An Additional Copy of the Homologous Region (hr1) Sequence in the Autographa californica Multinucleocapsid Polyhedrosis Virus Genome Promotes Hyperexpression of Foreign Genes ² Betapudi Venkaiah, ‡,§ Priya Viswanathan, ‡,| Saman Habib, ‡,⊥ and Seyed E. Hasnain* ,|,# Eukaryotic Gene Expression Laboratory, National Institute of Immunology, New Delhi 110067, India, Laboratory of Molecular and Cellular Biology, CDFD, Hyderabad 500076, India, and Jawaharlal Nehru Centre for AdVanced Scientific Research, Bangalore 560 064, India ReceiVed January 6, 2004; ReVised Manuscript ReceiVed April 15, 2004 ABSTRACT: The Autographa californica multinucleocapsid nuclear polyhedrosis virus genome contains nine homologous region (hr1, hr1a, hr2, hr2a, hr3, hr4a, hr4b, hr4c, and hr5) sequences that are thought to be involved in viral replication and activation of transcription. Our results show that the 750 bp hr1 sequence is capable of functioning as an enhancer of transcription of foreign genes from the homologous late polyhderin gene promoter and the heterologous Drosophila heat shock protein (hsp70) promoter in insect cells. Introduction of an additional copy of the complete hr1 element downstream to the polyhedrin locus in the viral genome, while not affecting the stability of the recombinant virus for at least 30 serial passages, led to hyperexpression of reporter genes. The enhancement in the expression levels of foreign genes varied from 40 to 90-fold depending on the promoter used. The baculovirus expression vector system (BEVS) utilizing Autographa californica multinucleocapsid nuclear poly- hedrosis virus (AcMNPV) 1 continues to be widely used for the production of recombinant proteins in insect cells (1- 6). This system employs the viral very late and powerful polyhedrin (polh) and p10 gene promoters that allow abundant expression of foreign genes in a temporally regulated manner. The circular supercoiled double-stranded DNA genome of 134 Kb encodes around 150 polypeptides (7, 8). AcMNPV expression undergoes tight regulation at the transcriptional level and occurs in an ordered fashion through early and very late phases (9, 10). The viral genome consists of nine homologous region (hr1, hr1a, hr2, hr2a, hr3, hr4a, hr4b, hr4c, and hr5) sequences of 120-800 bp in length (11). Because of their high A + T content, symmetric location throughout the viral genome, presence of imperfect palindromes, and the ability to serve as origin of replication (ori) in transient replication assays, the hrs are thought to be involved in viral replication (12). Whether any of these are indeed essential for and function as viral replication origins in vivo is still not known. The hrs have also been shown to act as transcriptional enhancers of reporter genes driven by the baculovirus early and delayed-early promoters (13-19). We previously demonstrated that hr1, located ∼3.7 kb upstream of the polh gene in the wild-type AcMNPV genome, enhances transcription from this very late viral promoter in a position and orientation independent manner in plasmid based transient expression assays (20, 21). Enhancement of expression was demonstrated to be a direct result of enhanced transcription from the polh promoter and was independent of the ori function of hr1. We also showed that a 38 kDa host protein, hr1-binding protein (hr1-BP), interacts with high specificity and affinity with functionality relevant motifs at multiple sites within hr1 (21). We have now analyzed the effect of hr1 on the expression of reporter genes driven by the Drosophila heat shock protein (hsp70) promoter and polh promoter using recombinant viruses carrying the hr1 element cloned downstream in the poly- hedrin locus in the baculovirus genome. A dramatic increase in foreign gene expression, driven by homologous and heterologous promoters is evident as a consequence of the presence of the extra copy of the hr1 element without affecting virus stability. This approach can be used for producing recombinant protein products of therapeutic inter- est (22) at much higher levels in the baculovirus insect cell system. EXPERIMENTAL PROCEDURES Virus and Cell Culture. Spodoptera frugiperda cells (Sf9 and Sf21) were maintained at 27 °C in TNMFH medium (Life Technologies Inc.) supplemented with 10% fetal calf serum (23). Cells were infected with virus at 0.1 plaque- forming units per cell (PFU/cell) for serial passage. For other ² This work was supported by the Department of Biotechnology, Government of India to CDFD. P.V. is a recipient of a Senior Research Fellowship from the Council of Scientific and Industrial Research, Government of India. * Corresponding author. Tel: 91-40-27155604. Fax: 91-40- 27155610. E-mail: ehtesham@cdfd.org.in. ‡ National Institute of Immunology. § Current address: Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106- 4970. | CDFD. ⊥ Current address: Membrane Biology Division, Central Drug Research Institute, Chattar Manzil, Lucknow-226001, India. # Jawaharlal Nehru Centre for Advanced Scientific Research. 1 Abbreviations: AcMNPV, Autographa californica multinucleo- capsid polyhedrosis virus; hr1, homologous region 1; polh, polyhedrin; hsp70, Drosophila heat shock protein 70; hpi, hours post infection. 8143 Biochemistry 2004, 43, 8143-8151 10.1021/bi049953q CCC: $27.50 © 2004 American Chemical Society Published on Web 05/29/2004