The Homologous Region Sequence (hr1) of Autographa californica Multinucleocapsid Polyhedrosis Virus Can Enhance Transcription from Non-baculoviral Promoters in Mammalian Cells

Abstract

The Autographa californica multinucleocapsid polyhedrosis virus homologous region sequence hr1 enhances transcription from the viral polyhedrin promoter in Spodoptera frugiperda insect cells and independently functions as an origin of replication (ori) sequence. The binding of the host nuclear protein, hr1-binding protein (hr1-BP), is crucial for the enhancer activity (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737–747 and Habib, S., and Hasnain, S. E. (1996) J. Biol. Chem. 271, 28250–28258). We demonstrate that hr1 can also enhance transcription from non-baculoviral promoters like cytomegalovirus and hsp70 in mammalian cells but does not support ori activity in these cells. Unlike insect cells, hr1 can also function in mammalian cells as an enhancer when present in trans. hr1 DNA sequence binds with high affinity and specificity to nuclear factors in the mammalian cells. The insect hr1-BP- and the hr1-BP-like proteins from mammalian cells (mhr1-BP) have different properties with respect to ion requirements, DNA groove binding, and molecular size. When mammalian cells are infected with a recombinant baculovirus containing two promoters, the baculovirus polyhedrin and Drosophila hsp70 gene promoter, the hsp70 gene promoter alone is active in these cells, and this activity is further enhanced by the presence of an additional hr1 in the recombinant virus. hr1 may thus also have a role in baculovirus-mediated gene delivery in mammalian cells. The Autographa californica multinucleocapsid polyhedrosis virus homologous region sequence hr1 enhances transcription from the viral polyhedrin promoter in Spodoptera frugiperda insect cells and independently functions as an origin of replication (ori) sequence. The binding of the host nuclear protein, hr1-binding protein (hr1-BP), is crucial for the enhancer activity (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737–747 and Habib, S., and Hasnain, S. E. (1996) J. Biol. Chem. 271, 28250–28258). We demonstrate that hr1 can also enhance transcription from non-baculoviral promoters like cytomegalovirus and hsp70 in mammalian cells but does not support ori activity in these cells. Unlike insect cells, hr1 can also function in mammalian cells as an enhancer when present in trans. hr1 DNA sequence binds with high affinity and specificity to nuclear factors in the mammalian cells. The insect hr1-BP- and the hr1-BP-like proteins from mammalian cells (mhr1-BP) have different properties with respect to ion requirements, DNA groove binding, and molecular size. When mammalian cells are infected with a recombinant baculovirus containing two promoters, the baculovirus polyhedrin and Drosophila hsp70 gene promoter, the hsp70 gene promoter alone is active in these cells, and this activity is further enhanced by the presence of an additional hr1 in the recombinant virus. hr1 may thus also have a role in baculovirus-mediated gene delivery in mammalian cells. Enhancers are genetic elements that up-regulate gene expression by enhancing transcription of genes, even when placed several thousand nucleotides away in a position or orientation-independent fashion. The SV40 enhancer, the first enhancer to be discovered (1Moreau P. Hen R. Wasylyk B. Everett R. Gaub M.P. Chambon P. Nucleic Acids Res. 1981; 9: 6047-6068Crossref PubMed Scopus (378) Google Scholar, 2Banerji J. Rusconi S. Schaffner W. Cell. 1981; 27: 299-308Abstract Full Text PDF PubMed Scopus (892) Google Scholar) and the most extensively studied, represents the best example of a prototypical enhancer that functions in a wide variety of cell types from different species. It is even active in non-mammalian systems like Xenopus laevis kidney cells or even in the green alga Acetabularia, and, in some cases, it can confer cell specificity or host-range response to the promoter (3Laimins L.A. Khoury G. Gorman C. Howard B. Gruss P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6453-6457Crossref PubMed Scopus (263) Google Scholar, 4de Villers J. Olson L. Tyndall C. Schaffner W. Nucleic Acids Res. 1982; 10: 7965-7976Crossref PubMed Scopus (80) Google Scholar). The baculovirus Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) 1The abbreviations used are: AcMNPVA. californica multinucleocapsid polyhedrosis virushr1homologous region 1hr1-BPhr1-binding proteinmhr1-BPhr1-binding protein from mammalian cellspolhpolyhedrinEMSAelectrophoretic mobility shift assayh.p.i.hours post infectionCMVcytomegalovirus.1The abbreviations used are: AcMNPVA. californica multinucleocapsid polyhedrosis virushr1homologous region 1hr1-BPhr1-binding proteinmhr1-BPhr1-binding protein from mammalian cellspolhpolyhedrinEMSAelectrophoretic mobility shift assayh.p.i.hours post infectionCMVcytomegalovirus. genome consists largely of unique sequences. Interspersed, however, are nine homologous region (hr) sequences called hr1, hr1a, hr2, hr2a, hr3, hr4a, hr4b, hr4c, and hr5 (5Ayers M.D. Howard S.C. Kuzio J. Lopez-Ferber M. Possee R.D. Virology. 1994; 202: 586-605Crossref PubMed Scopus (840) Google Scholar, 6Rasmussen C. Leisy D.J. Ho P.Y. Rohrmann G.F. Virology. 1996; 224: 235-245Crossref PubMed Scopus (22) Google Scholar). The hrs vary in length from 150 to 800 bp and have a modular structure containing two to eight imperfect, 30-bp palindromes with an EcoRI site at the center of each palindrome. Homologous region 1 (hr1) has been shown to be an enhancer for the immediate early gene ie-N and delayed early gene 39k (7Carson D.D. Summers M.D. Guarino L.A. J. Virol. 1991; 65: 945-951Crossref PubMed Google Scholar). We earlier reported that hr1 enhances transcription from the polyhedrin promoter in a classic enhancer-like manner (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar, 9Habib S. Hasnain S.E. J. Biol. Chem. 1996; 271: 28250-28258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 10Hasnain, S. E., Venkaiah, B., and Habib, S. (October 12, 1999) U. S. Patent 5,965,393Google Scholar). hr1-mediated enhancement is position- and orientation-independent and can activate transcription to about 10-fold in a plasmid-based transient expression system and to more than 90-fold when inserted as an additional copy within the viral genome (10Hasnain, S. E., Venkaiah, B., and Habib, S. (October 12, 1999) U. S. Patent 5,965,393Google Scholar). Enhancement mediated by hr1 follows the temporal profile of very late gene expression (20–60 h.p.i.) and was a direct function of the number of modules (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar). hr1 is bifunctional, i.e. it is both a transcriptional enhancer and an ori of replication (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar), and these functions of hr1 are independent of each other. Although the palindrome alone is sufficient for the ori function, the palindrome plus the flanking sequence is essential for the enhancer function. A host cell protein, named hr1-BP (9Habib S. Hasnain S.E. J. Biol. Chem. 1996; 271: 28250-28258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), which binds very specifically to the palindrome plus the flanking sequence, is required for the enhancer function. We also showed that hr1 can stimulate expression from other homologous promoters like the AcMNPV late promoter cor (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar) and very late promoter p10 and from heterologous promoters such as Drosophila hsp70 in insect cells. 2P. Viswanathan and S. E. Hasnain, unpublished observation.2P. Viswanathan and S. E. Hasnain, unpublished observation. A. californica multinucleocapsid polyhedrosis virus homologous region 1 hr1-binding protein hr1-binding protein from mammalian cells polyhedrin electrophoretic mobility shift assay hours post infection cytomegalovirus. A. californica multinucleocapsid polyhedrosis virus homologous region 1 hr1-binding protein hr1-binding protein from mammalian cells polyhedrin electrophoretic mobility shift assay hours post infection cytomegalovirus. In this study, we describe the ability of the baculovirus hr1 to function as an enhancer, both when placed in cis as well as trans, but not as an ori sequence in heterologous mammalian cells. These cells also contain protein factors that bind to hr1, but these binding proteins are distinct from hr1-BP present in insect cells. This enhancement of gene expression can be exploited for the development of improved vectors for baculovirus-mediated gene delivery in mammalian cells. Cells and Virus—Spodoptera frugiperda cells (Sf9) were grown in TNMFH medium (Invitrogen) containing 10% fetal bovine serum as described previously (11Chatterji U. Ahmad R. Venkaiah B. Hasnain S.E. Gene (Amst.). 1996; 171: 209-213Crossref PubMed Scopus (15) Google Scholar). All the mammalian cell lines used namely, Vero (African monkey kidney epithelial cell line), COS-1 (African green monkey kidney fibroblast-like, SV40-transformed cell line), HepG2 (human hepatocyte cell line), HeLa (human adenocarcinoma cell line), and PS-1 (porcine kidney cell line) cell lines were cultured in Dulbecco's modified Eagle's medium containing high glucose. The C6 strain of wild type AcMNPV was used in infection experiments. Recombinant viruses vAclacluc and vAclacluc-hr1 were constructed (10Hasnain, S. E., Venkaiah, B., and Habib, S. (October 12, 1999) U. S. Patent 5,965,393Google Scholar, 11Chatterji U. Ahmad R. Venkaiah B. Hasnain S.E. Gene (Amst.). 1996; 171: 209-213Crossref PubMed Scopus (15) Google Scholar) using pBacPAK8 (Clontech, Palo Alto, CA) baculovirus transfer vectors as per standard procedures. The recombinant viruses were purified and titrated, and viral infection was carried out at a multiplicity of infection of 10 for each virus. To ascertain that equal of viral DNA from the different the insect cells, equal of the were a the by with the and (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar). was constructed by a from the promoter, and SV40 the site within the site of to a was by with and was to a hr1 as from (9Habib S. Hasnain S.E. J. Biol. Chem. 1996; 271: 28250-28258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), to with different and number of hr1 and have hr1 to the promoter in the or as present in the viral genome to the polyhedrin and have two of hr1 in the or to the promoter, and have a hr1 placed to the in the or of insect cells was carried out using as described previously (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar). cell were using the of cells were the cells were with to of of was in of of was to and the was for This was to containing of in of medium and for and the DNA was to cells and the was to 1 1 of fetal bovine was to the cells by infection with wild type or recombinant viruses or with transfer of infected cells were at different and or expression were as described (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar, 11Chatterji U. Ahmad R. Venkaiah B. Hasnain S.E. Gene (Amst.). 1996; 171: 209-213Crossref PubMed Scopus (15) Google Scholar). All the both and viral were in at To out by of DNA the cells, it was that equal of DNA from the different the insect cells. equal of the the were a and with the DNA or DNA as by of the protein were from cells or the mammalian cells using a modified (9Habib S. Hasnain S.E. J. Biol. Chem. 1996; 271: 28250-28258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, S.E. S. Jain A. Burma S. B. 1996; PubMed Scopus Google Scholar). with nuclear from both insect and mammalian cells were carried out by of nuclear with of The was carried out in a containing 10 1 and 1 of at for The was a and at 150 in and 1 In a or of the was to the to the of the nuclear the of the binding, the containing the ion was to the at the required To the with and the hr1 was for at with of A or green and for binding by with the nuclear DNA was insect and mammalian cells, by infection with wild type AcMNPV virus or infection at as in the The cell DNA was at different post or infection as described (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar) and in of To assay for of DNA was with to the and with both and to for (9Habib S. Hasnain S.E. J. Biol. Chem. 1996; 271: 28250-28258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The DNA was and hr1 from the in a and in is a classic enhancer of transcription from the AcMNPV promoters in insect cells. To hr1 can enhance expression in mammalian cells, transient were The expression from Vero cells, with hr1 sequence, was with cells with the hr1 sequence. The presence of hr1 and in the to the promoter as in the viral genome or but in or in in in expression the two of the hr1 sequence in an or position not additional with the were in other mammalian cells. enhancement of expression was in HepG2 cells and PS-1 cells of the position and of hr1 to the promoter not When the were cells, a enhancement is not These that hr1 can function as an enhancer of promoter in both mammalian cells and cells in a classic enhancer-like fashion. hr1 from and in hr1-mediated transcriptional enhancement with other promoters and the was This the hsp70 and the baculovirus polyhedrin with a copy of the hr1 sequence The expression of the from cells with this was with a that not the hr1 sequence. In Vero cells, about in expression in the the is The enhancement by hr1 is more or of the used not COS-1 cells and HepG2 cells also hr1-mediated in to a with Vero cells not hr1 enhance expression of from hsp70 promoter in different heterologous host cells. hr1 When in in are DNA In the have been of of also in M. P. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). were to hr1, in insect cells, can also function in in mammalian cells. hr1 sequence but with the promoter gene was Vero cells with different of containing the hr1 in in was to the to of When Vero cells were with the of and and of an in expression is as a direct function of These demonstrate that hr1 sequence can as an enhancer when present in trans. this enhancement is a direct function of the of the in cells. hr1-BP-like in the with hr1 is a containing palindromes as in with EcoRI site at the center of each palindrome. A enhancer an EcoRI palindrome and flanking sequences. hr1 was to with number of palindromes and flanking sequences The was used in an electrophoretic mobility shift assay to for the presence of hr1-binding proteins in mammalian cells. A distinct in the of the can be when the is with mammalian Vero cell nuclear The binding of or more of the Vero cell nuclear factors to hr1 is and this is from the that the can be with hr1 but not with heterologous like even when present in high and of hr1 the binding and of heterologous has and the specificity of the and also bind with high specificity to the mammalian hr1-binding protein (mhr1-BP) present within the mammalian cell nuclear not These demonstrate that factors present in mammalian cells with high specificity with the hr1 of DNA the is a that binds to the groove of the green binds to the The hr1 was alone or with or green or with the of Vero cell nuclear with different of or green was by It be that of or green a of the shown by an this was more at of the both the and at the it that the of with the hr1 DNA both the the of hr1 to the an the Vero cell nuclear and the hr1 is with of a in the binding is shown by an than a of binding is with an than of binding is very however, at a of a very binding is not These demonstrate the the The or to hr1 in and Cells the AcMNPV hr1 binds to the insect cell host protein, and this binding is crucial for the enhancer function. shown that mammalian cells, even are not to AcMNPV contain or more factors that binds to hr1 we these to present in insect cells. In an the was with of nuclear and of Vero nuclear The in the two has different 1 with When of the Vero and are independent to by the are when binding were carried out of and each of and Vero the were This that the hr1-binding proteins in the two nuclear are of different molecular and different with hr1 bind to different within the hr1, not out each other. These the mammalian cells contain or more factors that bind to hr1, these factors are different from the hr1-BP in of molecular and sequence protein in such an hr1 as a in cells, hr1 also functions as an origin of The ori and enhancer are independent of each other and are using different (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar). The ori activity is by a standard which is the of the to which the site to be with L.A. Summers M.D. J. Virol. PubMed Google Scholar). which is within the site for and DNA be by the DNA that is in mammalian cells not be at sequence and be to can DNA from that which has in the DNA was from mammalian cells with and by AcMNPV viral infection a to the presence of viral factors required for ori function. A cells were with but not infected with virus was also to the hr1 ori activity can even in the of viral DNA was different 12, and or of and for In the cells, a high molecular is in cells with by viral infection 12, and cells with but not by viral infection not but were with and to Cells with by viral infection are also to and In a is not at in mammalian HepG2 cells, even in cells with by viral infection 12, and in the insect cells, the can support replication in the presence of viral infection hr1 to function as an origin of replication in mammalian cells. A Recombinant the hsp70 the not in mammalian cells but can the cells a A recombinant virus containing two promoters and two the of the promoter and the hsp70 promoter, was used to different mammalian cells and cells. different post infection and were In the cells activity follows the profile of is at from to and is very high at In mammalian cells, is activity not which is the that promoter baculovirus The activity follows a different In cells this as early as from to The activity in mammalian cells is very than in insect cells. In COS-1 cells and HepG2 cells the activity is at from to It is to that the insect cells mammalian cells is a of the replication of baculovirus in the insect cells as well as of the hsp70 The Drosophila hsp70 promoter is more active in a homologous insect cell than in a mammalian of an hr1 in the Recombinant the from the hsp70 in shown that the hr1 sequence can enhance transcription from hsp70 promoter in transient in mammalian cells, we for hr1-mediated enhancement when it is present as an additional copy in the virus. A recombinant virus was constructed by an additional copy of the hr1 enhancer the vAclacluc (10Hasnain, S. E., Venkaiah, B., and Habib, S. (October 12, 1999) U. S. Patent 5,965,393Google Scholar, 11Chatterji U. Ahmad R. Venkaiah B. Hasnain S.E. Gene (Amst.). 1996; 171: 209-213Crossref PubMed Scopus (15) Google Scholar). than enhancement of expression of in mammalian cells from hsp70 promoter was when vAclacluc-hr1 was used as with vAclacluc It is to insect cells, mammalian cells not support baculovirus replication and even hr1 enhance expression of a gene by a heterologous insect The baculovirus AcMNPV hr1, the enhancer study, is bifunctional, in that it can function both as an enhancer of transcription and an origin of Spodoptera frugiperda insect cells (Sf9) are the for which follows a temporal profile of gene expression and has been used to heterologous hr1 can to enhance the expression of the different of the immediate early gene ie-N the delayed early 39k gene (7Carson D.D. Summers M.D. Guarino L.A. J. Virol. 1991; 65: 945-951Crossref PubMed Google Scholar, L.A. Summers M.D. J. Virol. PubMed Google Scholar, L.A. Summers M.D. J. Virol. PubMed Google Scholar), the late gene cor and very late (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar). A number of such as the role of hr1, ability to enhance transcription from a wide of promoters within the host system by a host hr1-BP (9Habib S. Hasnain S.E. J. Biol. Chem. 1996; 271: 28250-28258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), the of baculovirus-mediated gene transfer mammalian cells, to the role of hr1 in mammalian cells and the of host factors that with A for sequence that with enhancer binding proteins and transcription factors in other viral and systems that a of these were present in The most were the with high to the enhancer by the of transcription factors in the enhancer of the virus A. J. Virol. PubMed Google Scholar). This was or to each palindrome in This is within the region in the flanking which high of We have that hr1 as a classic enhancer of transcription from the promoter in mammalian cells such as kidney cells and cells HeLa and cells Enhancement was also when the Drosophila hsp70 promoter is hr1-mediated was independent of the gene The enhancement was in Vero cells, a enhancement was in In cells, the binding of host hr1-BP to hr1 is for enhancer function. of nuclear factors present in mammalian cells (mhr1-BP) that bind with high affinity and specificity to different of hr1 was with the ability of hr1 to function in host and hr1-BP are distinct from each other in of molecular ion for binding, and the manner in which the Enhancers are DNA sequences that gene the of which is not several have been We have for the first that hr1 is of enhancing transcription even in trans, i.e. when it is present are that an enhancer can have DNA when present in M. P. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of ability to stimulate transcription In these are called In the called as a of the of present transcription from the promoter the Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Although hr1 can when present both in and in mammalian cells, it can in in insect cells. This be to the in the properties of the enhancer binding proteins in the two hr1-BP and by of may be to about enhancement of transcription from promoters present hr1-BP has or role in the ori function of hr1 (8Habib S. Pandey S. Chatterji U. Burma S. Ahmad R. Jain A. Hasnain S.E. ).DNA Cell Biol. 1996; 15: 737-747Crossref PubMed Scopus (28) Google Scholar, 9Habib S. Hasnain S.E. J. Biol. Chem. 1996; 271: 28250-28258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), the presence of hr1-BP like factors in mammalian cells not be of as as the ori function is This was the as was from the of the hr1 to support replication in mammalian cells. 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