Mol Cell Biol. with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo. Development of gene expression vectors based on subgenomic replicons of positive-strand RNA viruses has gained much attention over Closantel the last decade (11, 32). Genomes of the alphaviruses Semliki Forest virus (SFV) (7, 29), Sindbis (SIN) virus (1, 10, 15) and Venezuelan equine encephalitis virus (12, 37), as well as the poliovirus genome (34, 36), have all been used. An important characteristic feature of these systems is the ability of replicon RNA to self-replicate, thereby amplifying the input template in the host cell. This amplification in turn leads to increased production of encoded proteins. Replicon RNAs can be delivered into host cells by direct transfection with RNA transcripts produced in vitro from corresponding plasmid DNAs (20, 47) or by infection with virus-like particles (VLPs) containing encapsidated replicon RNAs (10, 12, 29, Rabbit Polyclonal to TIE2 (phospho-Tyr992) 37). Alternatively, they can be transcribed from transfected replicon-encoded plasmid DNAs utilizing cellular RNA polymerase II transcription machinery (1, 7, 14, 15). It was shown that replicon-based DNA vectors produced higher levels of encoded heterologous proteins than conventional plasmid DNA expression vectors and also elicited greatly enhanced immune responses (7, 19). Applications for most of the alphavirus and poliovirus replicon vectors have been limited to only short-term transient expression due to the cytopathic effects (CPE) induced by vector replication in mammalian cells (18). To address this problem, noncytopathic SIN virus replicon-based vectors containing the puromycin resistance gene were developed by isolation of SIN replicon mutants adapted to puromycin selection in BHK cells (1, 17). However, the use of these vectors is restricted by a number of limitations, such as a narrow host range, relatively low levels of heterologous gene (HG) expression, and some instability of expression in cell populations during passaging (1). We have been developing a gene expression system based on subgenomic replicons of another RNA virus, the flavivirus Kunjin (KUN), containing deletions in the structural region of the genome (25). In contrast to the alphavirus and poliovirus replicons, as well as full-length KUN RNA, replication of KUN replicons in mammalian cell cultures did not produce any apparent CPE (25). Recently we reported the construction and use of RNA-based KUN replicon vectors for HG expression in cell culture after the direct transfection of in vitro-synthesized recombinant KUN replicon RNAs or after infection with recombinant KUN VLPs (43). In this study we describe the development of DNA-based KUN replicon vectors and demonstrate their ability to direct high-level prolonged HG expression in a range of cell lines and in vivo. Moreover, we show the induction of antibody response against a KUN vector-encoded HG after immunization of mice with the corresponding KUN replicon DNA construct. These noncytopathic DNA-based KUN replicon expression vectors Closantel should be useful for a variety of applications both in vitro and in vivo. MATERIALS AND METHODS Cells. BHK21 (baby hamster kidney), Vero (green African monkey kidney), HepG2 (human hepatocarcinoma), HeLa (human cervical epitheloid carcinoma), A172 (human glioblastoma), and 293 (transformed human embryonal kidney) cells were grown in Dulbecco’s minimal essential medium (Gibco BRL) supplemented with 10% fetal bovine serum. HEp-2 (human larynx epidermoid carcinoma) cells were grown in RPMI 1640 medium supplemented with 15 mM HEPES (DNA polymerase (Stratagene). The sequences of primers used in preparation of KUN replicon vectors and constructs are shown in Table ?Table1.1. TABLE 1 Primers used in preparation of KUN replicon vectors and?constructs plasmid (26). The pKUNrep3 vector was constructed by cloning the puromycin -galactosidase (-Gal) gene, which was PCR amplified from the C20DX/-gal/2Arep construct (43) by using NsiLacZ_F and NsiLacZ_R primers with incorporated and immunization against heterologous pathogenes conferring puromycin resistance. Nucleic Acids Res. 1986;14:4617C4624. [PMC free article] [PubMed] [Google Scholar] 43. Varnavski A N, Khromykh A A. Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes. Virology. 1999;255:366C375. [PubMed] [Google Scholar] 44. Vilcek J, Sen G C. Interferons and other cytokines. In: Fields B N, Knipe D M, Howley P M, editors. Fields virology. 3rd ed. Philadelphia, Pa: Lippincott-Raven Publishers; 1996. pp. 375C399. [Google Scholar] 45. Westaway E G, Closantel Mackenzie J M, Kenney M T, Jones M K, Khromykh A A. Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with.