Constitutive Expression in Human Cells of Herpes Simplex Virus Type 1 Glycoprotein 6 Gene Cloned in an Episomal Eukaryotic Vector R. MANSERVIGI,* R. GUALANDRI,* M. NEGRINI,” L. ALBONICI,* G. MILANESI,t *Institute
of Ferrara, Via Luigi Borsari 46, l-44 100 Ferrara, and tlnstitute of Biochemical National Research Council, Via Abbiategrasso 187, I-27100 Pavia, Italy Received
April 8, 1988; accepted
July 15, 1988
Expression of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) was obtained in human cells from the gB-1 gene cloned in the episomal replicating vector pBK-1, which contains the origin of replication and early region of the human papovavirus BK. Selective systems for the TK+ phenotype in TK-143B cells and for resistance to G418 in adenovirus 5-transformed 293 cells were used to obtain stable transformants that produced gB-1. While gB-1 expression in 143B cells required induction by HSV-1 early proteins, constitutive gB1 production was observed in 293 cells, where endogenous transacting factors probably replace the need for early viral products in the activation of the cloned gB-1 gene. The amount of recombinant gB-1 was comparable to that produced during HSV-1 lytic infection in human cells, due to amplification of the inserted gene in the replicating episomal vector. Expression of gB-1 was induced by cadmium and zinc when the promoter of the mouse metallothionein-I gene was placed upstream of gB1 structural sequences. The inducible system where the gB-1 gene is under the control of its own promoter could be employed to clarify the role of early viral products in induction of gB-1 synthesis. Constitutive expression of gB-1 in human cells could provide useful material for diagnostic purposes and for the preparation of a subunit vaccine against HSV infections. o 198s Academic
Eukaryotic viral vectors can be distinguished according to their fate inside the transfected cells. In most cases the vector DNA integrates into cellular DNA in a small number of copies per cell. Episomal vectors based on simian virus 40 (SV40) (I, 2) or bovine papillomavirus (3) sequences have been used to express genes in monkey or mouse cells. Episomal vectors have several advantages over integration vectors. They can replicate to a high copy number, allowing amplification of the inserted gene which is released from chromosomal restraints. We have recently developed a viral vector (pBK-1) that replicates and persists episomally in human cells (4). It contains DNA sequences from the plasmid pML as well as the origin of replication and early region of the human papovavirus BK (BKV). Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) is essential for virus penetration, cell fusion, and pathogenic effect (5-8). It belongs to the group of late yl proteins, whose expression is coordinately regulated by early (Y and middle p gene products (9). The gB-1 gene has been recently cloned in Escherichia co/i (10) and expressed in yeast (1 I), in rodent and simian cells (12-15). We have inserted the gB-1 gene into pBK-1 and obtained its expression in human cells.
Three vectors were constructed: pBK-gB1 , pBK-TKgB1, and pBK-MT-gB1 (Fig. 1). In pBK-gB1 and pBK-TKgB1 transcription is directed by the gB-1 endogenous promoter, while in pBK-MT-gB1 the gB-1 gene is under control of the mouse metallothionein-I gene promoter. Human TK- 143B cells (16) were transfected with pBKTK-gB1 DNA by the calcium phosphate coprecipitation technique (17) and selected in HAT medium (18). Of 24 stable cell clones analyzed by immunoprecipitation with anti-gB-1 monoclonal antibody l-144, none showed detectable immunoprecipitated forms of gB1, indicating that the 143B TKf transformants do not express gB-1 constitutively. Induction of gB-1 expression was obtained in one of two 143B TK+ clonal cell lines (Fig. 2A) by infection with the HSV-1 ts mutant (HFEM)-tsB5 (7) which does not express gB-1 at the nonpermissive temperature. The amount of gB-1 synthesized by the positive clone was approximately the same at both permissive and nonpermissive temperatures. These results suggest that one or more functional HSV-1 early products are required to activate the expression of the cloned gB-1 gene in 143B-transformed cells. In search for heterologous transcriptional activators to induce the expression of the cloned gB-1 gene, we cotransfected pBK-gB1 and pSV2-neo into human 293 cells (19) and selected transformants in medium containing G418 (400 pg/ml). 293 cells express the adeno-
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Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved
out of twenty-five G418-resistant clones expressed gB-1. Two bands of 120 and 110 kDa, corresponding in size to the mature form of gB-1 and to its precursor, were clearly visible in immunoprecipitates of positive clones (Fig. 2B). Two proteins of identical size were immunoprecipitated from HSV-1 -infected 293 cells. Similar results were obtained in 293 cells cotransfected with pBK-MT-gB1 and pSV2-neo (Fig. 2C). In these experiments, 10 out of 25 clones expressed gB-1 and production was increased 1.3-to 2.5fold by treatment with cadmium chloride (2 @II!)or zinc chloride (100 pl\/1) (data not shown). No gB-1 was immunoprecipitated from the culture fluid of 143B and 293 cell clones. The amount of recombinant gB-1 was evaluated by ELISA carnal TK BayelI Barn 0 fragment
Acl4 cl7 i c
FIG. 1. In the upper left corner the HSV-1 genome is represented schematically. The BamHI G fragment, containing the gB-1 gene, is enlarged. The vector pBK-1 was constructed by ligating together the EcoRI-BarnHI fragments from the bacterial plasmid pML and from the human papovavirus BK. BK virus sequences comprise the early region and the origin of replication (Ori). The regions coding for BKV large T and small t antigens as well as for ampicillin resistance (Ap’) are indicated. pBK-BamG (15.3 kb) was constructed by inserting the 7.7.kb BamHl G fragment from the HSV-1 genome into the unique BarnHI site of the vector pBK-1. A 1.9.kb fragment, containing a BarnHI site, was removed from pBK-BamG by digestion with Sall, followed by circularization to obtain pBK-gB1 (13.4 kb). pBK-TK-gB1 (17.0 kb) was constructed by inserting the HSV-1 BarnHI Q fragment, containing the TK gene, into the remaining unique BarnHI site of pBKgB1. To construct pBK-MT-gB1 (12.7 kb), the promoter of the mouse metallothionein-I gene was excised as a 2.0.kb fragment from plasmid pJYMMT(E) clone 28 BarnHI (32) by digestion with EcoRl and Bglll. Endogenous gB-1 promoter sequences were eliminated from pBK-BamG by digestion with Xhol and Sall. After addition of Xhol linkers, the fragment containing the metallothionein promoter was directly ligated to the large Xhol-Sal1 fragment from pBK-BamG, containing the gB-1 coding sequences, because Xhol and SalI have compatible ends. Direction of transcription in the HSV-1 genome and in the different vector constructions is indicated by arrows. Black boxes mark gB-1 promoter and nonessential sequences and white boxes indicate gB-1 coding sequences. Stippled boxes indicate the TK gene, hatched boxes indicate the metallothionein-I gene promoter (pMT), and thin lines indicate BKV as well as pML sequences,
virus 5 immediate early proteins E1A and E1B and produce transacting factors that stimulate transcription from eukaryotic cellular and viral genes (20-22). Seven
143B+tsB5 34 39 & ; k
MP I 883 483 C
cl4+tsB5 34 39
cl7+tsB5 34 39 :taD:
3C4 3C5 3C51D * 14 h-4
FIG. 2. Radioimmunoprecipitation of recombinant gB-1 by monoclonal antibody l-144. Cells were labeled in methionine-free medium containing 50 &i/ml of [35S]methionine (sp act 1000 to 2000 Ci/ mmol). Cell extracts were prepared by lysing cells in 10 mM sodium phosphate buffer, pH 7.4, containing 1% Nonidet-P40,0.5% sodium deoxycholate, 1 Om4 M tosyl-L-phenylalanyl-chloromethyl ketone, and 1 OW3 M phenylmethylsulfonyl fluoride as protease inhibitors. Immunoprecipitation, electrophoresis in polyacrymide gels, and autoradiography were carried out as described (12). (A) Clones 4 and 7 of 143B cells transformed by pBK-TK-gB1 were analyzed uninfected or after infection with the gB-1 ts mutant HSV-1 (HFEM)-tsB5 and incubation at 34 and 39”. Ceils were labeled with [?S]methionine from 1.5 to 14 hr after infection and lysed for immunoprecipitation at the end of the labeling period. Control 1438 TK- cells were infected with the mutant and treated as the clones. The open and closed arrows indicate absence and presence, respectively, of gB-1 (gB) and its precursor (pgB). (B) Clones of 293 cells transformed by pBK-gB1 and (C) by pBK-MT-gB1. Cells were labeled with [35S]methionine for 4 hr. lmmunoprecipitates from control cultures include uninfected 293 cells (C) and 293 cells infected by HSV-1 strain MP (I). In (B) the left lane (MP) shows the glycoprotein pattern of 293 cells infected with MP and labeled with [‘4C]glucosamine (5 &i/ml, sp act >200 mCi/ mmol) from 4 to 18 hr after infection. In (C) the right lane (3C5/D) shows the dimeric forms of gB-1 and of its precursor (open arrows) produced by clone 3C5. To detect gB-1 dimers, the sample shown in this lane was not boiled before electrophoresis. The precursor (pgB) and mature (gB) forms of gB-1 are indicated by closed arrows.
QUANTITATIVE DETERMINATION OF RECOMBINANT gB-1 PRODUCED CONSTITUTIVELY BY 293 CELLTRANSFORMANTS ODw~100 protein
Cell Normal 293 HSV-1 F-infected 293 transformants 483 7B1 7B6 8B3 3c3 3c4 3c5 ID9 2D9 5D5
Vector copy number
1.14 0.72 0.78 0.45 2.00 1.31 >2.00 0.72 0.57 1.06
20 15 15 7 25 20 28 10 7 18
Note. ELISA tests for quantitative analysis of gB-1 were performed by a capture method as described previously (33). A gB-1 rabbit polyclonal antibody, immobilized on y-irradiated polystyrene microtiter plates, was reacted with cell lysates (prepared as described for immunoprecipitation, see legend to Fig. 2) followed by addition of I-1 44 anti-g&l mouse monoclonal antibody and a biotinylated antibody to mouse IgG. The reaction was developed with orthophenylendiamine and evaluated spectrophotometrically at 492 nm. Protein concentration was determined according to Lowry eta/. (34). Clones 483,781, 7B6, and 883 were transformed by pBK-gB1; clones 3C3,3C4,3C5, 1 D9,2D9, and 5D5 were transformed by pBK-MT-gB1. Vector copy number was determined by densitometric analysis of hybridization bands. ND, not done.
on cell lysates of 293 cell transformants. The results presented in Table 1 show that production is variable in different clones, but some clones express gB-1 as efficiently as in HSV-1 Iytic infection. The quantity produced correlates with vector copy number, suggesting that the critical factor for gB-1 expression is amplification of the recombinant DNA. Correct processing and proper maturation of recombinant gB-1 was assessed by two types of evidence. First, recombinant gB-1 produced dimers (Fig. 2C, lane 3C5/D), suggesting that it has a normal conformation. Second, infection of 293 clone 3C5 with gB-1 ts mutant (HFEM)-tsB5 at the nonpermissive temperature (39”) produced infectious virus at the titer of 105.’ PFU/ml, whereas normal 293 cells infected by the same viral mutant produced 102.4PFU/ml. Infection at the permissive temperature (34’) yielded 107.7 PFU/ml in clone 3C5 and 107.5 PFU/ml in normal 293 cells. These results indicate that recombinant gB-1 is functionally normal and competent to contribute infectivity to virions. The state of the recombinant DNA molecules in transformed cells was analyzed by Southern blot hy-
bridization of total cellular DNA to a pBK-TK-gB 1 probe. Figure 3 shows the results for three clones obtained in either 143B or 293 cells. Undigested DNA from each of the clones revealed molecules comigrating with supercoiled form I and circular form II of control vector DNA. Digestion of 143B clone 7 DNA with BarnHI, which cuts pBK-TK-gB1 twice, yielded two bands that comigrated with 3.6 kb (TK gene) and 13.4 kb (pBKgB1) marker fragments. When DNA from 293 clones 4B3 and 3C4 was digested with BarnHI, which cleaves pBK-gB1 or pBK-MT-gB1 at a single site, one band was detected corresponding to the full-length linear form of vector DNA. No other bands, suggestive of vector integrated sequences or rearrangements, were found, indicating that the recombinant plasmids are maintained in human cells as native molecules in a free, episomal state at 10 to 30 genome equivalents per cell, as estimated by densitometric analysis. Vector DNA extracted from both 143B and 293 cell clones was completely cleaved by A&ol, while it was resistant to digestion by Dpnl, demonstrating that the episomal recombinant molecules replicate in human cells. Mice immunized with cell lysates of 293 cell clone 3C4 produced neutralizing antibodies to HSV-1 strains F and MP and, with a lower titer, to HSV-2 strain G. They were protected from paralysis and death after a
FIG. 3. Detection of vector DNA in 143B clone 7, transformed by pBK-TK-gB1 ; in 293 clone 486, transformed by pBK-gB1 ; and in 293 clone 3C4, transformed by pBK-MT-gB1 Total cellular DNAwas analyzed undigested or digested with the indicated restriction endonucleases, electrophoresed in borate buffer through a 0.8% agarose gel, and transferred to nitrocellulose sheets that were blot-hybridized to a nick-translated ?labeled pBK-TK-gB1 probe. pBK-TK-gB1 DNA (50 and 10 genome equivalents) was included as control. Supercoiled form I (FI), relaxed form II (FII), and full-length linear form Ill (FIII) of the control are indicated.
SHORT TABLE IMMUNOGENICITYOF
In vitro neutralization (geometric mean titer (log*)) Antigen 293 clone 3C4 cell lysate 293 clone 3C4 culture medium Normal 293 cell lysate None
HSV-1 challenge (number of mice) Dead/Normal
fection by immunological tests. Although direct evidence is lacking, a critical role in immunogenicity may be attributed to the carbohydrate moiety of gB-1. Since reactions leading to gB-1 glycosylation and processing are carried out by cellular factors and enzymes (31), the glycosydic composition of the recombinant glycoprotein depends on the nature of the transfected cells. Expression in human cells would ensure that carbohydrate antigenic determinants of gB-1 are identical to those of the virion glycoprotein produced during natural infection in humans. ACKNOWLEDGMENTS
Note. Cell lysates were prepared as for immunoprecipitation (see legend to Fig. 2) and clarified by centrifugation at 15,000 g for 30 min. Culture fluid was concentrated 50-fold by ultrafiltration. Groups of eight 2-month-old female Balb/C mice were immunized with cell lysates or culture medium. Each animal received 12 mg of protein. For group 1 this amount is equivalent to 157 0Dag2 (ELISA) of recombinant gB-1. The antigen was mixed with an equal volume of complete Freund’s adjuvant and administered intraperitoneally. Animals receiving no antigen were injected with adjuvant only. A second immunization was repeated after 20 days and animals were bled 15 days later. Neutralization titers of sera were determined as 100% plaque reduction end-points on Vero cells infected by HSV-1 strains F and MP and by HSV-2 strain G. Mice were challenged 30 days after the second immunization by inoculation of the HSV-1 encephalitogenie strain 13 (500 LD& into the rear right foot pad. The dead mouse in group 1 had the lowest neutralization titer (1 :16) for HSV-1 F and MP.
lethal challenge with HSV-1. No antibody production and protection were detected in mice immunized with the concentrated culture fluid of 293 cell clone 3C4 or with cell lysate of normal 293 cells (Table 2). This episomal vector for gB-1 expression in human cells could be useful to study the effect of mutations in the gB-1 gene without interference by neighboring cellular sequences. The inducible 1438 system could elucidate which HSV-1 early proteins are necessary to activate expression of the gB-1 gene in human cells. gB-1 is immunogenic, eliciting both humoral and cellmediated immune responses (23, 24) and anti-gB antibodies are protective against HSV infection in experimental animals (25-27). Protection of mice immunized with recombinant gB-1 produced in human cells suggests a possible development for preparation of a subunit vaccine. Cross-reaction of gB-1 with equivalent glycoproteins of HSV-2 (28) and varicella-zoster virus (29, 30) could be exploited to confer protection against these infections. The system could also provide diagnostic material useful to detect and monitor HSV-1 in-
This work was supported by the Italian National Research Council (Progetti Finalizzati “Biotecnologie e Biostrumentazione” e “Oncologia”), by North Atlantic Treaty Organization Research Grant 86170, and by Associazione ltaliana per la Ricerca sul Cancro (A.I.R.C.). We thank A. Bevilacqua and M. Bonazzi for excellent technical assistance and for preparing the manuscript.
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