VIRUS GENES 6:4, 379-386, 1992 © Kluwer Academic Publishers, Manufactured in The Netherlands

Transient Expression Assay in a Baculovirus System using Firefly Luciferase Gene as a Reporter TAMARA N. KOPYLOVA-SVIRIDOVA, ~ VALENTINA I. KRAUZOVA, I TATYANA M. TIMIRYASOVA, ~ TATYANA V. GORELOVAfl NIKOLAI G. SHUPPE, 2 AND ISTVAN FODOR x'3

1Laboratory of Genetic Engineering, Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchino, Moscow region, USSR; 21nstitutefor General Genetics, USSR Academy of Sciences, Moscow, USSR; 31nstitutefor Biochemistry and Protein Research, Agricultural Biotechnology Center, GOd6llb Hungary Received October 17, 1991 Accepted December 30, 1991 Requests for reprints should be addressed to I. Fodor, Institute for Biochemistry and Protein Research, Agricultural Biotechnology Center, P.O. Box 170, G6d/5116, Hungary.

Key words: recombinant DNA, gene expression, polyhedrin gene promoter, expression vector, DNA sequencing

Abstract

Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.

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Introduction

To use the baculovirus as a cloning and expression vehicle, transfer plasmid vectors have been designed that contain foreign gene under the control of baculovirus-specific promoter and flanking DNA segments involved in homologous recombination in vivo. Cotransfection of insect cells with a transfer (integrative) plasmid and viral DNA produces recombinant baculoviruses expressing foreign genes (1). Similar procedures for construction of recombinant viruses by homologous recombination have been earlier elaborated for herpes-, adeno-, and poxviruses (2-6). In studies with poxviruses very useful gene expression assays have been recently described (7-9) that allow monitoring of the transient (temporal) expression of foreign genes from the transfecting plasmids, prior to construction of recombinant viruses. However, a transient expression system for baculoviruses has not been reported. To analyze gene expression various reporter genes can be used: E. coli chloramphenicol acetyl transferase (cat), [3-galactosidase (lacZ), 13-glucuronidase (gus), neomycin phosphotransferase (neo), and xanthine-guanine phosphoribosyl transferase (Ecogpt). Recently the firefly luciferase reporter gene (luc) has been successfully employed in bacterial (I0), plant (11), mammalian (12), and viral (9,1315) systems. The firefly Photinus pyralis luciferase (Luc) is an enzyme catalyzing D(-) luciferin oxidation in the presence of ATP, Mg 2+ , and 02, yielding oxyluciferin, accompanied by the emission of yellow-green bioluminescence at 562 nm. Compared with other reporter genes, the luc gene assay is very specific and is 1000fold more sensitive than the cat and lacZ assays (12,13). Luc activity quantitatively can be measured using a luminometer; qualitative photodetection can be also obtained by exposing using special films. Here we describe a transient expression assay system for baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) using the luc gene as a reporter. It can be used to assess the functionality of promoters and the accuracy of promoter-gene fusions prior to construction of recombinant viruses.

Materials and Methods

Viruses and cell cultures A continuous cell lines BmN-4 derived from silkworm Bombyx mori, Sf-9 of the moth Spodoptera frugiperda and CV-t from green monkey kidney were obtained from the collection of the cell cultures in the Institute of General Genetics of the USSR Academy of Sciences in Moscow. Insect cells were maintained in TNM-

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FH medium supplemented with 10% fetal calf serum (16,17); CV-1 cells were grown in DMEM medium containing 5% fetal bovine serum and 10 mM Hepes. BmNPV and AcNPV were obtained from S. Maeda and M.D. Summers, respectively. Vaccinia virus (strain LIVP), originally obtained from the Institute of Viral Preparation in Moscow, was propagated and purified as reported previously (9).

Transfection procedure Transfection, plaque assay, and virus propagation selection of occlusion-negative plaques were performed basically according to Summers and Smith (17). BmN-4 or Sf-9 cells were seeded 2-3 hr prior to the transfection at a density of 5 x 106 cells per I0 cm plate. Two hours prior to the experiment the cell culture medium was aspirated and cells were cotransfected with 20-30 Ixg of plasmid and 10-20 Ixg of BmNPV or AcNPV DNAs in 1 ml/10 cm plate using the calcium phosphate procedure (18). After 30 min adsorption fresh FNM-FH medium with 10% fetal calf serum was added, and cells were incubated for 4 hr at 27°C. The transfection mixture was removed and 10 ml of fresh FNM-FH medium was added to each plate. Cells were harvested by scraping at different incubation times. Transfection of VV-infected CV-I cells has been described earlier (9).

Measurement of luciferase gene expression Luc gene expression was observed by measuring the Luc activity in the extract of cotransfected cells. Cells from I0 cm plates were collected by centrifugation and then suspended in 0.1-0.2 ml Hepes buffer (pH 7.4) given below, freezethawed, and dispersed by vortex. Luciferase activity was measured on a 1250LKB luminometer. The reaction mixture contained 25 mM Hepes (pH 7.4), 136 mM NaC1, I mM EDTA, 6 mM MgSO4, 5 mM ATP, and 1-100 ILl extract. The enzyme activity was initiated by the addition of 10 txm D(-) luciferin (Sigma) and was estimated in relative light units (mV) per 5 x l06 cells collected from a 10-cm plate or per milligram of protein (19). The measurement of Luc activity in the extract of CV-1 cells has been described earlier (9).

Plasmids Transfer vectors pBE274 (16) of BmNPV and pAc610 (17) of AcNPV were provided by S. Maeda and M.D. Summers, respectively. The construction of the plasmids pSCL47 and p19L4 was reported earlier (9,14). Firefly luciferase gene (pJD201) was obtained from Dr. D.R. Helinski.

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Results For transient expression studies a series of recombinant plasmids were designed. For the BmNPV system, plasmids pBE274L258 and pBE274L27 were constructed by inserting the HindIII-SmaI fragment of p19L4 containing a luc gene into the EcoRI site of pBE274. Thus, in these plasmids the luc gene was placed under the control of the BmNPV potyhedrin gene (polh) promoter in direct (pBE274L258) or reverse (pBE274L27) orientation relative to the promoter (Fig. 1A). Promoter-luc sequences in these plasmids were flanked with BmNPV D N A fragments adjacent to polh. For the construction of the plasmid pl9PPL13, the HpaI-BamHI fragment of pBE274L258 carrying the luc gene under the control of the BmNPV polh promoter was inserted between the HincII and BamHI sites

of pUCI9 (20). For the AcNPV system, plasmid p610L298 was constructed by inserting the BamHI fragment of pSCL47 into the BamHI site of the AcNPV transfer vector pAc610. In the resulting plasmid the luc gene was flanked with the 3' terminal

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portion (63 bp) of polh and adjacent AcNPV sequences--from the 5' end, and with the polh promoter and adjacent viral D N A - - f r o m the 3' end (Fig. 1B). Plasmid p610L44 differs from p610L298 by a 0,5 kb insert from pSCll (21) containing the vaccinia virus (VV) late promoter P11 at the 5' end of the luc gene. Deletion of the EcoRI-EcoRV fragment (99 bp) of p610L298 comprising the transcription start point and a significant part of the polh promoter sequences resulted in the plasmid p610PPLI2. In contrast to transfer plasmids pBE274L258, pBE274L27, and p610L298, which contain flanking sequences of the viral DNA involved in homologous recombination with the viral DNA, plgPPL13 is a nonintegrative vector, since it lacks flanking baculovirus-specific sequences. Transient expression of a foreign gene in a baculovirus system requires the simultaneous presence of the plasmid containing the foreign gene and the replicating virus in the same cell, In BmNPV system insect cells, Bm-4 were cotransfected with plasmids carrying the luc gene and BmNPV DNA. Similarly, in AcNPV system Sf-9 cells were cotransfected with plasmids carrying luc and AcNPV DNA. The efficacy of transient expression in both systems were analyzed by measuring luc gene expression. Experiments showed that the maximal transient gene expression in both baculovirus systems was established at 6 days of incubation. Data regarding BnNPV gene expression assay with the plasmid pBE274L258 are presented in Fig. 2. Therefore, in an experiment described in Fig. 1, where all plates were transfected in an identical manner, the luc expression was measured at 6 days after transfection. In the BmNPV transient expression system, high luc gene expression was achieved with the plasmids p19PPL13 and pBE274L258 carrying luc gene under the control of the polh promoter (Fig. 1A). Plasmid pBE274L27 in the same expression system resulted also in a significant decrease in luc gene expression, apparently due to the reverse orientation of the luc gene and the polh promoter. A low level of Luc activity was observed in the control experiments with the plasmids pSCL47 and p19L4, which did not contain virus-specific promoter sequences (Fig. IC). In the AcNPV transient expression system, high luc gene expression was achieved with the plasmid p610L298 (Fig. IB). Interestingly, luc gene expression in this plasmid was directed from the AcNPV promoter located on the 3'-terminus to the polh DNA segment or adjacent AcNPV sequences. Insertion of the VV late promoter at the 5'-end of the luc gene (p610L44) caused a high level of luc expression in the VV transient expression system and, compared with p610L298, a sixfold decrease in the AcNPV system (Fig. 1B). For comparison, plasmid pSCL47 containing the luc gene under the control of the VV early (P7.5) promoter induced efficient Luc expression in the VV system, but a very low level of expression in the baculovirus system (Fig. 1C). Surprisingly, deletion of the authentic AcNPV polh promoter (p610PPL12) compared to p610L298 reduced luc gene expression eightfold (Fig. 1B). In several

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attempts we failed to construct a plasmid carrying the luc gene under the control of the AcNPV polh promoter. A low level of Luc activity in Sf-9 cells was observed with the plasmids pSCL47and p19L4 (Fig. 1C). In control experiments without transfecting plasmids, Luc activity was not observed at all in two insect cell lines transfected with corresponding viral DNA, nor in CV-1 cells infected with VV.

Discussion

The usefulness of the transient expression assay for cytoplasmic poxviruses has been previously demonstrated (7-9). Here we describe how cotransfection of plasmid and viral DNA can be applied for transient expression studies in nuclear baculovirus systems as well. Two baculoviruses, BmNPV and AcNPV, have been successfully tested in the expression assay using reporter firefly luciferase gene. Application of the luc gene reporter significantly increases the sensitivity of the expression systems, as has been observed earlier by others (13) and in our lab (9). The luc gene of the nonintegrative plasmid pl9PPLI3 with the polh promoter was expressed in BmN-4 cells by four orders of magnitude higher than the plasmid p19L4 carrying bacterial promoter. Since PI9PPL13 does not contain the baculovirus-specific sequences required for homologous recombination in vivo,

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luc gene expression from the newly formed recombinant viruses is excluded. Thus, the Luc should be expressed entirely from the transfected plasmid pl9PPLI3. Since the difference in luc gene expression at transfection of BmN-4 cells with pI9PPL13 and integrative plasmid pBE274L258 was insignificant, we suggest that the transient expression assay can be applied for both transfer and nonintegrative plasmids. In the AcNPV transient expression system we have found that the luc gene of the plasmid p610L298 is expressed under the control of a promoter located at the 3'-terminal coding region of polh or adjacent sequences. Insertion of the VV late promoter between AcNPV sequences and luc gene (p610L44), as we expected, reduced the efficiency of luc expression. Interestingly, we found that the polh promoter located at the 3'-end of the luc gene enhanced the expression of the gene, as was demonstrated with the plasmid p610PPLI2: Deletion of a substantial part (99bp) from the promoter region significantly decreased luc expression (Fig.

1B). Thus, the transient expression assays for baculoviruses BmNPV and AcNPV using luc as a reporter allow the estimation of functional activity of the construction so that the tested recombinant plasmids might be further employed for the construction of integrative plasmids or recombinant viruses. This assay should also be useful in studies on early events of in vivo recombination between transfected plasmid and viral DNAs, and recombinant baculovirus reproduction in a cell culture.

Acknowledgments We thank Drs. S. Maeda, M.D. Summers, and D.R. Helinski for providing us with the viruses and plasmids mentioned in Materials and Methods.

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