Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors

Abstract

Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366-375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of beta-galactosidase (beta-Gal) in several mammalian cell lines, and expression remained high ( approximately 150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by beta-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-beta-Gal antibody response after intramuscular inoculation of the beta-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined 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.

FIG. 1

Incorporation of the HDVr sequence into KUN replicons. (A) RNA-based KUN replicon constructs. Filled boxes represent translated regions of the KUN replicon, as for the previously described C20DX2Arep plasmid (43). SP6, the SP6 RNA polymerase promoter. 5′UTR and 3′UTR represent the KUN 5′ and 3′ untranslated regions, respectively. Ub, mouse Ubiquitin gene (16). Hatched boxes indicate the FMDV-2A sequence (40). HDVr, the HDVr sequence (35) with its cleavage site indicated by an arrow. pA, the SV40-derived polyadenylation signal sequence. Restriction sites used in construct preparation are as shown. Sequence of the 3′ UTR-HDVr junction, confirmed by sequencing, is indicated. (B) IF analysis with KUN anti-NS3 antibodies of BHK21 cells 24 h after transfection with equal amounts of C20Ubrep or C20UbHDVrep in vitro-transcribed RNAs. In vitro transcription and transfection of KUN replicon RNA, as well as anti-NS3 IF analysis, were performed as described previously (24, 25). (C) RT-PCR analysis of the KUN RNA transcripts. The RNA templates were either in vitro transcribed from the C20UbHDVrep plasmid DNA (as described in reference with omission of the synthetic cap analogue; lanes 2 and 3) or extracted from purified KUN virus grown in Vero cells and decapped (; lane 4). The RNA templates were purified by phenol-chloroform extraction and ethanol precipitation and were self-ligated with T4 RNA ligase (Pharmacia) at 17°C for 12 h in the reaction buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 10 mM ATP, 60 μg of bovine serum albumin/ml) (lanes 2 and 4). The resulting circular RNAs were then RT-PCR amplified across the 3′ end-5′ end junction by using One-Step RT-PCR System (Gibco BRL), essentially as described by the manufacturer. The primers for RT-PCR were forward primer 5′-GCTGCGAAGTGATCCATGTAA-3′ (representing nucleotides 10582 to 10603 of the KUN 3′UTR sequence; 24) and reverse primer 5′-GGGCCCTCCTGGTTTCTT-3′ (complementary to nucleotides 119 to 102 of the KUN core-5′UTR region; 24). The size of the HDVr-pA sequence was 279 bp, and the expected sizes of RT-PCR products were 561 bp and/or 840 bp, depending on whether the HDVr cleavage occurred or not. Lane 3, the product of RT-PCR from unligated in vitro-transcribed C20UbHDVrep RNA (negative control). The ∼450- and ∼700-bp fragments in lane 3 represent nonspecific amplification products. Lane 4 (see above), a positive control for the experiment; lane 1, 1-kb Plus DNA molecular size marker (Gibco BRL).

FIG. 2

DNA-based KUN replicon vectors. Most of the designations are as in Fig. 1. CMV, the eukaryotic CMV-derived immediate-early enhancer/promoter region (8). Ub- and FMDV-2A-mediated cleavages are indicated by arrows. Sequence of the CMV-5′UTR junction, confirmed by sequencing, is shown. dGDD with an arrow (in pKUNrep2) indicates the position of the deletion of the RNA polymerase motif GDD (26). PAC, the PAC gene (42). IRES, the sequence of the encephalomyelocarditis virus IRES. Restriction sites used in construct preparation are as shown.

FIG. 3

Comparison of RNA and protein syntheses from the RNA replication-competent and RNA replication-defective KUN replicon DNA vectors. (A) Northern blot hybridization analysis of total RNA isolated from BHK21 cells 36 h after transfection with equal amounts of pKUNrep2 (lane 1) or pKUNrep2(dGDD) (lane 2) plasmid DNAs, or untransfected cells (lane 3). The probe was a [³²P]dCTP-labeled cDNA fragment representing the last 761 nucleotides of the KUN genome (24). (B) RIP analysis with KUN anti-NS3 antibodies of BHK21 cells radiolabeled for 1 h at 30 h after transfection with pKUNrep2 (lanes 1 and 2) or pKUNrep2(dGDD) (lanes 3 and 4) plasmid DNAs. Shown are RIP samples from cells labeled in the absence (−) (lanes 1 and 3) or presence (+) (lanes 2 and 4) of ACD. The specificity of KUN anti-NS3 antibodies in RIP and IF analyses was demonstrated previously (45). Relative phosphorimager counts of the radiolabeled NS3 bands in corresponding RIP samples are shown with the background level deducted. DNA transfection, Northern blot hybridization, protein labeling, ACD treatment, and RIP procedures were performed as described in Materials and Methods.

FIG. 4

Comparative analyses of transient β-Gal expression from the KUN replicon [pKUNβrep2, pKUNβrep2(dGDD); see Materials and Methods], pSCAβ (SFV replicon-based; 14), and pCMVβ (conventional plasmid; Clontech) DNA constructs. BHK21 cell monolayers (∼1.3 × 10⁵ cells in 16-mm-diameter wells of 24-well plates) were transfected with ∼0.8 μg of corresponding DNAs and incubated until reaching confluency (∼2 days posttransfection). The cells were then trypsinized and transferred into larger plates to allow continuous cell division. Forty percent of the trypsinized cells were transferred into 35-mm-diameter plates, allowed to propagate, and assayed for β-Gal activity at day 3 after initial transfection. Fifty percent of the trypsinized cells were transferred into 60-mm-diameter plates, allowed to propagate, and assayed for β-Gal activity at day 5 after initial transfection. U*, the amounts of biochemically active β-Gal protein, in units of the enzymatic activity, produced in the total volume of each cell lysate collected from 16-mm-diameter wells (days 1 and 2), 35-mm-diameter plates (day 3), and 60-mm-diameter plates (day 5). The values are the means ± standard deviation for triplicate experiments.

FIG. 5

Expression of β-Gal in different cell lines using DNA-based KUN replicon vectors. (A) Transient β-Gal expression in indicated cell lines, detected by X-Gal staining 30 to 40 h after transfection with the pKUNβrep2 DNA. (B) Stable β-Gal expression in different cell lines at indicated passages after transfection with the pKUNβrep3 (BHK21, Vero, 293, HEp-2 cells) or pKUNβrep4 (A172 cells) DNAs and subsequent puromycin selection. DNA transfection and puromycin selection of cell cultures were performed as described in Materials and Methods.

FIG. 6

β-Gal expression in mouse lung epithelial cells at indicated times after intranasal inoculation with the DNA-based KUN replicon construct pKUNβrep2. Control panel shows a lung section of a mouse inoculated with the pKUNrep2 DNA. DNA administration and preparation of lung sections were performed as described in Materials and Methods.

FIG. 7

Induction of β-Gal-specific antibodies in mice after i.m. immunization with β-Gal-encoding plasmid DNA constructs. (A) β-Gal-specific IgG responses in a 1/800 dilution of sera from BALB/c mice 4, 8, and 11 weeks after i.m. immunization with pKUNrep2, pKUNβrep2, pSCAβ, and pCMVβ plasmid DNA constructs (as per Materials and Methods). ELISA readings for individual mouse sera are shown by open squares (preimmune sera [pr]) and filled squares (immunized sera; 4, 8, and 11 weeks postpriming). Horizontal lines show the averages for each group. (B) β-Gal-specific IgG responses induced in individual sera of BALB/c mice 8 weeks post i.m. immunization (3 weeks postboost) with the pKUNβrep2 and control pKUNrep2 plasmid DNAs. OD₄₅₀, optical density at 450 nm.