VIROLOGY

185,428-431

(1991)

Expression

of Epstein-Barr WILLIAM

Virus Nuclear Antigen

H. SCHUBACH,’

GARY HORVATH,

2 in insect Ceils from a Baculovirus

Vector

BRENT SPOTH, AND JANET C. HEARING

Division of Oncology, Department of Medicine, HSC T- 17,080, State University of New York at Stony Brook, Stony Brook, New York 1179@8 174 Received May 20, 199 1; accepted August 15, 199 1 The open reading frame encoding the Epstein-Barr virus nuclear antigen 2 (EBNA-2) has been expressed in a recombinant baculovirus vector. The resulting product migrates with the same apparent molecular weight as EBNA-2 from latently infected or converted B cell lines. Rabbit antisera derived from the innoculation of this material immunoprecipitated EBNA-2 from cell extracts of EBV-containing cells. The high level of protein expression obtained in insect cells stands in sharp contrast to that seen in a number of mammalian cell lines using a variety of promoters including the endogenous EBNA-2 promoter, the Moloney MuLV LTR, the murine immunoglobulin heavy chain promoter, the Q 19~1 Academic PMS, IIN. human cytomegalovirus immediate early promoter, and the adenovirus major late promoter.

Epstein-Barr virus (EBV) establishes a latent infection in human B lymphocytes which can then be propagated under routine culture conditions (I). This in vitro growth transformation probably reflects the in vivo occurrence of circulating, latently infected B lymphocytes in previously infected persons and is related to the well-documented observation of EBV-associated lymphoproliferative diseases in immunosuppressed humans (2) and in scid mice reconstituted with human lymphocytes from EBV-positive donors (3). Several studies have demonstrated that the EBV nuclear antigen 2 (EBNA-2) is necessary for 6 cell immortalization (4, 5). It is likely that the immortalization function of EBNA-2 is mediated by its activity as a transcriptional transactivator, since it has been shown that EBNA-2 can activate transcription of the viral terminal protein (6) and the latent membrane protein (7) genes and either alone (8) or in combination with the viral latent membrane protein (9, 70) can activate the expression of the lymphocyte receptor for IgE (CD23). The DNA sequence of the EBNA-2 open reading frame (or-f) of the M-ABA strain of EBV predicts a polypeptide of 490 amino acids containing a stretch of 44 prolines, an acidic region near the carboxyl terminus, and an internal basic region (11, our unpublished data). The low level of expression of EBNA-2 in latently infected cells has precluded some important functional studies, prompting us to seek a suitable system for its high level expression. In order to express EBNA-2, the plasmid pE2 was constructed by removing the or-f encoding EBNA-2 contained in the Thai-Dral fragment of ~780-28 (72), ligating &/II linkers, and cloning this into a unique Bglll

site of a derivative of PBS, pVZ1. The EBNA-2 or-f was excised from pE2 with Xbal, which flanked the or-f in pE2, and was cloned into the Nhel site of the baculovirus expression plasmid pJVP1 OZ (13) by blunt-end ligation to create pJE2. This vector drives expression of EBNA-2 from the viral polyhedrin promoter and the fscherichia co/i fl-galactosidase gene from the PlO promoter in divergent directions. pJE2 contains 21 bp of EBV sequences upstream from the ATG initiating the EBNA-2 orf and its orientation was verified by sequence analysis. Recombination of the vector containing EBNA-2 into the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome resulted in the generation of virions expressing both EBNA-2 and the E. co/i P-galactosidase gene, the latter being detectable by cleavage of the chromogenic substrate x-gal. The methods for construction of recombinant baculoviruses expressing EBNA-2 were essentially those described by Summers and Smith (14). Candidate viral clones were plaque purified through three rounds to yield stable recombinant virus stocks. These were picked and grown on Sf9 cells in small cultures for 3 days, and the infected cells were harvested, lysed, and screened by immunoblot analysis using human serum recognizing EBNA-2. Figure 1A shows a Coomassiestained gel of lysates of Sf9 cells infected with four independently cloned stocks of the EBNA-2 recombinant baculovirus strain, AcE2. A prominent band representing approximately 10% of the total cellular protein is present in AcE2-infected cells, but absent from uninfected (data not shown) and AcMNPV-infected Sf9 cells and is indicated by an arrow. Figure 1 B shows the results of an immunoblot of the same lysates probed with human serum recognizing EBNA-2. The expressed protein was immunoprecipitated with a rabbit serum directed against the carboxyl-terminal

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B. 1234

5

FIG. 1. Expression of EBNA-2 in lysates of Sf9 cells infected with clonal isolates of AcE2. (A) Coomassie stain of SDS-PAGE (15) of nonrecombinant AcMNPV and (lane 1) four clones (lanes 2-5) derived from the initial screen of candidate recombinant viruses. The arrow in the first sample lane indicates the polyhedrin polypeptide of the nonrecombinant virus. The arrow on the right designates the immunoreactive EBNA-2 protein expressed in cells infected with donal isolates of AcE2. (B) lmmunoblot of the same samples shown in A probed with a human serum recognizing EBNA-2. The unmarked lane contains molecular weight markers: myosin, phosphorylase b, bovine serum albumin, olvalbumin, and carbonic anhydrase.

100 amino acid residues of EBNA-2 (76) (data not shown). The recombinant protein was used to immunize rabbits to generate antiserum Rb422. The immunogen was prepared by four cycles of freezing and thawing A&2-infected cells, followed by boiling in 5% ,&mercaptoethanol and 1% SDS, and dialysis against 10% sucrose. This antiserum was used in furtheranalysis of EBNA-2 expression and it precipitated EBNA-2 from EBV-infected cell lines as shown in Fig. 2. This serum fails to precipitate immunoreactive material from the EBV-negative cell line BL41 or from BL41/ P3HR-1 (17) cells. The latter have been converted in vitro with the variant of EBV, P3HR-1, that has suffered a deletion of the EBNA-2 orf (4). The serum also fails to precipitate immunoreactive material from uninfected Sf9 cells. Prominent immunoreactive bands are found in IB4 (18) cells, BL41/B95 (17) cells, and the JIJOYE (19) cells which express a low molecular weight variant of EBNA-2 (20). The same EBNA-2 sequences that were cloned into pJE2 were cloned into several other expression vectors resulting in either low or undetectable levels of EBNA-2 expression. The retroviral plasmid pSVE2 (Fig. 3) was derived from pSVx(S)Zl (21) by removing the EBNA-2 or-f from pE2 by Bglll digestion and ligation into the unique BarnHI site of pSVx(S)Zl. After calcium phosphate-mediated transfection (22) into NIH/3T3 cells and selection of G418-resistant clones, the majority were found to express levels of EBNA-2 that were detectable by immunoblotting (Fig. 4A). When the same vector was used in an effort to achieve transient ex-

429

pression in BL4 1/P3HR- 1 ceils following efectroporation (IO), no EBNA-2 expression was detectableby immunoblotting of an immune precipitation (data not shown). In the latter experiment we monitored transfection efficiency by assay of chloramphenicof acetyltransferase activity using the plasmid pSV2CAT (23). In all experiments we found 7.5-l 0% conversion of chloramphenicol to the acetylated form. A plasmid, pU430/23 (IO), which utilizes the endogenous promoter of EBNA-2 expressed Ic+v levels of EBNA-2 protein detectable by immune precipitation in a transient expression assay following efectroporation of BL4 l/ P3HR-1 cells (data not shown). The expression plasmid pHlg was made by cloning the immunoglobulin promoter-enhancer cassette from pLNlL (K. Marcu, unpub4ished) into the C/al site of pHEBo (24). The EBNA-2 orf was excised from pE2 by cleavage with Bglll and ligated into the Ss-/ll site of pHlg to create pHIE2 (Fig. 3). The EBNA-2 or-f was placed into the plasmid p359 (B. Sugden, unpublished) under the control of the human cytomegalovirus immediate early promoter by excision of the EBNA-2 orf from pE2 with SalI and 8g/ll, ligation into p359 modified by removal of vector sequences encoding dihydrofolate reductase, replacing this with a Xhol-BarnHI linker, cleavage, and religation to create pCE2 (Fig. 3). Both of these constructs failed to direct expression of EBNA-2 following either transient or stable transfection. For transient expression we performed immune precipitation 48 hr after electroporation of BL4l/P3HR-1. In this case, transfection efficiency was monitored by CAT assays. For stable transfection, D98/HRl (25) cells were transfected by the calcium phosphate technique

abc

de

f

g

FIG. 2. lmmunoblot of immune precipitations of several ceil lines using antiserum Rb422. Approximately 10’ celts or 0.5 Bg of partially purified EBNA-2 from an extract of AcE2lnfected Sf9 odb (lane g) was immune precipitated using serum Rb422 and immunoblotted with human serum recognizing EBNA-2. Immune compi@xes were detected with ?-protein A (ICN, IO &i/&g). (a) Uninfected Sf9; (b) BL41; (c) BL41/P3HR-1; (d) BL41/B95; (e) IB4; (f) lijoye; (g) 0.5 Ng of baculovirus-expressed EBNA-2.

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FIG. 3. Expression constructs encoding EBNA-2. pU430/23 has been previously described (70). Indicated on the map are EBV BarnHI fragments DW, W, DYl , and DHl (11). pSVE2 contains the SV40 origin of replication and the MuLV-LTR. pHIE2 contains the IgH promoter and the IgH enhancer, and pCE2 contains the CMV promoter. Both pHIE2 and pCE2 contain the EBV oriP. pNLE2 contains the adenovirus Ela enhancer, the major late promoter (MLP), the tripartite leader sequence (TPL), and the El b polyadenylation signal.

followed by ,selection of hygromycin-resistant cell lines. D98/HRl is a hybrid epithelial cell line that expresses EBNA-1 and thus permits the replication of the EBV oriP-containing constructs pCE2 and pHIE2. In these cases, the presence of plasmid DNA was detected in transfectants by Southern analysis (data not shown). Figure 4C shows the results of stable transfection of D98/HRl cells with pHIE2. The same results

A 1234567

FIG. 4. Analysis of EBNA2 expression in expression vectors depicted in Fig. 3. (A) lmmunoblot of NIH/3T3 cells probed with human serum after transfection with pSVE2. Lane (1) Ramos, an EBV-negative Burkitt lymphoma cell line (29); (2) Raji cells (30); (3) BL41/B95 cells; (4) untransfected NIH/3T3 cells; (5) NIH13T3 cells transfected with vector alone; (6) NIH/3T3 cells transfected with the vector having EBNA-2 in the antisense orientation; (7) NIH/3T3 cells transfected with the vector having EBNA-2 in the sense orientation. (B) lmmunoblot using human serum to demonstrate expression of EBNA-2 by recombinant adenovirus. (1) Ramos cells: (2) Raji cells; (3) BL41/B95 cells; (4) uninfected 293 cells; (5) 293 infected with d1309; (6) 293 cells infected with EBNA-2-containing adenovirus recombinant. (C) lmmunoblot of immune precipitations of D98HR-1 cells transfected with pHIE2. Lane (1) BL41/B95 cells; (2) D98HR-1 cells; (3) D98HR-1 cells transfected with vector alone; (4) D98HR-1 cells transfected with pHIE2.

were obtained with pCE2 (data not shown). In addition, RNA blot analyses failed to detect any EBNA-2 mRNA in transfected D98/HRl following introduction of pCE2 and pHIE2 constructs, whereas EBNA-2 RNA was readily detected following introduction of pU430/23 (data not shown). Recombinant, helper-free adenovirus expressing EBNA-2 (26) was generated by excising the EBNA-2 orf from pE2 by SalI and Bglll digestion followed by ligation into the vector pNL3C (R. Schneider, unpublished) previously digested by the same enzymes to create pNLE2 (Fig. 3). The plasmid pNLE2 was linearized with EcoRl and cotransfected into 293 cells by the calcium phosphate technique with C/al and Xbal cleaved Ad5dl309. Resulting plaques were harvested and recombinant virus stocks identified by restriction analysis. Intermediate levels of EBNA-2 expression were seen from this construct which expresses EBNA-2 from the major late promoter (Fig. 4B). The wide variation in the levels of EBNA-2 expression achievable with the various vectors used in the present study suggests a feature of the biology of EBNA-2. The RNA is expressed at fairly low levels in latently infected cells (18). Furthermore, several lines of evidence suggest that EBNA-2 acts either directly or in cooperation with other factors as a transcriptional transactivator. Thus high level expression in mammalian cells in general, and in human B cells in particular, may be detrimental to the expressing cell as a result of indiscriminant activation of genes which downregulate cell growth, resulting in selection against EBNA-2 expressing ceils. An alternative mechanism accounting for the low levels of expression of EBNA-2 in stably transfected cells would be titration of essential transcription factors necessary for cell growth by the stoichiometric excess of EBNA-2, the phenomenon termed “squelching” (27). This model would account for the absence of EBNA-2 expression in cells transfected with the CMV and immunoglobulin promoter constructs, both of which would be predicted to spec-

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ifY high levels of expression in human B cells. Consistent with this explanation is our finding of a potent transcriptional activation domain in EBNA-2 when assayed in yeast cells (Horvath and Schubach, in preparation); however, the failure to detect EBNA-2 mRNA following transient transfection of the nonexpressing constructs argues against this mechanism. It is possible that the low expressing vectors fail to produce significant levels of EBNA-2 because of the failure of processing or export of the mRNA. By contrast, the retroviral construct directs low levels of expression in stably transfected NIH/3T3 cells. Perhaps the context, murine cells rather than human B cells, does not limit EBNA-2 expression by the same constraints as a result of the evolutionary divergence of transcription factors. The high levels of expression seen in insect cells may result from several factors: The efficient polyhedrin promoter of A. californica is utilized; expression is transient, occurring maximally during an interval between 60 and 80 hr (data not shown); and the cellular environment may be sufficiently divergent from that of human B cells to avoid the potential deleterious effects of EBNA-2 overexpression or squelching. The analysis of recombinant EBNA-2 presented here is limited by the lack of a functional assay for the protein. We have succeeded in biochemically purifying the protein by conventional chromatographic methods, and the resultant material will prove valuable for structural and functional studies of EBNA-2 since it permits us to purify large amounts of the protein. It may also prove to be of clinical utility since it can specifically detect anti-EBNA-2 antibodies. The latter have been shown to rise early in the course of infectious mononucleosis and to remain high in some cases of chronic EBV infection (28). ACKNOWLEDGMENTS This work was supported by grants Al25893. We thank Christine Olsen for We also thank 0. Lenoir, C. Richardson, Bornkamm, R. Schneider, B. Sugden, K. and R. Glaser for cells and plasmids.

from the NIH, Al29466 and expert technical assistance. F. Grasser, M. Summers, G. Marcu, M. Viola, P. Hearing,

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