRF1 C   αβγ UL26.5        
P03203 gi:119113 EBNA-3B nuclear protein (EBNA-4) BERF2a/BERF2b L ×     Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]), Young & Rickinson (2004[Young, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757-768.])      
P03204 gi:119114 EBNA-3C nuclear protein (EBNA-6, EBNA-4B) (−) effect on transactivation, EBNA-2 and cell cycle, essential for immortalization BERF3/BERF4 L ×     Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]), Young & Rickinson (2004[Young, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757-768.])      
P03184 gi:136878 Role in DNA packaging, cytosolic zinc-binding protein, cysteine-rich BFLF1 P   αβγ UL32 Chang et al. (1996[Chang, Y. E., Poon, A. P. & Roizman, B. (1996). J. Virol. 70, 3938-3946.])      
P03183 gi:136873 Nuclear membrane phosphoprotein, part of intracellular virions, egress protein, complex with BFRF1 BFLF2 M   αβγ UL31 Lake & Hutt-Fletcher (2004[Lake, C. M. & Hutt-Fletcher, L. M. (2004). Virology, 320, 99-106.]), Gonnella et al. (2005[Gonnella, R., Farina, A., Santarelli, R., Raffa, S., Feederle, R., Bei, R., Granato, M., Modesti, A., Frati, L., Delecluse, H. J., Torrisi, M. R., Angeloni, A. & Faggioni, A. (2005). J. Virol. 79, 3713-3727.]) 2 Insoluble
P03185 gi:136886 Nuclear membrane protein p38, transmembrane with large cytoplasm domain, complex with BFLF2 BFRF1 M   βγ hcmvUL50 Lake & Hutt-Fletcher (2004[Lake, C. M. & Hutt-Fletcher, L. M. (2004). Virology, 320, 99-106.])      
P14347 gi:140660 Unknown function, not included in virions BFRF2 U   βγ hcmvUL49 Bellows et al. (2002[Bellows, D. S., Howell, M., Pearson, C., Hazlewood, S. A. & Hardwick, J. M. (2002). J. Virol. 76, 2469-2479.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P14348 gi:139195 Smallest capsid protein (sCP) on outer capsid surface, HSV1-VP26 homologue, KSHV ORF 65 homologue BFRF3 C   αβγ UL35 Nealon et al. (2001[Nealon, K., Newcomb, W. W., Pray, T. R., Craik, C. S., Brown, J. C. & Kedes, D. H. (2001). J. Virol. 75, 2866-2878.]), Bowman et al. (2003[Bowman, B. R., Baker, M. L., Rixon, F. J., Chiu, W. & Quiocho, F. A. (2003). EMBO J. 22, 757-765.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
AAA45868 gi:330334 DNA-cleavage and packaging protein, part of the DNA-packaging machinery (BFRF0.5, HS4BAM) BFRF4 P   αβγ UL33 Alba (2002[Alba, M. M. (2002). VIDA Database. https://www.biochem.ucl.ac.uk/bsm/virus_database/ .]), Beard & Baines (2004[Beard, P. M. & Baines, J. D. (2004). Virology, 324, 475-482.])      
P03222 gi:136833 Tegument protein (gp115) BGLF1 T   γ   Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.]) 1 Insoluble
P03221 gi:114952 Tegument protein, MyrPBP BGLF2 T   αβγ UL16 Boeckmann et al. (2003[Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Gasteiger, E., Martin, M. J., Michoud, K., O'Donovan, C., Phan, I., Pilbout, S. & Schneider, M. (2003). Nucleic Acids Res. 31, 365-370.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03220 gi:136988 Homologue to HSV-1 tegument protein, but not included in virion, gp118 BGLF3 U   αβγ UL14 Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P13288 gi:125627 Ser/Thr kinase, phosphorylation of nucleoside analogues, tegument protein BGLF4 N   αβγ UL13 Smith & Smith (1989[Smith, R. F. & Smith, T. F. (1989). J. Virol. 63, 450-455.]), Marschall et al. (2002[Marschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van den Bogaard, M. & Stamminger, T. (2002). J. Gen. Virol. 83, 1013-1023.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.]) 4 Insoluble
P03217 gi:119691 Alkaline exonuclease, involved together with BALF2 in DNA recombination BGLF5 R   αβγ UL12 Cheng et al. (1980[Cheng, Y. C., Chen, J. Y., Hoffmann, P. J. & Glaser, R. (1980). Virology, 100, 334-338.]), Reuven et al. (2004[Reuven, N. B., Willcox, S., Griffith, J. D. & Weller, S. K. (2004). J. Mol. Biol. 342, 57-71.])      
P03219 gi:23893636 DNA-packaging protein, terminase small subunit BGRF1/BDRF1 P × αβγ UL15 Alba (2002[Alba, M. M. (2002). VIDA Database. https://www.biochem.ucl.ac.uk/bsm/virus_database/ .])      
P03181 gi:140778 Proline-rich protein LF3, unknown function, tandem repeats, NotI repeat (125 bp), homologue in rhesus LCV BHLF1 U       Laux et al. (1985[Laux, G., Freese, U. K. & Bornkamm, G. W. (1985). J. Virol. 56, 987-­995.]), Rivailler et al. (2002[Rivailler, P., Jiang, H., Cho, Y. G., Quink, C. & Wang, F. (2002). J. Virol. 76, 421-426.]), Farell (2005[Farell, P. J. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 263-287. Norwich: Caister Academic Press.])      
P03182 gi:119102 Anti-apoptotic factor bcl-2 homologue, early antigen protein R (EA-R), nuclear antigen BHRF1 S   γ   Huang et al. (2003[Huang, Q., Petros, A. M., Virgin, H. W., Fesik, S. W. & Olejniczak, E. T. (2003). J. Mol. Biol. 332, 1123-1130.]) 2 †† PDB 1q59 (NMR)
P03208 gi:138777 G-protein coupled receptor (G-PCR), 7 transmembrane helices, 6 glycosylation sites, 2 disulfide bridges gP64 BILF1 M   γ   Hutt-Fletcher (2005[Hutt-Fletcher, L. M. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 359-378. Norwich: Caister Academic Press.]), Paulsen et al. (2005[Paulsen, S. J., Rosenkilde, M. M., Eugen-Olsen, J. & Kledal, T. N. (2005). J. Virol. 79, 536-546.])      
P03218 gi:138183 Membrane glycoprotein gp55/80, Ig-like, gp78 BILF2 M       Mackett et al. (1990[Mackett, M., Conway, M. J., Arrand, J. R., Haddad, R. S. & Hutt-Fletcher, L. M. (1990). J. Virol. 64, 2545-2552.]), Boeckmann et al. (2003[Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Gasteiger, E., Martin, M. J., Michoud, K., O'Donovan, C., Phan, I., Pilbout, S. & Schneider, M. (2003). Nucleic Acids Res. 31, 365-370.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03211 gi:119110 Latent nuclear protein EBNA-1, assures EBV episome maintenance replication, Gly-rich domain, essential for immortalization BKRF1 L       Bochkarev et al. (1996[Bochkarev, A., Barwell, J. A., Pfuetzner, R. A., Bochkareva, E., Frappier, L. & Edwards, A. M. (1996). Cell, 84, 791-800.])     PDB 1b3t , 1vhi
P03212 gi:140976 Glycoprotein L precursor, gp25, in gL–gH complex involved in viral fusion together with gB BKRF2 M   αβγ UL1 Hutt-Fletcher (2005[Hutt-Fletcher, L. M. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 359-378. Norwich: Caister Academic Press.]), Spear & Longnecker (2003[Spear, P. G. & Longnecker, R. (2003). J. Virol. 77, 10179-10185.])      
P12888 gi:137034 Uracil-DNA glycosylase BKRF3 N   αβγ UL2 Winters & Williams (1993[Winters, T. A. & Williams, M. V. (1993). Virology, 195, 315-326.]) 10 § Publication in preparation
P30117 gi:267499 Tegument phosphoprotein BKRF4 T   γ   Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03200 gi:138368 Envelope glycoprotein gp350 (gp340) initial cell binding through complement receptor 2 (CR2, CD21) BLLF1 M   αγ   Spear & Longnecker (2003[Spear, P. G. & Longnecker, R. (2003). J. Virol. 77, 10179-10185.]), Hutt-Fletcher (2005[Hutt-Fletcher, L. M. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 359-378. Norwich: Caister Academic Press.])      
AAA45880 gi:330361 Envelope glycoprotein gp220, obtained through in-frame splicing from BLLF1 BLLF1b M × αγ   Beisel et al. (1985[Beisel, C., Tanner, J., Matsuo, T., Thorley-Lawson, D., Kezdy, F. & Kieff, E. (1985). J. Virol. 54, 665-674.])      
P03199 gi:140999 Unknown function BLLF2 U   γ          
P03195 gi:118952 dUTP pyrophosphatase, dUTPase BLLF3 N   αγ UL50 Sommer et al. (1996[Sommer, P., Kremmer, E., Bier, S., Konig, S., Zalud, P., Zeppezauer, M., Jones, J. F., Mueller-Lantzsch, N. & Grasser, F. A. (1996). J. Gen. Virol. 77, 2795-2805.]), Tarbouriech et al. (2005[Tarbouriech, N., Buisson, M., Seigneurin, J. M., Cusack, S. & Burmeister, W. P. (2005). Structure, 13, 1299-1310.]) 2 § PDB 2bsy , 2bt1
P03196 gi:141001 Membrane glycoprotein gN, part of the gM–gN complex, part of the envelope–tegument interaction BLRF1 M   βγ hcmvUL73 Lake & Hutt-Fletcher (2004[Lake, C. M. & Hutt-Fletcher, L. M. (2004). Virology, 320, 99-106.])      
P03197 gi:141002 Tegument protein BLRF2 T   γ   Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])   ‡‡ Soluble
P12977 gi:119112 EBNA-3A nuclear protein (EBNA-3), (−) effect on transactivator EBNA-2 and cell cycle, essential for immortalization BLRF3/BERF1 L ×     Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]), Waltzer et al. (1996[Waltzer, L., Perricaudet, M., Sergeant, A. & Manet, E. (1996). J. Virol. 70, 5909-5915.]), Young & Rickinson (2004[Young, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757-768.]) 1 No expression
P03191 gi:119098 Processivity factor, sliding clamp, early antigen protein D (EA-D, polymerase accessory protein) BMRF1 R   αβγ UL42 Tsurumi et al. (1993[Tsurumi, T., Kobayashi, A., Tamai, K., Daikoku, T., Kurachi, R. & Nishiyama, Y. (1993). J. Virol. 67, 4651-4658.])      
P03192 gi:141066 Receptor for cellular integrins, needed for infection of epithelial cells, 10 TM helices, RGD motif BMRF2 M   γ   Modrow et al. (1992[Modrow, S., Hoflacher, B. & Wolf, H. (1992). Arch. Virol. 127, 379-­386.]), Tugizov et al. (2003[Tugizov, S. M., Berline, J. W. & Palefsky, J. M. (2003). Nature Med. 9, 307-314.])      
P03230 gi:126373 Latent membrane protein 1 (LMP-1), interferes with signalling, TRAF-binding through CTAR1 and 2, essential for immortalization BNLF1 L × γ   Young & Rickinson (2004[Young, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757-768.])      
B28918 gi:23893667 Potential membrane protein BNLF2a U       Rivailler et al. (2002[Rivailler, P., Jiang, H., Cho, Y. G., Quink, C. & Wang, F. (2002). J. Virol. 76, 421-426.]), Farell (2005[Farell, P. J. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 263-287. Norwich: Caister Academic Press.])      
A28918 gi:7460898 Potential gp141 BNLF2b U       Rivailler et al. (2002[Rivailler, P., Jiang, H., Cho, Y. G., Quink, C. & Wang, F. (2002). J. Virol. 76, 421-426.]), Farell (2005[Farell, P. J. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 263-287. Norwich: Caister Academic Press.])      
P03179 gi:139165 Major tegument protein (MTP), viral surface protein involved in B lymphocyte binding BNRF1 T   γ   Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.]), Lopez et al. (2005[Lopez, R., Urquiza, M., Patino, H., Suarez, J., Reyes, C., Patarroyo, M. A. & Patarroyo, M. E. (2005). Biochimie, 87, 985-992.])      
P03189 gi:136893 Large tegument protein-binding protein (LTPBP) BOLF1 T   αβγ UL37 Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03187 gi:139172 Minor capsid protein-binding protein (mCP-BP), Triplex protein HSV-1 VP19C homologue BORF1 C   γ UL38 Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03190 gi:132602 Ribonucleoside reductase, large 140 kDa subunit BORF2 N   αβγ UL39 Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]) 2 No expression
P03186 gi:135574 Large tegument protein (LTP) BPLF1 T   αβγ UL36 Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03209 gi:115130 Transcription activator, R transactivator, Rta, dimeric (TAF50) BRLF1 S   γ   Hardwick et al. (1988[Hardwick, J. M., Lieberman, P. M. & Hayward, S. D. (1988). J. Virol. 62, 2274-2284.]), Gruffat et al. (1990[Gruffat, H., Manet, E., Rigolet, A. & Sergeant, A. (1990). Nucleic Acids Res. 18, 6835-6843.]) 3 Insoluble
Not in DB Not in DB Spliced BRLF1-BZLF1 protein, repressor of BZLF1, RAZ BRLF1/BZLF1 S ×     Manet et al. (1989[Manet, E., Gruffat, H., Trescol-Biemont, M. C., Moreno, N., Chambard, P., Giot, J. F. & Sergeant, A. (1989). EMBO J. 8, 1819-1826.]), Furnari et al. (1994[Furnari, F. B., Zacny, V., Quinlivan, E. B., Kenney, S. & Pagano, J. S. (1994). J. Virol. 68, 1827-1836.]), Segouffin et al. (1996[Segouffin, C., Gruffat, H. & Sergeant, A. (1996). J. Gen. Virol. 77, 1529-1536.])      
Not in DB Not in DB Hypothetical BRLF1-BZLF1 splice variant BRLF1/BZLF1b S ×     Farell (2005[Farell, P. J. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 263-287. Norwich: Caister Academic Press.])      
P03207 gi:141395 Enhancement of the induction of the lytic cycle BRRF1 S   γ   Segouffin-Cariou et al. (2000[Segouffin-Cariou, C., Farjot, G., Sergeant, A. & Gruffat, H. (2000). J. Gen. Virol. 81, 1791-1799.]), Hong et al. (2004[Hong, G. K., Delecluse, H. J., Gruffat, H., Morrison, T. E., Feng, W. H., Sergeant, A. & Kenney, S. C. (2004). J. Virol. 78, 4983-4992.]) 1 No expression
P03210 gi:141396 Tegument protein, unknown function BRRF2 T   γ   Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03193 gi:136937 Primase, subunit of the helicase–primase complex, part of the replication machinery BSLF1 R   αβγ UL52 Yokoyama et al. (1999[Yokoyama, N., Fujii, K., Hirata, M., Tamai, K., Kiyono, T., Kuzushima, K., Nishiyama, Y., Fujita, M. & Tsurumi, T. (1999). J. Gen. Virol. 80, 2879-2887.])   No expression
Q04360 gi:1708410 mRNA-export factor (EB2, Mta,SM), mRNA splicing, interaction with human Spen proteins, IE63 (ICP27) homologue BSLF2/BMLF1 S × αβγ UL54 Boeckmann et al. (2003[Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Gasteiger, E., Martin, M. J., Michoud, K., O'Donovan, C., Phan, I., Pilbout, S. & Schneider, M. (2003). Nucleic Acids Res. 31, 365-370.]), Hiriart et al. (2005[Hiriart, E., Gruffat, H., Buisson, M., Mikaelian, I., Keppler, S., Meresse, P., Mercher, T., Bernard, O. A., Sergeant, A. & Manet, E. (2005). J. Biol. Chem. 280, 36935-36945.]), Swaminathan (2005[Swaminathan, S. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 631-649. Norwich: Caister Academic Press.]) 6 In crystallization
P03194 gi:141432 Palmitoylated tegument protein (PalmP) BSRF1 T   αβγ UL51 Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P30119 gi:267575 Capsid maturation, capsid-associated BTRF1 C   αγ UL21 Wagenaar et al. (2001[Wagenaar, F., Pol, J. M., de Wind, N. & Kimman, T. G. (2001). Vet. Res. 32, 47-54.])      
P03233 gi:136861 Portal plug (EC-RF2), capsid-associated tegument protein, seals DNA inside capsid BVRF1 C   αβγ UL25 Sheaffer et al. (2001[Sheaffer, A. K., Newcomb, W. W., Gao, M., Yu, D., Weller, S. K., Brown, J. C. & Tenney, D. J. (2001). J. Virol. 75, 687-698.]), Johannsen et al. (2004[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.])      
P03234 gi:139231 Protease/assemblin BVRF2 C   αβγ UL26 Buisson et al. (2002[Buisson, M., Hernandez, J. F., Lascoux, D., Schoehn, G., Forest, E., Arlaud, G., Seigneurin, J. M., Ruigrok, R. W. & Burmeister, W. P. (2002). J. Mol. Biol. 324, 89-103.]) 2 PDB 1o6e
P03177 gi:1170666 Thymidine kinase BXLF1 N   αγ UL32 Littler et al. (1986[Littler, E., Zeuthen, J., McBride, A. A., Trost Sorensen, E., Powell, K. L., Walsh-Arrand, J. E. & Arrand, J. R. (1986). EMBO J. 5, 1959-1966.]) 3 Insoluble
P03231 gi:138312 Glycoprotein gp85, gH, part of gHgLgp42, fusion BXLF2 M   αβγ UL22 Spear & Longnecker (2003[Spear, P. G. & Longnecker, R. (2003). J. Virol. 77, 10179-10185.]), Hutt-Fletcher (2005[Hutt-Fletcher, L. M. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 359-378. Norwich: Caister Academic Press.])      
P03232 gi:136852 Nucleoprotein BXRF1 U   αβγ UL24 Pearson & Coen (2002[Pearson, A. & Coen, D. M. (2002). J. Virol. 76, 10821-10828.]) 2 Insoluble
P12978 gi:119111 EBNA-2 nuclear protein, transcription factor, interacts with RBPJ-K, essential for immortalization BYRF1 L ×     Henkel et al. (1994[Henkel, T., Ling, P. D., Hayward, S. D. & Peterson, M. G. (1994). Science, 265, 92-95.]), Waltzer et al. (1994[Waltzer, L., Logeat, F., Brou, C., Israel, A., Sergeant, A. & Manet, E. (1994). EMBO J. 13, 5633-5638.]), Young & Rickinson (2004[Young, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757-768.])      
P03206 gi:115196 Trans-activator ZEBRA, origin binding protein (EB1, Zta), bZip similar to CCAAT/enhancer binding protein α BZLF1 S × γ   Chevallier-Greco et al. (1986[Chevallier-Greco, A., Manet, E., Chavrier, P., Mosnier, C., Daillie, J. & Sergeant, A. (1986). EMBO J. 5, 3243-3249.]), Rooney et al. (1988[Rooney, C., Taylor, N., Countryman, J., Jenson, H., Kolman, J. & Miller, G. (1988). Proc. Natl Acad. Sci. USA, 85, 9801-9805.]), Giot et al. (1991[Giot, J. F., Mikaelian, I., Buisson, M., Manet, E., Joab, I., Nicolas, J. C. & Sergeant, A. (1991). Nucleic Acids Res. 19, 1251-1258.]), Petosa et al. (2006[Petosa, C., Morand, P., Baudin, F., Moulin, M., Artero, J. B. & Muller, C. W. (2006). Mol. Cell, 21, 565-572.])     PDB 2c9n , 2c9l
QQBE27 gi:73982 Function unknown, BZLF1 splice variant BZLF1b U ×     Countryman & Miller (1985[Countryman, J. & Miller, G. (1985). Proc. Natl Acad. Sci. USA, 82, 4085-4089.]), Farell (2005[Farell, P. J. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 263-287. Norwich: Caister Academic Press.])      
P03205 gi:141578 gp42, MHC class II binding protein, part of gHgLgp42 complex BZLF2 M   γ   Mullen et al. (2002[Mullen, M. M., Haan, K. M., Longnecker, R. & Jardetzky, T. S. (2002). Mol. Cell, 9, 375-385.]), Ressing et al. (2003[Ressing, M. E., van Leeuwen, D., Verreck, F. A., Gomez, R., Heemskerk, B., Toebes, M., Mullen, M. M., Jardetzky, T. S., Longnecker, R., Schilham, M. W., Ottenhoff, T. H., Neefjes, J., Schumacher, T. N., Hutt-Fletcher, L. M. & Wiertz, E. J. (2003). Proc. Natl Acad. Sci. USA, 100, 11583-11588.])     PDB 1kg0
P03235 gi:140616 EC-RF4 (ECRF4) protein ECRF4 U       Rivailler et al. (2002[Rivailler, P., Jiang, H., Cho, Y. G., Quink, C. & Wang, F. (2002). J. Virol. 76, 421-426.]), Ressing et al. (2003[Ressing, M. E., van Leeuwen, D., Verreck, F. A., Gomez, R., Heemskerk, B., Toebes, M., Mullen, M. M., Jardetzky, T. S., Longnecker, R., Schilham, M. W., Ottenhoff, T. H., Neefjes, J., Schumacher, T. N., Hutt-Fletcher, L. M. & Wiertz, E. J. (2003). Proc. Natl Acad. Sci. USA, 100, 11583-11588.])      
Q8AZJ5 gi:23893655 Protein LF1, contains a dUTPase like domain, γ-herpes ORF10 family LF1 U   γ   Rivailler et al. (2002[Rivailler, P., Jiang, H., Cho, Y. G., Quink, C. & Wang, F. (2002). J. Virol. 76, 421-426.]), Davison & Stow (2005[Davison, A. J. & Stow, N. D. (2005). J. Virol. 79, 12880-12892.]), Farell (2005[Farell, P. J. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 263-287. Norwich: Caister Academic Press.])      
Q99306 gi:23893654 Protein LF2, contains a dUTPase like domain, γ-herpes ORF11 family LF2 U   γ   Rivailler et al. (2002[Rivailler, P., Jiang, H., Cho, Y. G., Quink, C. & Wang, F. (2002). J. Virol. 76, 421-426.]), Davison & Stow (2005[Davison, A. J. & Stow, N. D. (2005). J. Virol. 79, 12880-12892.]), Farell (2005[Farell, P. J. (2005). Epstein-Barr Virus, edited by E. S. Robertson, pp. 263-287. Norwich: Caister Academic Press.])      
Not in DB Not in DB RK-BARF0, interaction with notch RK-BARF0 S ×     Fries et al. (1997[Fries, K. L., Sculley, T. B., Webster-Cyriaque, J., Rajadurai, P., Sadler, R. H. & Raab-Traub, N. (1997). J. Virol. 71, 2765-2771.]), Kusano & Raab-Traub (2001[Kusano, S. & Raab-Traub, N. (2001). J. Virol. 75, 384-395.])      
P13285 gi:126379 LMP-2A, interference with protein kinase signalling, gene terminal protein, essential for immortalization   L ×     Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]), Young & Rickinson (2004[Young, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757-768.])      
Q8AZK9 gi:23893578 LMP-2B, negative regulator of LMP-2A   L ×     Kieff (1996[Kieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343-2396. Philadelphia: Lipincott-Raven.]), Young & Rickinson (2004[Young, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757-768.])      
†Cloned.
‡Expressed.
§Structure solved.
¶Purified protein.
††Crystals.
‡‡Soluble protein.
[Figure 1]
Figure 1
(a) Classification of the EBV proteins according to function. (b) Outcome for the proteins entering into the structural genomics project.

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[Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Gasteiger, E., Martin, M. J., Michoud, K., O'Donovan, C., Phan, I., Pilbout, S. & Schneider, M. (2003). Nucleic Acids Res. 31, 365-370.]) and VIDA (Alba, 2002[Alba, M. M. (2002). VIDA Database. https://www.biochem.ucl.ac.uk/bsm/virus_database/ .])] 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[link], Fig. 1[link]a). 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[Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286-16291.]).

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[link]). 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[Deleage, G., Blanchet, C. & Geourjon, C. (1997). Biochimie, 79, 681-­686.]), small size and a high stability index according to the ExPASy ProtParam tool (Gasteiger et al., 2005[Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. & Bairoch, A. (2005). The Proteomics Protocols Handbook, edited by J. M. Walker, pp. 571-607. Totowa, NJ, USA: Humana Press.]). Known membrane proteins, surface glycoproteins 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[Cserzo, M., Wallin, E., Simon, I., von Heijne, G. & Elofsson, A. (1997). Protein Eng. 10, 673-676.]) and the TMHMM server (Krogh et al., 2001[Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. (2001). J. Mol. Biol. 305, 567-580.]) 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[Studier, F. W. (2005). Protein Expr. Purif. 41, 207-234.]). 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 eluate 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 light scattering (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. 1[link]b, Table 1[link]), 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[Deleage, G., Blanchet, C. & Geourjon, C. (1997). Biochimie, 79, 681-­686.]) and ClustalW-based alignments (Thompson et al., 1994[Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). Nucleic Acids Res. 22, 4673-4680.]) 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[link]), 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[Otterlei, M., Haug, T., Nagelhus, T. A., Slupphaug, G., Lindmo, T. & Krokan, H. E. (1998). Nucleic Acids Res. 26, 4611-4617.]). 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[Tarbouriech, N., Buisson, M., Seigneurin, J. M., Cusack, S. & Burmeister, W. P. (2005). Structure, 13, 1299-1310.]). Work on the EBV protease domain predated the SPINE project (Buisson et al., 2002[Buisson, M., Hernandez, J. F., Lascoux, D., Schoehn, G., Forest, E., Arlaud, G., Seigneurin, J. M., Ruigrok, R. W. & Burmeister, W. P. (2002). J. Mol. Biol. 324, 89-103.]). Structural determination of BHRF1 was abandoned despite the existence of small crystals when an NMR structure was reported (Huang et al., 2003[Huang, Q., Petros, A. M., Virgin, H. W., Fesik, S. W. & Olejniczak, E. T. (2003). J. Mol. Biol. 332, 1123-1130.]). BARF1 was obtained through an external collaboration and expressed in eukaryotic cells (de Turenne-Tessier et al., 2005[Turenne-Tessier, M. de, Jolicoeur, P., Middeldorp, J. M. & Ooka, T. (2005). Virus Res. 109, 9-18.]) before entering our structure-determination pipeline. Protein purification using an N-terminal His6 tag together with a TEV protease cleavage site, sometimes including size-exclusion chromatography, 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[Fogg, M. J. et al. (2006). Acta Cryst. D62, 1196-1207.]). 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[link]). Crystallographic details for each EBV structure are given in Table 2[link] and further details on the structure determinations and refinement have been or will be published elsewhere.

Table 2
Crystallographic results

Protein reference EBV ORF, PDB code Construct Inhibitor Protein buffer Precipitant Space group Unit-cell parameters (Å) Resolution (Å), method Structure
Protease§ BVRF2, 1o6e GSHMAS + (2–235) + RS Diisopropylfluorophosphate 100 mM NaCl, 20 mM Tris–HCl pH 7.5, 1 mM EDTA, 10 mM β-mercaptoethanol 1.4 M sodium formate, 100 mM sodium acetate pH 4.6, 5 mM EDTA P3121, twinned a = b = 52.8, c = 330.5 2.3, MR Fig. 2[link](a)
dUTPase BLLF3, 2bsy GAMGSGIP + (1–278) dUMP 250 mM NaCl, 20 mM imidazole, 10 mM MgCl2, 20 mM HEPES pH 7.5 20% PEG 3350, 200 mM LiSO4, 100 mM Tris–HCl pH 8.5 P212121 a = 56.7, b = 55.8, c = 81.1 1.5, SAD Fig. 2[link](b)
  2bt1 GAMGSGIP + (1–278) α,β-Imino-dUTP 250 mM NaCl, 20 mM imidazole, 10 mM MgCl2, 20 mM HEPES pH 7.5 3 M NaCl, 100 mM Bis-Tris–HCl pH 6.5 P622 a = b = 146.6, c = 77.1 2.7, MR  
BARF1†† BARF1, 2ch8 (21–221) 100 mM NaCl, 20 mM HEPES–NaOH pH 7.5 1 M (NH4)2SO4, 2% PEG 3350, 100 mM Bis-Tris–HCl pH 6.0 H3, twinned a = b = 179.3, c = 95.7 2.3, SAD Fig. 2[link](c)
Uracil DNA glycosylase‡‡ BKRF3, N/A GAM + (25–255) UGI of bacteriophage, PBS-2 100 mM NaCl, 20 mM Tris pH 7.510 mM DTT 20% PEG 3350, 50 mM NH4Cl C2221 a = 62.8, b = 83.5, c = 273.4 2.3, MR Fig. 2[link](d)
†Extra residues arising from cloning are given; the residues of the viral ORF are indicated in parentheses.
‡Method used for structure determination. MR, molecular replacement; SAD, single-wavelength anomalous dispersion on a heavy-atom derivative.
§Buisson et al. (2002[Buisson, M., Hernandez, J. F., Lascoux, D., Schoehn, G., Forest, E., Arlaud, G., Seigneurin, J. M., Ruigrok, R. W. & Burmeister, W. P. (2002). J. Mol. Biol. 324, 89-103.]).
¶Tarbouriech et al. (2005[Tarbouriech, N., Buisson, M., Seigneurin, J. M., Cusack, S. & Burmeister, W. P. (2005). Structure, 13, 1299-1310.]).
††Tarbouriech et al. (2006[Tarbouriech, N., Ruggiero, F., de Turenne-Tessier, M., Ooka, T. & Burmeister, W. P. (2006). J. Mol. Biol. 359, 667-678.]).
‡‡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.

[Figure 2]
Figure 2
Protein structures (see Table 2[link]).

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.

References

First citationAlba, M. M. (2002). VIDA Database. https://www.biochem.ucl.ac.uk/bsm/virus_database/Google Scholar
First citationAltschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Nucleic Acids Res. 25, 3389–3402.  CrossRef CAS PubMed Web of Science Google Scholar
First citationBeard, P. M. & Baines, J. D. (2004). Virology, 324, 475–482.  Web of Science CrossRef PubMed CAS Google Scholar
First citationBeisel, C., Tanner, J., Matsuo, T., Thorley-Lawson, D., Kezdy, F. & Kieff, E. (1985). J. Virol. 54, 665–674.  CAS PubMed Web of Science Google Scholar
First citationBellows, D. S., Howell, M., Pearson, C., Hazlewood, S. A. & Hardwick, J. M. (2002). J. Virol. 76, 2469–2479.  Web of Science CrossRef PubMed CAS Google Scholar
First citationBochkarev, A., Barwell, J. A., Pfuetzner, R. A., Bochkareva, E., Frappier, L. & Edwards, A. M. (1996). Cell, 84, 791–800.  CrossRef CAS PubMed Web of Science Google Scholar
First citationBoeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Gasteiger, E., Martin, M. J., Michoud, K., O'Donovan, C., Phan, I., Pilbout, S. & Schneider, M. (2003). Nucleic Acids Res. 31, 365–370.  Web of Science CrossRef PubMed CAS Google Scholar
First citationBorza, C. M. & Hutt-Fletcher, L. M. (1998). J. Virol. 72, 7577–7582.  Web of Science CAS PubMed Google Scholar
First citationBowman, B. R., Baker, M. L., Rixon, F. J., Chiu, W. & Quiocho, F. A. (2003). EMBO J. 22, 757–765.  Web of Science CrossRef PubMed CAS Google Scholar
First citationBuisson, M., Hernandez, J. F., Lascoux, D., Schoehn, G., Forest, E., Arlaud, G., Seigneurin, J. M., Ruigrok, R. W. & Burmeister, W. P. (2002). J. Mol. Biol. 324, 89–103.  Web of Science CrossRef PubMed CAS Google Scholar
First citationCabras, G., Decaussin, G., Zeng, Y., Djennaoui, D., Melouli, H., Broully, P., Bouguermouh, A. M. & Ooka, T. (2005). J. Clin. Virol. 34, 26–34.  Web of Science CrossRef PubMed CAS Google Scholar
First citationChang, Y. E., Poon, A. P. & Roizman, B. (1996). J. Virol. 70, 3938–3946.  CAS PubMed Web of Science Google Scholar
First citationCheng, Y. C., Chen, J. Y., Hoffmann, P. J. & Glaser, R. (1980). Virology, 100, 334–338.  CrossRef CAS PubMed Web of Science Google Scholar
First citationChevallier-Greco, A., Manet, E., Chavrier, P., Mosnier, C., Daillie, J. & Sergeant, A. (1986). EMBO J. 5, 3243–3249.  CAS PubMed Web of Science Google Scholar
First citationCoen, D. M. & Schaffer, P. A. (2003). Nature Rev. Drug Discov. 2, 278–288.  Web of Science CrossRef CAS Google Scholar
First citationCountryman, J. & Miller, G. (1985). Proc. Natl Acad. Sci. USA, 82, 4085–4089.  CrossRef CAS PubMed Web of Science Google Scholar
First citationCserzo, M., Wallin, E., Simon, I., von Heijne, G. & Elofsson, A. (1997). Protein Eng. 10, 673–676.  CrossRef CAS PubMed Web of Science Google Scholar
First citationDavison, A. J. & Stow, N. D. (2005). J. Virol. 79, 12880–12892.  Web of Science CrossRef PubMed CAS Google Scholar
First citationDecaussin, G., Leclerc, V. & Ooka, T. (1995). J. Virol. 69, 7309–7314.  CAS PubMed Web of Science Google Scholar
First citationDeleage, G., Blanchet, C. & Geourjon, C. (1997). Biochimie, 79, 681–­686.  Web of Science CrossRef CAS PubMed Google Scholar
First citationFarell, P. J. (2005). Epstein–Barr Virus, edited by E. S. Robertson, pp. 263–287. Norwich: Caister Academic Press.  Google Scholar
First citationFogg, M. J. et al. (2006). Acta Cryst. D62, 1196–1207.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationFries, K. L., Sculley, T. B., Webster-Cyriaque, J., Rajadurai, P., Sadler, R. H. & Raab-Traub, N. (1997). J. Virol. 71, 2765–2771.  CAS PubMed Web of Science Google Scholar
First citationFurnari, F. B., Zacny, V., Quinlivan, E. B., Kenney, S. & Pagano, J. S. (1994). J. Virol. 68, 1827–1836.  CAS PubMed Web of Science Google Scholar
First citationGasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. & Bairoch, A. (2005). The Proteomics Protocols Handbook, edited by J. M. Walker, pp. 571–607. Totowa, NJ, USA: Humana Press.  Google Scholar
First citationGilligan, K. J., Rajadurai, P., Lin, J. C., Busson, P., Abdel-Hamid, M., Prasad, U., Tursz, T. & Raab-Traub, N. (1991). J. Virol. 65, 6252–6259.  PubMed CAS Web of Science Google Scholar
First citationGiot, J. F., Mikaelian, I., Buisson, M., Manet, E., Joab, I., Nicolas, J. C. & Sergeant, A. (1991). Nucleic Acids Res. 19, 1251–1258.  CrossRef PubMed CAS Web of Science Google Scholar
First citationGong, M., Ooka, T., Matsuo, T. & Kieff, E. (1987). J. Virol. 61, 499–­508.  CAS PubMed Web of Science Google Scholar
First citationGonnella, R., Farina, A., Santarelli, R., Raffa, S., Feederle, R., Bei, R., Granato, M., Modesti, A., Frati, L., Delecluse, H. J., Torrisi, M. R., Angeloni, A. & Faggioni, A. (2005). J. Virol. 79, 3713–3727.  Web of Science CrossRef PubMed CAS Google Scholar
First citationGruffat, H., Manet, E., Rigolet, A. & Sergeant, A. (1990). Nucleic Acids Res. 18, 6835–6843.  CrossRef CAS PubMed Web of Science Google Scholar
First citationHardwick, J. M., Lieberman, P. M. & Hayward, S. D. (1988). J. Virol. 62, 2274–2284.  CAS PubMed Web of Science Google Scholar
First citationHenkel, T., Ling, P. D., Hayward, S. D. & Peterson, M. G. (1994). Science, 265, 92–95.  CrossRef CAS PubMed Web of Science Google Scholar
First citationHiriart, E., Gruffat, H., Buisson, M., Mikaelian, I., Keppler, S., Meresse, P., Mercher, T., Bernard, O. A., Sergeant, A. & Manet, E. (2005). J. Biol. Chem. 280, 36935–36945.  Web of Science CrossRef PubMed CAS Google Scholar
First citationHitt, M. M., Allday, M. J., Hara, T., Karran, L., Jones, M. D., Busson, P., Tursz, T., Ernberg, I. & Griffin, B. E. (1989). EMBO J. 8, 2639–2651.  CAS PubMed Web of Science Google Scholar
First citationHong, G. K., Delecluse, H. J., Gruffat, H., Morrison, T. E., Feng, W. H., Sergeant, A. & Kenney, S. C. (2004). J. Virol. 78, 4983–4992.  Web of Science CrossRef PubMed CAS Google Scholar
First citationHsu, D. H., de Waal Malefyt, R., Fiorentino, D. F., Dang, M. N., Vieira, P., de Vries, J., Spits, H., Mosmann, T. R. & Moore, K. W. (1990). Science, 250, 830–832.  CrossRef CAS PubMed Web of Science Google Scholar
First citationHuang, Q., Petros, A. M., Virgin, H. W., Fesik, S. W. & Olejniczak, E. T. (2003). J. Mol. Biol. 332, 1123–1130.  Web of Science CrossRef PubMed CAS Google Scholar
First citationHutt-Fletcher, L. M. (2005). Epstein–Barr Virus, edited by E. S. Robertson, pp. 359–378. Norwich: Caister Academic Press.  Google Scholar
First citationHwang, J. S. & Bogner, E. (2002). J. Biol. Chem. 277, 6943–6948.  Web of Science CrossRef PubMed CAS Google Scholar
First citationJohannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proc. Natl Acad. Sci. USA, 101, 16286–16291.  Web of Science CrossRef PubMed CAS Google Scholar
First citationKieff, E. (1996). Fields Virology, edited by B. N. Fields, pp. 2343–2396. Philadelphia: Lipincott–Raven.  Google Scholar
First citationKrogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. (2001). J. Mol. Biol. 305, 567–580.  Web of Science CrossRef PubMed CAS Google Scholar
First citationKusano, S. & Raab-Traub, N. (2001). J. Virol. 75, 384–395.  Web of Science CrossRef PubMed CAS Google Scholar
First citationLake, C. M. & Hutt-Fletcher, L. M. (2000). J. Virol. 74, 11162–11172.  Web of Science CrossRef PubMed CAS Google Scholar
First citationLake, C. M. & Hutt-Fletcher, L. M. (2004). Virology, 320, 99–106.  Web of Science CrossRef PubMed CAS Google Scholar
First citationLaux, G., Freese, U. K. & Bornkamm, G. W. (1985). J. Virol. 56, 987–­995.  CAS PubMed Web of Science Google Scholar
First citationLittler, E., Zeuthen, J., McBride, A. A., Trost Sorensen, E., Powell, K. L., Walsh-Arrand, J. E. & Arrand, J. R. (1986). EMBO J. 5, 1959–1966.  CAS PubMed Web of Science Google Scholar
First citationLopez, R., Urquiza, M., Patino, H., Suarez, J., Reyes, C., Patarroyo, M. A. & Patarroyo, M. E. (2005). Biochimie, 87, 985–992.  Web of Science CrossRef PubMed CAS Google Scholar
First citationMackett, M., Conway, M. J., Arrand, J. R., Haddad, R. S. & Hutt-Fletcher, L. M. (1990). J. Virol. 64, 2545–2552.  CAS PubMed Web of Science Google Scholar
First citationManet, E., Gruffat, H., Trescol-Biemont, M. C., Moreno, N., Chambard, P., Giot, J. F. & Sergeant, A. (1989). EMBO J. 8, 1819–1826.  CAS PubMed Web of Science Google Scholar
First citationMarschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van den Bogaard, M. & Stamminger, T. (2002). J. Gen. Virol. 83, 1013–1023.  Web of Science PubMed CAS Google Scholar
First citationMarshall, W. L., Yim, C., Gustafson, E., Graf, T., Sage, D. R., Hanify, K., Williams, L., Fingeroth, J. & Finberg, R. W. (1999). J. Virol. 73, 5181–5185.  Web of Science PubMed CAS Google Scholar
First citationModrow, S., Hoflacher, B. & Wolf, H. (1992). Arch. Virol. 127, 379–­386.  CrossRef PubMed CAS Web of Science Google Scholar
First citationMullen, M. M., Haan, K. M., Longnecker, R. & Jardetzky, T. S. (2002). Mol. Cell, 9, 375–385.  Web of Science CrossRef PubMed CAS Google Scholar
First citationNealon, K., Newcomb, W. W., Pray, T. R., Craik, C. S., Brown, J. C. & Kedes, D. H. (2001). J. Virol. 75, 2866–2878.  Web of Science CrossRef PubMed CAS Google Scholar
First citationNewcomb, W. W., Juhas, R. M., Thomsen, D. R., Homa, F. L., Burch, A. D., Weller, S. K. & Brown, J. C. (2001). J. Virol. 75, 10923–10932.  Web of Science CrossRef PubMed CAS Google Scholar
First citationOtterlei, M., Haug, T., Nagelhus, T. A., Slupphaug, G., Lindmo, T. & Krokan, H. E. (1998). Nucleic Acids Res. 26, 4611–4617.  Web of Science CrossRef CAS PubMed Google Scholar
First citationPaulsen, S. J., Rosenkilde, M. M., Eugen-Olsen, J. & Kledal, T. N. (2005). J. Virol. 79, 536–546.  Web of Science CrossRef PubMed CAS Google Scholar
First citationPearson, A. & Coen, D. M. (2002). J. Virol. 76, 10821–10828.  Web of Science CrossRef PubMed CAS Google Scholar
First citationPetosa, C., Morand, P., Baudin, F., Moulin, M., Artero, J. B. & Muller, C. W. (2006). Mol. Cell, 21, 565–572.  Web of Science CrossRef PubMed CAS Google Scholar
First citationRaab-Traub, N. (2005). Epstein–Barr Virus, edited by E. S. Robertson, pp. 71–92. Norwich: Caister Academic Press.  Google Scholar
First citationRessing, M. E., van Leeuwen, D., Verreck, F. A., Gomez, R., Heemskerk, B., Toebes, M., Mullen, M. M., Jardetzky, T. S., Longnecker, R., Schilham, M. W., Ottenhoff, T. H., Neefjes, J., Schumacher, T. N., Hutt-Fletcher, L. M. & Wiertz, E. J. (2003). Proc. Natl Acad. Sci. USA, 100, 11583–11588.  Web of Science CrossRef PubMed CAS Google Scholar
First citationReuven, N. B., Willcox, S., Griffith, J. D. & Weller, S. K. (2004). J. Mol. Biol. 342, 57–71.  Web of Science CrossRef PubMed CAS Google Scholar
First citationRickinson, A. & Kieff, E. (1996). Fields Virology, edited by B. N. Fields, Vol. 2, pp. 2397–2446. Philadelphia: Lippincott–Raven.  Google Scholar
First citationRivailler, P., Jiang, H., Cho, Y. G., Quink, C. & Wang, F. (2002). J. Virol. 76, 421–426.  Web of Science CrossRef PubMed CAS Google Scholar
First citationRobertson, E. S., Ooka, T. & Kieff, E. D. (1996). Proc. Natl Acad. Sci. USA, 93, 11334–11340.  CrossRef CAS PubMed Web of Science Google Scholar
First citationRooney, C., Taylor, N., Countryman, J., Jenson, H., Kolman, J. & Miller, G. (1988). Proc. Natl Acad. Sci. USA, 85, 9801–9805.  CrossRef CAS PubMed Web of Science Google Scholar
First citationSavva, C. G., Holzenburg, A. & Bogner, E. (2004). FEBS Lett. 563, 135–140.  Web of Science CrossRef PubMed CAS Google Scholar
First citationSegouffin-Cariou, C., Farjot, G., Sergeant, A. & Gruffat, H. (2000). J. Gen. Virol. 81, 1791–1799.  Web of Science PubMed CAS Google Scholar
First citationSegouffin, C., Gruffat, H. & Sergeant, A. (1996). J. Gen. Virol. 77, 1529–1536.  CrossRef CAS PubMed Web of Science Google Scholar
First citationSheaffer, A. K., Newcomb, W. W., Gao, M., Yu, D., Weller, S. K., Brown, J. C. & Tenney, D. J. (2001). J. Virol. 75, 687–698.  Web of Science CrossRef PubMed CAS Google Scholar
First citationSmith, R. F. & Smith, T. F. (1989). J. Virol. 63, 450–455.  CAS PubMed Web of Science Google Scholar
First citationSommer, P., Kremmer, E., Bier, S., Konig, S., Zalud, P., Zeppezauer, M., Jones, J. F., Mueller-Lantzsch, N. & Grasser, F. A. (1996). J. Gen. Virol. 77, 2795–2805.  CrossRef CAS PubMed Web of Science Google Scholar
First citationSpear, P. G. & Longnecker, R. (2003). J. Virol. 77, 10179–10185.  Web of Science CrossRef PubMed CAS Google Scholar
First citationStrockbine, L. D., Cohen, J. I., Farrah, T., Lyman, S. D., Wagener, F., DuBose, R. F., Armitage, R. J. & Spriggs, M. K. (1998). J. Virol. 72, 4015–4021.  Web of Science CAS PubMed Google Scholar
First citationStudier, F. W. (2005). Protein Expr. Purif. 41, 207–234.  Web of Science CrossRef PubMed CAS Google Scholar
First citationSwaminathan, S. (2005). Epstein–Barr Virus, edited by E. S. Robertson, pp. 631–649. Norwich: Caister Academic Press.  Google Scholar
First citationTarbouriech, N., Buisson, M., Seigneurin, J. M., Cusack, S. & Burmeister, W. P. (2005). Structure, 13, 1299–1310.  Web of Science CrossRef PubMed CAS Google Scholar
First citationTarbouriech, N., Ruggiero, F., de Turenne-Tessier, M., Ooka, T. & Burmeister, W. P. (2006). J. Mol. Biol. 359, 667–678.  Web of Science CrossRef PubMed CAS Google Scholar
First citationThompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). Nucleic Acids Res. 22, 4673–4680.  CrossRef CAS PubMed Web of Science Google Scholar
First citationTrus, B. L., Heymann, J. B., Nealon, K., Cheng, N., Newcomb, W. W., Brown, J. C., Kedes, D. H. & Steven, A. C. (2001). J. Virol. 75, 2879–2890.  Web of Science CrossRef PubMed CAS Google Scholar
First citationTsurumi, T., Kobayashi, A., Tamai, K., Daikoku, T., Kurachi, R. & Nishiyama, Y. (1993). J. Virol. 67, 4651–4658.  CAS PubMed Web of Science Google Scholar
First citationTugizov, S. M., Berline, J. W. & Palefsky, J. M. (2003). Nature Med. 9, 307–314.  Web of Science CrossRef PubMed CAS Google Scholar
First citationTurenne-Tessier, M. de, Jolicoeur, P., Middeldorp, J. M. & Ooka, T. (2005). Virus Res. 109, 9–18.  Web of Science CrossRef PubMed Google Scholar
First citationWagenaar, F., Pol, J. M., de Wind, N. & Kimman, T. G. (2001). Vet. Res. 32, 47–54.  CrossRef PubMed CAS Google Scholar
First citationWaltzer, L., Logeat, F., Brou, C., Israel, A., Sergeant, A. & Manet, E. (1994). EMBO J. 13, 5633–5638.  CAS PubMed Web of Science Google Scholar
First citationWaltzer, L., Perricaudet, M., Sergeant, A. & Manet, E. (1996). J. Virol. 70, 5909–5915.  CAS PubMed Web of Science Google Scholar
First citationWei, M. X. & Ooka, T. (1989). EMBO J. 8, 2897–2903.  CAS PubMed Web of Science Google Scholar
First citationWinters, T. A. & Williams, M. V. (1993). Virology, 195, 315–326.  CrossRef CAS PubMed Web of Science Google Scholar
First citationYokoyama, N., Fujii, K., Hirata, M., Tamai, K., Kiyono, T., Kuzushima, K., Nishiyama, Y., Fujita, M. & Tsurumi, T. (1999). J. Gen. Virol. 80, 2879–2887.  Web of Science PubMed CAS Google Scholar
First citationYoung, L. S. & Rickinson, A. B. (2004). Nature Rev. Cancer, 4, 757–768.  Web of Science CrossRef CAS Google Scholar

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