Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain
2009; Elsevier BV; Volume: 284; Issue: 26 Linguagem: Inglês
10.1074/jbc.m109.005728
ISSN1083-351X
AutoresTatiana Gianni, Michele Amasio, Gabriella Campadelli‐Fiume,
Tópico(s)Virus-based gene therapy research
ResumoHerpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. gD serves as a receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in an N-terminal domain carrying the receptor-binding sites and a C-terminal domain carrying the profusion domain, required for fusion but not receptor binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other, we searched for biochemical defined complexes in infected and transfected cells and in virions. Previously, interactions were detected in transfected whole cells by split green fluorescent protein complementation (Atanasiu, D., Whitbeck, J. C., Cairns, T. M., Reilly, B., Cohen, G. H., and Eisenberg, R. J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 18718–18723; Avitabile, E., Forghieri, C., and Campadelli-Fiume, G. (2007) J. Virol. 81, 11532–11537); it was not determined whether they led to biochemical complexes. Infected cells harbor a gD-gH complex (Perez-Romero, P., Perez, A., Capul, A., Montgomery, R., and Fuller, A. O. (2005) J. Virol. 79, 4540–4544). We report that gD formed complexes with gB in the absence of gH/gL and with gH/gL in the absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry and did not increase at virus entry into the cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the amino acids 240–260 portion and downstream with Thr304-Pro305 as critical residues and of the binding site to gH/gL at the amino acids 260–310 portion with Pro291-Pro292 as critical residues. The results indicate that gD carries composite-independent binding sites for gB and gH/gL, both of which are partly located in the profusion domain. Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. gD serves as a receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in an N-terminal domain carrying the receptor-binding sites and a C-terminal domain carrying the profusion domain, required for fusion but not receptor binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other, we searched for biochemical defined complexes in infected and transfected cells and in virions. Previously, interactions were detected in transfected whole cells by split green fluorescent protein complementation (Atanasiu, D., Whitbeck, J. C., Cairns, T. M., Reilly, B., Cohen, G. H., and Eisenberg, R. J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 18718–18723; Avitabile, E., Forghieri, C., and Campadelli-Fiume, G. (2007) J. Virol. 81, 11532–11537); it was not determined whether they led to biochemical complexes. Infected cells harbor a gD-gH complex (Perez-Romero, P., Perez, A., Capul, A., Montgomery, R., and Fuller, A. O. (2005) J. Virol. 79, 4540–4544). We report that gD formed complexes with gB in the absence of gH/gL and with gH/gL in the absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry and did not increase at virus entry into the cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the amino acids 240–260 portion and downstream with Thr304-Pro305 as critical residues and of the binding site to gH/gL at the amino acids 260–310 portion with Pro291-Pro292 as critical residues. The results indicate that gD carries composite-independent binding sites for gB and gH/gL, both of which are partly located in the profusion domain. Herpes simplex virus (HSV) 2The abbreviations used are: HSVherpes simplex virusaaamino acidHVEMherpesvirus entry mediatorWBWestern blotpAbpolyclonal antibodymAbmonoclonal antibodyPFUplaque-forming unit(s)ORFopen reading frameBACbacterial artificial chromosomewtwild typeCELISAcell enzyme-linked immunosorbent assay. entry into the cell occurs by fusion, and requires a multipartite apparatus made of a glycoprotein quartet: gD, gB, and the heterodimer gH/gL; for reviews, see Refs. 1Campadelli-Fiume G. Amasio M. Avitabile E. Cerretani A. Forghieri C. Gianni T. Menotti L. Rev. Med. Virol. 2007; 17: 313-326Crossref PubMed Scopus (116) Google Scholar, 2Rey F.A. EMBO Rep. 2006; 7: 1000-1005Crossref PubMed Scopus (52) Google Scholar, 3Spear P.G. Cell Microbiol. 2004; 6: 401-410Crossref PubMed Scopus (462) Google Scholar). When ectopically expressed, the four glycoproteins mediate cell-cell fusion (4Turner A. Bruun B. Minson T. Browne H. J. Virol. 1998; 72: 873-875Crossref PubMed Google Scholar). gD serves as the receptor-binding glycoprotein and interacts with three alternative receptors, nectin1, herpesvirus entry mediator (HVEM) and modified heparan sulfate (5Montgomery R.I. Warner M.S. Lum B.J. Spear P.G. Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 6Geraghty R.J. Krummenacher C. Cohen G.H. Eisenberg R.J. Spear P.G. Science. 1998; 280: 1618-1620Crossref PubMed Scopus (782) Google Scholar, 7Cocchi F. Menotti L. Mirandola P. Lopez M. Campadelli-Fiume G. J. Virol. 1998; 72: 9992-10002Crossref PubMed Google Scholar, 8Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (888) Google Scholar). It also represents the key player in the triggering of fusion, i.e. in inducing fusion execution by gB and gH/gL (9Cocchi F. Fusco D. Menotti L. Gianni T. Eisenberg R.J. Cohen G.H. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7445-7450Crossref PubMed Scopus (122) Google Scholar, 10Fusco D. Forghieri C. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9323-9328Crossref PubMed Scopus (82) Google Scholar, 11Carfi A. Willis S.H. Whitbeck J.C. Krummenacher C. Cohen G.H. Eisenberg R.J. Wiley D.C. Mol. Cell. 2001; 8: 169-179Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 12Krummenacher C. Supekar V.M. Whitbeck J.C. Lazear E. Connolly S.A. Eisenberg R.J. Cohen G.H. Wiley D.C. Carfí A. EMBO J. 2005; 24: 4144-4153Crossref PubMed Scopus (220) Google Scholar). These are among the most highly conserved proteins across the Herpesviridae family and constitute the fusion core apparatus. Their respective roles in fusion execution are unclear at present. Thus, gH carries elements typical of fusion glycoproteins, i.e. hydrophobic regions able to interact with target cells or artificial membranes and two heptad repeats potentially able to form a coiled coil (13Gianni T. Martelli P.L. Casadio R. Campadelli-Fiume G. J. Virol. 2005; 79: 2931-2940Crossref PubMed Scopus (75) Google Scholar, 14Gianni T. Menotti L. Campadelli-Fiume G. J. Virol. 2005; 79: 7042-7049Crossref PubMed Scopus (63) Google Scholar, 15Gianni T. Piccoli A. Bertucci C. Campadelli-Fiume G. J. Virol. 2006; 80: 2216-2224Crossref PubMed Scopus (45) Google Scholar, 16Galdiero S. Falanga A. Vitiello M. Browne H. Pedone C. Galdiero M. J. Biol. Chem. 2005; 280: 28632-28643Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 17Galdiero S. Vitiello M. D'Isanto M. Falanga A. Collins C. Raieta K. Pedone C. Browne H. Galdiero M. J. Gen. Virol. 2006; 87: 1085-1097Crossref PubMed Scopus (55) Google Scholar, 18Galdiero S. Falanga A. Vitiello M. Raiola L. Fattorusso R. Browne H. Pedone C. Isernia C. Galdiero M. J. Biol. Chem. 2008; 283: 29993-30009Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Peptides mimicking the hydrophobic regions of gH promote fusion of artificial membranes (19Gianni T. Fato R. Bergamini C. Lenaz G. Campadelli-Fiume G. J. Virol. 2006; 80: 8190-8198Crossref PubMed Scopus (36) Google Scholar, 20Galdiero S. Falanga A. Vitiello M. D'Isanto M. Cantisani M. Kampanaraki A. Benedetti E. Browne H. Galdiero M. Peptides. 2008; 29: 1461-1471Crossref PubMed Scopus (45) Google Scholar). With respect to gB, the crystal structure shows a trimer with a central coiled-coil and an overall structure similar to that of the fusion glycoprotein G of vesicular stomatitis virus in its postfusion conformation (21Heldwein E.E. Lou H. Bender F.C. Cohen G.H. Eisenberg R.J. Harrison S.C. Science. 2006; 313: 217-220Crossref PubMed Scopus (478) Google Scholar, 22Roche S. Bressanelli S. Rey F.A. Gaudin Y. Science. 2006; 313: 187-191Crossref PubMed Scopus (358) Google Scholar). Recently, a receptor for gB was described (23Satoh T. Arii J. Suenaga T. Wang J. Kogure A. Uehori J. Arase N. Shiratori I. Tanaka S. Kawaguchi Y. Spear P.G. Lanier L.L. Arase H. Cell. 2008; 132: 935-944Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Of note, although for virus-to-cell entry and for cell-cell fusion, fusion requires the simultaneous presence of gB and gH/gL, fusion of perinuclear virions with the outer nuclear membranes appears to necessitate either gH/gL or gB (24Farnsworth A. Wisner T.W. Webb M. Roller R. Cohen G. Eisenberg R. Johnson D.C. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10187-10192Crossref PubMed Scopus (149) Google Scholar). Furthermore, it has been reported that HSV fusion may be preceded by a hemifusion intermediate mediated by gD and gH/gL (25Subramanian R.P. Geraghty R.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2903-2908Crossref PubMed Scopus (141) Google Scholar). herpes simplex virus amino acid herpesvirus entry mediator Western blot polyclonal antibody monoclonal antibody plaque-forming unit(s) open reading frame bacterial artificial chromosome wild type cell enzyme-linked immunosorbent assay. A major focus of current research is definition of protein-protein interactions. With respect to HSV entry/fusion, to understand how the four glycoproteins cross-talk to each other, it is pivotal to detect which complexes are formed by the glycoproteins in their prefusion and fusion-active conformations, and, ultimately, the chain of interactions that signal the gD encounter with its cellular receptor and culminate in activation of gB and gH/gL. The proposed model of gD-activated entry/fusion (2Rey F.A. EMBO Rep. 2006; 7: 1000-1005Crossref PubMed Scopus (52) Google Scholar, 9Cocchi F. Fusco D. Menotti L. Gianni T. Eisenberg R.J. Cohen G.H. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7445-7450Crossref PubMed Scopus (122) Google Scholar, 10Fusco D. Forghieri C. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9323-9328Crossref PubMed Scopus (82) Google Scholar, 11Carfi A. Willis S.H. Whitbeck J.C. Krummenacher C. Cohen G.H. Eisenberg R.J. Wiley D.C. Mol. Cell. 2001; 8: 169-179Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 12Krummenacher C. Supekar V.M. Whitbeck J.C. Lazear E. Connolly S.A. Eisenberg R.J. Cohen G.H. Wiley D.C. Carfí A. EMBO J. 2005; 24: 4144-4153Crossref PubMed Scopus (220) Google Scholar, 26Campadelli-Fiume G. Menotti L. Arivin A. Campadelli-Fiume G. Mocarski E. Moore P.S. Roizman B. Whitley R. Yamanishi K. Human Herpesviruses Biology, Terapy, and Immunoprophylaxis. Cambridge Press, Cambridge, UK2007: 93-111Google Scholar, 27Lazear E. Carfi A. Whitbeck J.C. Cairns T.M. Krummenacher C. Cohen G.H. Eisenberg R.J. J. Virol. 2008; 82: 700-709Crossref PubMed Scopus (49) Google Scholar) envisions that (i) gD ectodomain is organized in two functionally and topologically distinct regions, the N-terminal one, spanning amino acids (aa) 1 to ∼240/260, carrying the receptor binding sites, and the C-terminal one (aa 240/260–310), carrying the profusion domain required for fusion but not for receptor binding; (ii) the unliganded gD adopts an auto-inhibited conformation, whereby the C-terminal domain folds around the N-terminal one and occupies or hinders the receptor-binding sites; (iii) gD undergoes a closed-to-open switch in conformation, whereby the C-terminal domain is dislodged from its binding site and exposes the profusion domain; and (iv) the active form of gD ultimately leads to the activation of gH/gL and gB. It was hypothesized that gB and gH/gL activation occurs through their recruitment to activated gD and that the C-terminal profusion domain carries the actual binding sites for gB and gH/gL (9Cocchi F. Fusco D. Menotti L. Gianni T. Eisenberg R.J. Cohen G.H. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7445-7450Crossref PubMed Scopus (122) Google Scholar, 12Krummenacher C. Supekar V.M. Whitbeck J.C. Lazear E. Connolly S.A. Eisenberg R.J. Cohen G.H. Wiley D.C. Carfí A. EMBO J. 2005; 24: 4144-4153Crossref PubMed Scopus (220) Google Scholar). An alternative possibility is that the C-terminal profusion domain simply enables the conformational changes in gD but does not carry the actual binding sites for gB and gH/gL. Efforts to validate the model prompted the search of interactions among the glycoprotein quartet. So far, it was found that HSV-infected cells harbor a co-immunoprecipitable complex made of HVEM, gD, and gH (28Perez-Romero P. Perez A. Capul A. Montgomery R. Fuller A.O. J. Virol. 2005; 79: 4540-4544Crossref PubMed Scopus (33) Google Scholar). A number of interactions were detected in transfected whole cells by split green fluorescent protein (or variations thereof) complementation assay (29Atanasiu D. Whitbeck J.C. Cairns T.M. Reilly B. Cohen G.H. Eisenberg R.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 18718-18723Crossref PubMed Scopus (153) Google Scholar, 30Avitabile E. Forghieri C. Campadelli-Fiume G. J. Virol. 2007; 81: 11532-11537Crossref PubMed Scopus (95) Google Scholar). The interactions were gD-gH/gL, gD-gB, and gB-gH/gL. The former two were found in cells transfected with two or three glycoproteins and are believed to mirror interactions that take place before gD activation. In contrast, the gB-gH/gL interaction was detected at fusion. Whether such interactions occur in infected cells or are seen only in transfected cells under conditions of high overexpression and whether they lead to biochemically defined complexes have not been investigated so far. In addition, inasmuch as the split green fluorescent protein complementation irreversibly stabilizes weak and transients interactions, it tends to overemphasize interactions. For other herpesviruses, evidence is accumulating of complex formation among the glycoproteins involved in virus entry. Thus, Epstein-Barr virus gH and gL form a complex with the receptor-binding glycoprotein gp42; human cytomegalovirus and murine γ-herpesvirus 68 gH and gL form a complex with gB (31Gillet L. Stevenson P.G. J. Virol. 2007; 81: 13082-13091Crossref PubMed Scopus (24) Google Scholar, 32Kirschner A.N. Lowrey A.S. Longnecker R. Jardetzky T.S. J. Virol. 2007; 81: 9216-9229Crossref PubMed Scopus (48) Google Scholar, 33Patrone M. Secchi M. Bonaparte E. Milanesi G. Gallina A. J. Virol. 2007; 81: 11479-11488Crossref PubMed Scopus (42) Google Scholar). The objective of this work was to provide biochemical evidence for complex formation among the glycoprotein quartet, to verify whether complexes are present in infected cells and in virions and whether they are formed at virus entry into the cell. We analyzed the composition of the complexes by two approaches, co-immunoprecipitation and a pulldown assay that exploits the ability of One-strep-tagged proteins to be specifically retained by the Strep-Tactin resin. Complexes with undistinguishable composition were detected in infected and transfected cells and in virions prior to entry into the cell. A panel of mutants enabled the preliminary location of part of the gD regions critical to gB- and gH/gL-binding sites at the profusion domain. The cells were grown in Dulbecco's modified minimum essential medium containing 5–20% fetal calf serum. HSV-1(F) was described (34Ejercito P.M. Kieff E.D. Roizman B. J. Gen. Virol. 1968; 2: 357-364Crossref PubMed Scopus (639) Google Scholar). The ΔgD F-gDβ (35Ligas M.W. Johnson D.C. J. Virol. 1988; 62: 1486-1494Crossref PubMed Google Scholar), ΔgB-KΔT (36Cai W.Z. Person S. Warner S.C. Zhou J.H. DeLuca N.A. J. Virol. 1987; 61: 714-721Crossref PubMed Google Scholar), ΔgH SCgHZ (37Forrester A. Farrell H. Wilkinson G. Kaye J. Davis-Poynter N. Minson T. J. Virol. 1992; 66: 341-348Crossref PubMed Google Scholar), and ΔgL (38Roop C. Hutchinson L. Johnson D.C. J. Virol. 1993; 67: 2285-2297Crossref PubMed Google Scholar) HSV mutants were grown and titrated in the respective complementing cells. R8 polyclonal antibody (pAb) to gD and BD80 monoclonal antibody (mAb) to aa 264–275 epitope of mature gD were generously provided by Dr. G. H. Cohen and Dr. R. Eisenberg; mAbs HD1, HC1, and H233 were a gift of Dr. L. Pereira. pAbs to gH and gL were a gift from Dr. H. Browne (Cambridge, UK) and D. Johnson (Portland). mAb H170 (reactive to aa 1–23 epitope), H1817, and H633 were purchased from Goodwin Institute. mAbs 52S, 53S, 30, and 5E1 were described (39Stefan A. Secchiero P. Baechi T. Kempf W. Campadelli-Fiume G. J. Virol. 1997; 71: 5758-5763Crossref PubMed Google Scholar, 40Showalter S.D. Zweig M. Hampar B. Infect. Immun. 1981; 34: 684-692Crossref PubMed Google Scholar, 41Brandimarti R. Huang T. Roizman B. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5406-5410Crossref PubMed Scopus (36) Google Scholar). mAb 52S reacts to a conformational-dependent epitope. mAb 53S reacts to a conformation-dependent and gL-dependent epitope. V5 mAb was from Invitrogen. pAb to gM was described (42Baines J.D. Roizman B. J. Virol. 1993; 67: 1441-1452Crossref PubMed Google Scholar). A pAb to gH/gL was derived to a soluble form of gH truncated at aa 789/gL produced in insect cells. 3T. Gianni and G. Campadelli-Fiume, manuscript in preparation. Vero cells grown in 12-well plates were infected with the indicated viruses at 0.1 PFU/cell for 90 min at 37 °C. The inoculum was removed, and unpenetrated virions were inactivated by means of an acidic wash (40 mm citrate acid, 10 mm KCl, 135 mm NaCl, pH 3). Replicate cultures were frozen at indicated times, i.e. 3, 24, and 48 h after infection. The progeny virus was titrated in Vero cells. The mammalian expression plasmids encoding gH, gL, and gB in MTS vector, and gD in pcDNA3.1, all under the cytomegalovirus promoter, were described (43Avitabile E. Lombardi G. Campadelli-Fiume G. J. Virol. 2003; 77: 6836-6844Crossref PubMed Scopus (45) Google Scholar). Plasmids encoding HVEM (pBEC) (5Montgomery R.I. Warner M.S. Lum B.J. Spear P.G. Cell. 1996; 87: 427-436Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar), HER2 (human epidermal growth factor receptor 2) (44Rovero S. Amici A. Carlo E.D. Bei R. Nanni P. Quaglino E. Porcedda P. Boggio K. Smorlesi A. Lollini P.L. Landuzzi L. Colombo M.P. Giovarelli M. Musiani P. Forni G. J. Immunol. 2000; 165: 5133-5142Crossref PubMed Scopus (306) Google Scholar), gDΔPFD (herein renamed gDΣ260–310), gDTP, and gDPP were described (9Cocchi F. Fusco D. Menotti L. Gianni T. Eisenberg R.J. Cohen G.H. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7445-7450Crossref PubMed Scopus (122) Google Scholar, 10Fusco D. Forghieri C. Campadelli-Fiume G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9323-9328Crossref PubMed Scopus (82) Google Scholar). To enable detection or retention to resin, gH, gL, and gC were tagged with heterologous epitopes, as follows. The 5E1 epitope consists of a 27-aa-long sequence recognized by mAb 5E1, initially derived to human herpesvirus 7 (39Stefan A. Secchiero P. Baechi T. Kempf W. Campadelli-Fiume G. J. Virol. 1997; 71: 5758-5763Crossref PubMed Google Scholar). The 5E1 epitope was inserted in gH (gH5E1), and the thrombin plus 5E1 and His (polyhistidine) epitopes were inserted in gL (gL5E1.His), just upstream of the stop codon. To generate gH5E1, a SphI restriction site was inserted in the cytoplasmic tail of gH, in place of the stop codon, by site-directed mutagenesis with oligonucleotides 5′-CCG TTT TTT TGG AGA CGC ATG CAA AGT GGG CGT GAA TTC GGC CGT TTC TCC GCC C-3′ and 5′-GGG CGG AGA AAC GGC CGA ATT CAC GCC CAC TTT GCA TGC GTC TCC AAA AAA ACG G-3′. The oligonucleotides contained EcoRI restriction site for screening and introduced a single mutation, E838M. Two annealing oligonucleotides, encoding the 5E1 epitope 5′-ACA TGC ATG CAT GTT TCC AGA CCA GGA AGC ACT ACA CCC TCT GGG AAC TCT GCA AGA TAT GGG-3′ and 5′-GGA AGA TCT GGT ACC TTA CGG AGT TAT ACT TCT AGG TGT GTT ATT CCC ATA TCT TGC AGA GTT CCC-3′ were ligated to SphI/BglII-digested gH. The oligonucleotides contained the Asp718 restriction site for easiness of screening. The aa sequence of gH5E1 cytoplasmic tail was modified to ILKVLRTSVPFFWRRMHVSRPGSTTPSGNSARYGNNTPRSITP. To generate gHst.V5, indicated below also as gHst, we followed a similar strategy; the annealing oligonucleotides encoding the Factor Xa protease cleavage site, followed by V5 and One-strep-tag epitopes, were 5′-GGA GAC GCA TGC TAA TCG AAG GGC GAG GTA AGC CTA TCC CTA ACC CTC TCC TAG GCC TCG ATT CTA CGA GCG CTT GGA GCC ACC CGC AGT TCG AGA AAG G-3′ and 5′-GGT AGT AGA TCT CAT TTT TCG AAC TGC GGG TGG CTC CAC GAT CCA CCT CCC GAT CCA CCT CCG GAA CCT CCA CCT TTC TCG AAC TGC GGG TGG CTC CAA G-3′. They were ligated to the SphI/BglII-digested gH. The aa sequence of gHst.V5 cytoplasmic tail became ILKVLRTSVPFFWRRMLIEGRGLPIPNPLLGLDSTSAWSHPQFELGGGSGGGSGGGSWSHPQFEL. To generate gL5E1.His, we followed essentially a similar strategy and inserted the NheI restriction site by site-directed mutagenesis, in place of the stop codon, by means of oligonucleotides 5′-CCC CAC TCC CGG CGC CTG CTA GCA TGA ATT CAC GGA AAC CCG TCC GGG TTC GGG-3′ and 5′-CCC GAA CCC GGA CGG GTT TCC GTG AAT TCA TGC TAG CAG GCG CCG GGA GTG GGG-3′. The annealing oligonucleotides encoding the trombin-5E1-His epitopes were 5′-CTA TAG CTA GCC CTG GTT CCG CGT GGA TCC TCC AGA CCA GGA AGC ACT ACA CCC TCT GGG AAC TCT GCA AGA TAT GGG AA-3′ and 5′-GGA AGA TCT TCA ATG GTG ATG GTG ATG ATG CGG AGT TAT ACT TCT AGG TGT GTT ATT CCC ATA TCT TGC AGA GTT CCC-3′ and were ligated to NheI/BglII digested gL. The aa sequence of the gL C terminus was changed from SRRL to SRRLLALVPRGSSRPGSTTPSGNSARYGNNTPRSITPHHHHHH. To generate gLV5.His, the annealing oligonucleotides encoding the V5 and His epitopes were 5′-ATG CTC GCT AGC TGG TAA GCC TAT CCC TAA CCC TCT CCT CGG TCT CGA TTC TAC GC-3′ and 5′-TTA GCG AGA TCT CAA TGG TGA TGA TGG TGA TGA TGA ACG GTA CGC GTA GAA TCG AGA C-3′ and were ligated to NheI/BglII-digested gL. The aa sequence of gL C terminus was changed from SRRL to SRRLLAGLPIPNPLLGLDSTRTVHHHHHHH. gCV5 was generated as follows. The gC ORF was PCR-amplified from HSV-1(F) DNA with primers encoding the V5 epitope 5′-AGA TCT AGG CCT ATG GCC CCG GGG CGG GTG GGC CTT GCC G TG GTC CTG TGG AGC CTG-3′ and 5′-GCA CGG GGC GGC CGC TTA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG GAT AGG CTT ACC GGC TAG CCG CCG ATG ACG CTG CCG CGA CTG TGA TGT GCG G-3′. The StuI-NotI-digested gC amplimer was ligated to MTS vector. For all plasmids, the ORF was sequenced. HSV1(BAC)-gDst was generated by bacterial artificial chromosome (BAC) "galK recombineering," employing the Escherichia coli strain SW102 and galK (galactokinase) positive/negative selectable marker, kindly provided by Dr. N. G. Copeland) (45Warming S. Costantino N. Court D.L. Jenkins N.A. Copeland N.G. Nucleic Acids Res. 2005; 33: e36Crossref PubMed Scopus (960) Google Scholar). pYEbac102 HSV-BAC was provided by Y. Kawaguchi and carries the BAC sequences inserted between UL3 and UL4 genes. The galK cassette was recombined in HSV-1(BAC) to replace the gD stop codon. To this end, the cassette was PCR-amplified from pGalK plasmid with primers that annealed in their 5′ end to sequences upstream and downstream of gD stop codon. The oligonucleotides used for amplification were 5′-CCC ACA TCC GGG AAG ACG ACC AGC CGT CCT CGC ACC AGC CCT TGT TTT ACC CTG GTG ACA ATT AAT CAT CGGCA-3′ and 5′-CAT CCC AAC CCC GCA GAC CTG ACC CCC CCG CAC CCA TTA AGG GGG GGT ATT CAG CAC TGT CCT GCT CCTT-3′. The amplimer was recombined into the HSV-1 BAC, and the recombinant bacteria were selected on minimal media containing galactose as the only carbon source. Next, the galK cassette was substituted with sequences encoding the One-strep-tag by means of a double-stranded oligonuocletide that carried flanking sequences homologous to gD sequences upstream and downstream of the stop codon. The double-stranded oligonucleotide was generated by annealing-extension of two synthetic oligonucleotides 5′-CCC ACA TCC GGG AAG ACG ACC AGC CGT CCT CGC ACC AGC CCT TGT TTT ACA GCG CTT GGA GCC ACC CGC AGT TCG AGA AAG GTG GAG GTT CCG GAG GTG GAT CCG GAG GT-3′ and 5′-CAT CCC AAC CCC GCA GAC CTG ACC CCC CCG CAC CCA TTA AGG GGG GGT ATT CAT TTT TCG AAC TGC GGG TGG CTC CAC GAT CCA CCT CCG GAT CCA CCT CCG GAA CC-3′. Its homologous recombination into the HSV-BAC was achieved by selecting against the galK cassette, i.e. by resistance to 2-deoxy-galactose on minimal plates containing glycerol as carbon source. Chloramphenicol selection was kept throughout, to maintain the HSV-BAC sequences. The recombinant HSV1(BAC)-gDst DNA was extracted from bacteria and transfected into 293 T cells to reconstitute the virus. The 3′ of gD gene was sequenced for accuracy in DNA extracted from bacteria and in virus stock. The linear map of the gD constructs employed in this study is shown in Fig. 6. To generate gDΣ240–260, the starting plasmid contained an Asp718 restriction site at aa 260 (46Cocchi F. Menotti L. Di Ninni V. Lopez M. Campadelli-Fiume G. J. Virol. 2004; 78: 4720-4729Crossref PubMed Scopus (18) Google Scholar). An additional Asp718 site was inserted at aa 240 by site-directed mutagenesis by means of oligonucleotides 5′-CAG CTT GAA GAT CGC GGT ACC GAA GCT TCC CAA GGC CCC ATA CAC GAG CAC CC-3′ and 5′-GGG TGC TCG TGT ATG GGG CCT TGG GAA GCT TCG GTA CCG CGA TCT TCA AGC TG-3′. Two synthetic annealing oligonucleotides encoding a 18-aa-long Ser-Gly linker 5′-PHO-GTA CCC AGT AGT GGC GGT GGC TCT GGA TCC GGC TCG AGC GGA GGC GGT AGC GGG-3′ and 5′-PHO-GGT CAT CAC CGC CAC CGA GAC CTA GGC CGA GCT CGC CTC CTC CAT CGC CCC ATG-3′ were ligated into Asp718-digested gD. Orientation was screened by colony PCR and confirmed by sequence. To generate gDΣ218–240, we followed essentially a similar strategy. Two Asp718 restrictions sites were introduced in gD plasmid at aa 218 and 240 by simultaneous double site-directed mutagenesis. The Asp718 site in aa 218 was inserted by means of oligonucleotides 5′-TGA CGG TGG ACA GCA TGG TAC CGC TGC CCC GCT TCA TCC-3′ and 5′-GGA TGA AGC GGG GCA GCG GTA CCA TGC TGT CCA CCG TCA-3′; the Asp718 site at aa 240 was inserted by means of the oligonucleotides described above for gDΣ240–260. The DNA encoding the 18-aa-long Ser-Gly linker was generate as above and ligated with Asp718-digested gD. Orientation was screened by colony PCR and confirmed by sequence. The starting plasmid for gDΔ61–218 was generated by Dr. L. Menotti in the course of an independent study. 4Menotti, L., Nicoletti, G., Gatta, V., Croci, S., Landuzzi, L., De Giovanni, C., Nanni, P., Lollini, P. L., and Campadelli-Fiume, G. (2009) Proc. Natl. Acad. Sci. U. S. A. 106, in press. Briefly, the gD ORF, engineered in a vector designed to enable homologous recombination of mutant gD into HSV-BAC, was mutagenized by insertion of NdeI restriction sites at aa 60 and 218. Two synthetic annealing oligonucleotides encoding a 18-aa Ser-Gly linker (5′-PHO-TAG TAG TGG CGG TGG CTC TGG ATC CGG CTC GAG CGG AGG CGG TAG CGG GAG TGG-3′ and 5′-pho-TACC CAC TCC CGC TAC CGC CTC CGC TCG AGC CGG ATC CAG AGC CAC CGC CAC TAC-3′) were ligated into the NdeI-digested gD. Finally, the SacII/HindIII fragment containing the partially deleted ORF was subcloned in pcDNA containing gD ORF. Orientation was screened by colony PCR and confirmed by sequence. To generate gDΣ240–310, Asp718 restriction sites were engineered at aa 240 and 310 of wt gD, respectively, by site-directed mutagenesis by means of oligonucleotides 5′-CAG CTT GAA GAT CGC GGT ACC GAA GCT TCC CAA GGC CCC ATA CAC GAG CAC CC 3′ and 5′-CAT CCC CCG GCG GTA CCG AAC AAC ATG GGC CTG 3′. A CD8 amplimer obtained with oligonucleotides 5′-CGG GGT ACC CAG TGG CGG TAG TTC GGC CCT GAG CAA CTC CATC3′ and 5′GCGC CCA GAT GTA GAT AGG TAC CGC GAA GTC CAG CCC CCT CGT GTGC 3′ and digested with Asp718 was then ligated into Asp718-digested gD. To generate gDΔ6–60, two EcoRI restrictions sites were introduced in pcDNA-gD plasmid by simultaneous double site-directed mutagenesis with prior elimination of the EcoRI site present in the vector. The EcoRI site at aa 6 was introduced by means of oligonucleotides 5′-CAA ATA TGC CTT GGC GGA GAA TTC TCT CAA GAT GGC CG-3′ and 5′-CGG CCA TCT TGA GAG AAT TCT CCG CCA AGG CAT ATT TG-3′; the EcoRI site at aa 59 was introduced by means of oligonucleotides 5′-CAC GGT TTA CTA CGC GAA TTC GGA GCG CGC CTG CCG-3′ and 5′-CGGC AG GC GCG CTC CGA ATT CGC GTA GTA AAC CGTG-3′. The EcoRI-digested gD was relegated, thus collapsing the EcoRI fragment. For all of the plasmids, the ORF was sequenced. Co-immunoprecipitation was carried out from infected or transfected cells. Infected 293 T cells received 5 PFU/cell of one of the following viruses, HSV-1(F), ΔgD, ΔgB, ΔgH, or ΔgL HSV, as titrated in Vero or their respective complementing cells. To enable detection of gH and gL, the cells were transfected with gH5E1 and gL5E1.His (5 and 3 μg of DNA/flask, respectively) 6 h prior to infection with HSV-1(F), ΔgD, and ΔgB viruses. Infected cells were harvested 18 h after infection. U251, human embryonic lung, and human foreskin fibroblast cells were infected with HSV-1(F) or ΔgD virus (5 PFU/cell). We used 3-fold higher amount of cells than in the experiments with infected 293T cells. To enable detection of gH and gL, the cells were transfected with gH5E1 and gL5E1.His (15 and 9 μg of DNA/T7
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