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Journal of Virology, July 2000, p. 6564-6569, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Cytoplasmic RNA Vector Derived from Nontransmissible Sendai Virus with Efficient Gene Transfer and Expression

Hai-Ou Li,1 Ya-Feng Zhu,1 Makoto Asakawa,1 Hidekazu Kuma,1 Takahiro Hirata,1 Yasuji Ueda,1 Yun-Sik Lee,1 Masayuki Fukumura,1 Akihiro Iida,1,* Atsushi Kato,2,dagger Yoshiyuki Nagai,2,Dagger and Mamoru Hasegawa1

DNAVEC Research Inc., Tsukuba-shi, Ibaraki 305-0856,1 and Department of Viral Infection, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo 108-8639,2 Japan

Received 18 January 2000/Accepted 6 April 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the Paramyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 × 108 to 1.0 × 108 cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome of 15,384 nucleotides and is a member of the family Paramyxoviridae. The SeV genome contains six major genes, which are lined up in tandem on a single negative-strand RNA. Three virus-derived proteins, the nucleoprotein (NP), phosphoprotein (P), and large protein (L; the catalytic subunit of the polymerase) form a ribonucleoprotein complex (RNP) with the SeV genomic RNA, and the RNP acts as a template for transcription and replication. Matrix protein (M) engages in the assembly of viral particles. Two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion protein (F), mediate the attachment of virions and penetration of RNPs into infected cells. F protein is synthesized as an inactive precursor protein F0 and split into F1 and F2 by proteolytic cleavage of a trypsin-like enzyme. SeV replication is independent of nuclear functions and does not have a DNA phase. Therefore, it does not transform cells by integrating its genetic information into the cellular genome (16).

Methods to rescue infectious viruses entirely from cloned cDNA have been established for segmented and nonsegmented negative-strand RNA viruses (6, 22, 23, 26). Such reverse genetics technology has enabled the construction of genetically engineered viruses which carry additional foreign genes and opened the way for the development of gene transfer vectors from RNA viruses of this type (24). The vectors prepared by this method have shown a high efficiency of gene transfer and expression of foreign proteins in vitro (3, 12, 18, 21, 28, 32, 36). However, the recombinant paramyxoviruses constructed to date have contained all the viral structural genes and thus are replication competent, giving rise to fully infectious progeny capable of spreading in the body.

Here we report the development of a novel SeV vector that is capable of self-replication but incapable of infecting neighboring cells. The vector does not encode F protein, which is one of the endogenous envelope proteins, but instead incorporates it expressed in trans. We further show that an inserted enhanced green fluorescent protein (EGFP) reporter gene is vigorously expressed from this SeV vector in cells of various origins in culture, including human smooth muscle cells, hepatocytes, and lung microvascular endothelial cells, in primary cultures of rat cerebral cortex cells, and in the lateral ventricles and hippocampus of the rat brain. Thus, this F-defective vector appears to represent the important first step toward human gene therapy and vaccine delivery using SeV replicons.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Virus. The attenuated SeV Z strain was used as a basis for the genome used in this study. Recombinant vaccinia virus vTF7-3 (9) expressing T7 RNA polymerase which had been inactivated with psoralen and long-wave UV light (34) was used for RNP recovery experiments. Recombinant adenovirus AxCANCre (14) expressing Cre recombinase was used for induction of F protein from LLC-MK2/F7 cells.

Cell culture. A rhesus monkey kidney cell line, LLC-MK2, was cultured in minimal essential medium (MEM) (Gibco-BRL, Rockville, Md.) supplemented with 10% heat-inactivated fetal calf serum (FCS). For virus propagation, LLC-MK2/F7 cells were cultured in MEM containing cytosine arabinoside (araC) (Sigma, St. Louis, Mo.) at 40 µg/ml and trypsin (Gibco-BRL) at 7.5 µg/ml. Normal human smooth muscle cells, normal human hepatocyte cells, and normal human lung microvascular endothelial cells (Cell Systems Corp., Kirkland, Wash.) were cultured in SFM CS-C medium (Cell Systems Corp.). All cells were cultured at 37°C in a humidified 5% CO2 atmosphere.

Plasmid construction. To replace the F gene of SeV cDNA clone with the EGFP reporter gene, the 6.0-kb SacI fragment of pSeV18+b(+) (12) which contained the F gene was cloned into pUC18 (Stratagene, La Jolla, Calif.) to generate pUC18/Sac. A 1,698-bp fragment of the total open reading frame of the F gene in pUC18/Sac was deleted by a combination of PCR and ligation. For an upstream fragment of the F gene, the primer pair FF-1 (5'-GTTGAGTACTGCAAGAGC-3') and FR-1 (5'-TTTGCCGGCATGCATGTTTCCCAAGGGGAGAGTTTTGCAACC-3') was used, and for a downstream fragment, the primer pair FF-2 (5'-AAAATGCATGCCGGCAGATGATCACGACCATTATCAGATGTCTTG-3') and FR-2 (5'-CTAAAGTACCGCGCGACC-3') was used (see Fig. 1A). The two amplified fragments were digested with BsmI-EcoT22I and Eco22TI-BglII, respectively, and ligated with the BsmI-BglII fragment of pUC18/Sac to generate pUC18/SacDelta F. The EGFP gene was amplified by PCR from pEGFP-N1 (Clontech, Palo Alto, Calif.) using a pair of NsiI- or NgoMIV-tagged primers (5'-ATGCATATGGAGATGCGGTTTTGGCAGTAC-3' [sense] and 5'-TGCCGGCTAATTATTACTTGTACAGCTCGTC-3' [antisense]). The amplified fragment of EGFP was digested with NsiI and NgoMIV and cloned into the NsiI-NgoMIV sites of pUC18/SacDelta F to generate pUC18/SacDelta F-EGFP. The 3.4-kb DraIII fragment of pUC18/SacDelta F-EGFP was replaced with the 4.4-kb DraIII fragment of pSeV18+b(+) to generate pSeV18+b(+)/Delta F-EGFP. For the plasmid expressing F protein by the Cre/loxP-inducible expression system (1), the 1.8-kb StyI-BstUI fragment of pSeV18+b(+) containing the F gene was blunt ended and inserted into the SwaI site of pCALNdLw (1) to generate pCALNdLw/F.

Establishment of F-expressing LLC-MK2/F7 cells. LLC-MK2 cells were transfected with pCALNdLw/F using the mammalian transfection kit (Stratagene) as specified by the manufacturer. G418 (400 µg/ml)-resistant clones were selected after 3 weeks. Expression of F protein was confirmed by infecting the clones with AxCANCre at a multiplicity of infection (MOI) of 3 and analyzed by Western blotting with anti-F monoclonal antibody (MAb) f236 (30) after 3 days. F protein expression on the cell surface was analyzed by flow cytometry after immunostaining with anti-F MAb and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G.

Recovery and amplification of the F-defective SeV vector. Approximately 107 LLC-MK2 cells seeded in a 10-cm-diameter dish were infected with psoralen- and long-wave UV-treated vTF7-3 at an MOI of 2. After a 1-h incubation at room temperature, the cells were washed three times with MEM and transfected at room temperature with a plasmid mixture containing pSeV18+b(+)/Delta F-EGFP (12 µg), pGEM-NP (4 µg), pGEM-P (2 µg), and pGEM-L (4 µg) (7) in 110 µl of Superfect transfection reagent (Qiagen, Tokyo, Japan). The transfected cells were maintained for 3 h in 3 ml of OptiMEM (Gibco-BRL) plus 3% FCS, washed three times with MEM, and incubated for 60 h in MEM containing araC (40 µg/ml). GFP expression by the transfected cells was examined by fluorescence microscopy to validate the formation of RNPs inside of the cells. The transfected cells were collected by centrifugation at 1,000 × g for 5 min, resuspended in OptiMEM (107 cells/ml), and lysed by three cycles of freezing and thawing. Subsequent RNP transfection was performed by mixing the lysate (106 cells/100 µl) with 75 µl of OptiMEM and 25 µl of DOSPER (Boehringer Mannheim, Germany) for 15 min at room temperature and then transfecting it into F-expressing LLC-MK2/F7 cells in a 24-well plate. At 24 h after the transfection, the cells were washed three times with MEM and incubated for 3 to 6 days in MEM containing araC (40 µg/ml) and trypsin (7.5 µg/ml). The spread of GFP-expressing cells to neighboring cells was examined by fluorescence microscopy. Virus yield is expressed in PFU and cell infectious units (CIU) (15).

Analysis of viral genomic RNA. Total viral RNA from the F-defective SeV vector or wild-type SeV was isolated using a QIAamp viral RNA mini kit (Qiagen), separated on a 2.2 M formaldehyde-1% agarose gel, transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech, Tokyo, Japan), and hybridized with an F or HN DNA probe generated with a DIG DNA labeling and detection kit (Boehringer). The probes for the F or HN gene were prepared from a 1.8-kb StyI-BstUI or a 1.8-kb HhaI-DraI fragment of SeV18+b(+), respectively.

Immunoelectron microscopy. Virus obtained by ultracentrifugation at 10,000 × g for 30 min was resuspended in phosphate-buffered saline (PBS) as 109 PFU/ml, dropped onto microgrids, dried at room temperature, and fixed with 3.7% formaldehyde for 15 min. Then the grids were treated with anti-F or anti-HN (HN-2) (20) MAb for 60 min, washed three times with PBS, and reacted with gold colloid-labeled anti-mouse immunoglobulin G for 60 min. Treated grids were then washed with PBS, dried, and stained with 4% uranium acetate for 2 min for electron microscopic examination with a JEM-1200EXII instrument (Nippon Denshi, Tokyo, Japan).

Gene transfer to primary cultures of rat cerebral cortex cells. Primary cultures of rat cortical neurons were prepared from E18.5 embryos as described previously (2, 11). Dissociated cells were plated at a density of 80,000 or 100,000/well in eight-well culture slides coated with poly-D-lysine (Becton Dickinson Labware, Bedford, Mass.). The cells were cultured at 37°C in a 5% CO2 atmosphere for 5 days in neural basal medium enriched with B27 supplement (Gibco-BRL). The F-defective SeV vector was infected at an MOI of 5 and incubated for 3 days. To identify neuronal cells, cells were fixed with 2% paraformaldehyde at room temperature for 15 min and immunostained with anti-MAP2 MAb (Boehringer-Mannheim). Immunocytochemistry was performed by indirect-immunofluorescence microscopy (10) with a confocal microscope system (MRC 1024; Nippon Bio-Rad, Tokyo, Japan) using a 470- to 500-nm and 510- to 550-nm excitation band-pass filter on an inverted microscope (Diaphot 30; Nikon, Tokyo, Japan).

Vector injection into rat brain. Female rats, F334/DuCrj (6 weeks old) (Charles River, Ontario, Canada) were anesthetized by intraperitoneal injection of Nembutal (5 mg/kg) and secured on a stereotaxic frame (model 900; David Koph Instruments, Tujunga, Calif.). For intraventricular injection, the burr hole was opened at 5.2 mm off the interaural line toward the bregma and 2.0 mm off lambda toward the right ear. The needle (30 gauge) was inserted 3.6 mm below the surface of the dura. A 20-µl volume of vector suspension (2 × 107 CIU) was injected into the lateral ventricle or hippocampus region.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Construction of F-defective SeV cDNA. F-defective SeV cDNA was constructed by replacing the F gene with an EGFP reporter gene (Fig. 1A). GFP expression was detectable in a single living cell, which allowed us to confirm the successful recovery of RNPs of F-defective SeV inside of such cells.


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  		FIG. 1.   System for generating the F-defective SeV vector from a cloned SeV cDNA. (A) Schematic representation of the organization of the plasmids pSeV18+b(+), carrying full-length SeV cDNA, and pSeV18+b(+)/Delta F-EGFP, carrying an F-defective SeV cDNA with an EGFP reporter gene. The restriction sites used for construction of pSeV18+b(+)/Delta F-EGFP are indicated. Primers used for PCR amplification are indicated by arrows. T7, T7 promoter; Rbz, hepatitis deltavirus ribozyme sequence; nt, nucleotides. (B) Schematic representation of the two-step procedure for recovery of the F-defective SeV vector. (Panel 1) In the first step, the functional RNPs are recovered in LLC-MK2 cells by using the four plasmids driven by a recombinant vaccinia virus expressing T7 RNA polymerase which had been inactivated with psoralen and long-wave UV light (UV-vTF7-3). (Panel 2) In the second step, RNPs are introduced via a cationic liposome to F-expressing LLC-MK2 cells (LLC-MK2/F7) and produce infectious F-defective virions.

Construction of a packaging cell line that expresses SeV F protein. SeV F protein is required for the formation of infectious SeV particles. Therefore, recovery of SeV from the RNA genome lacking F gene must be complemented with this gene in trans. We therefore constructed an F-expressing packaging LLC-MK2 cell line with a Cre/loxP-inducible expression system. LLC-MK2 cells were transfected with plasmid pCALNdLw/F, where the F gene is located under the stuffer neo sequence flanked by a pair of loxP sequences, and stable Neor clones were isolated. To these Neor clones, a recombinant adenovirus vector, AxCANCre (14), that expresses Cre recombinase was added. Of 15 clones, 7 expressed F protein inducibly; the clone that showed the highest F protein expression (Fig. 2A) was designated LLC-MK2/F7 and used as a packaging cell line for the F-defective SeV vector. Flow cytometry analysis showed the presence of F protein on the surface of LLC-MK2/F7 cells (Fig. 2B). The amount of this protein was approximately one-seventh of that on LLC-MK2 cells infected with wild-type SeV under the same experimental conditions.


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  		FIG. 2.   Inducible expression of F protein in LLC-MK2/F7 packaging cells. (A) Western blot analysis using anti-SeV F (f-236) MAb. Lanes: 1, LLC-MK2 infected with wild-type SeV (MOI = 1) for 24 h; 2, LLC-MK2/F7; 3, LLC-MK2/F7 infected with adenovirus AxCANCre (MOI = 3) and incubated for 3 days. (B) Flow cytometry analysis of cell surface proteins. Expression of F protein on the packaging cells was examined with the anti-SeV F (f-236) MAb. LLC-MK2/F7 without induction (top panel), LLC-MK2/F7 infected with AxCANCre (middle panel), and LLC-MK2 infected with wild-type SeV (bottom panel) are shown.

Recovery of functional RNPs from an F-defective cDNA. Conventionally, recombinant SeV with the wild-type genome were recovered from cloned cDNAs after infectious particles were rescued in cultured cells and further amplified in embryonated hen eggs or in cultured cells (15). Since infectious particles were not generated from cDNA lacking the F gene in non-F-expressing cells, we have devised a novel rescue procedure which consists of two steps (Fig. 1B). The first step was to recover RNPs of the F-defective RNA genome in LLC-MK2 cells by using an F-defective cDNA clone and the three plasmids expressing NP, P, and L proteins. GFP-expressing cells were the only RNP-expressing cells on the plate, because such cells were observed only when these four materials were cotransfected into LLC-MK2 cells. The second step was to transfect RNP into the F-expressing packaging cell line and to collect infectious particles from the supernatants. To raise the efficiency of recovery of RNPs in the first step, we adapted a vaccinia virus vTF7-3 (9) treated with psoralen and long-wave UV irradiation. This treatment inactivated the replication capability of the viruses without impairing their infectivity and T7 RNA polymerase expression. We estimated the recovery frequency by using wild-type SeV cDNA and inoculating the diluted lysates of transfected cells into embryonic hen eggs. With a previous recovery procedure, 1 CIU was detected from 105 transfected cells (15). However, with the improved protocol, 1 CIU was detected from only 103 cells, indicating an improvement of nearly 100-fold. As for the F-defective SeV cDNA, the numbers of GFP-expressing cells were scored to estimate the efficiency of recovery of functional RNP. Under these conditions, these cells were detected in approximately 1 in 105 transfected cells.

The F-defective SeV vector is specifically propagated in a packaging cell line in a trypsin-dependent manner. The lysates containing functional RNPs were obtained by freeze-thaw cycles, mixed with cationic liposome, and transfected into LLC-MK2/F7 or LLC-MK2 cells. The transfected cells were cultured in the presence or absence of trypsin. The infectious virus particles were recovered only from LLC-MK2/F7 cells cultured with trypsin, suggesting the rescue of infectious virus particles in these cells. The efficiency of recovery at this point was at least 1 CIU from 105 transfected cells. In LLC-MK2/F7 cells cultured in the absence of trypsin or in LLC-MK2 cells, GFP-expressing cells were detected but did not spread to neighboring cells (Fig. 3). These results showed that the propagation of the F-defective SeV vector and the formation of infectious virus particles are specific to the F-expressing packaging cells and are dependent on trypsin-cleavage. The infectious titer of particles recovered from supernatants of the packaging cells ranged from 0.5 × 108 to 1.0 × 108 CIU/ml.


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  		FIG. 3.   Specific production of the F-defective SeV vector in F-expressing packaging cells in a trypsin-dependent manner. LLC-MK2 cells (A) or AxCANCre-infected LLC-MK2/F7 cells (B and C) were infected with the F-defective SeV vector and incubated in the presence (A and C) or absence (B) of trypsin. GFP expression by the infected cells was observed by fluorescence microscopy 3 days after infection.

Confirmation of the genome structure and ultrastructure of the F-defective SeV vector. To examine the genome structure, total RNA from the F-defective SeV vector or wild-type SeV was prepared and analyzed by Northern blot analysis. Probing with the HN gene detected a clear genomic RNA in both F-defective SeV vector and wild-type SeV, but the F-defective SeV vector was smaller than the wild type. When the F gene was used as a probe, no signal was obtained from the F-defective SeV vector but a clear signal was obtained from wild-type SeV (Fig. 4A). The reverse transcription-PCR analysis confirmed the existence of the EGFP gene in the F-deleted region of the F-defective SeV vector (data not shown). These results confirmed that the F-defective SeV vector contains an RNA genome lacking the F gene. Electron microscopic examination of the F-defective SeV vector revealed internally located helical RNP-like structure and an envelope studded with spike-like structures (Fig. 4B). Immunoelectron microscopic examination located the F and HN proteins on the surface of the F-defective SeV vector (Fig. 4C and D).


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  		FIG. 4.   Structural characterization of the F-defective SeV vector. (A) Northern blot analysis of the RNA genome structure. RNAs from wild-type SeV (wt) and the F-defective SeV vector (Delta F) were prepared and hybridized with cDNA probes of HN (left panel) or F (right panel). The positions of 28S and 18S rRNA are shown. (B to D) Electron microscopic ultrastructure of viral particles. The F-defective SeV vector was negatively stained with phosphotungstic acid (B). The ultrastructure of virus particles after labeling with anti-F (C) or anti-HN (D) MAb and gold-conjugated goat anti-mouse immunoglobulin G is shown.

The F-defective SeV vector efficiently delivers and expresses the EGFP gene in variety of cell types. When primary cultures of neuronal cells derived from fetal rat cerebral cortex were infected with the F-defective SeV vector carrying the EGFP reporter gene at an MOI of 5, nearly 100% of the microtubule-associated protein 2 (MAP2)-positive cells expressed the EGFP reporter gene (Fig. 5A to C). Also, the vector infected and strongly expressed the EGFP gene in almost 100% of normal human hepatocytes, lung microvascular endothelial cells, and smooth muscle cells at an MOI of 3 (Fig. 5D to I). EGFP fluorescence of the infected cells was seen at least from 10 h to 10 days after vector infection. Furthermore, GFP expression was observed in nondividing neuronal cells or ependymal cells of the lateral ventricle when the vector was stereotaxically injected into the hippocampal region or an intraventricular region of rat brain, respectively (Fig. 6). Gene introduction into ependymal cells is of value, since it was reported recently that these cells could be neural stem cells that generate migratory neuronal precursor cells (13). These results showed that the F-defective SeV vector is capable of efficient infection and strong expression of foreign genes in a wide spectrum of cells and tissues.


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  		FIG. 5.   Introduction and expression of the EGFP gene by the F-defective SeV vector in a variety of cell types in vitro. (A to C) GFP expression by primary neuronal cells derived from rat cerebral cortex 5 days after infection with the vector at an MOI of 5 at lower (A) and higher (C) magnification and immunostained with anti-MAP2 antibody (B). (D to I) Normal human hepatocytes (D and G), normal human lung microvascular endothelial cells (E and H), and normal human smooth muscle cells (F and I) were infected with the F-defective SeV vector at an MOI of 3. GFP expression was observed 3 days after infection (G to I).


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  		FIG. 6.   Gene introduction into the rat central nervous system. The F-defective SeV vector carrying the EGFP gene was injected into rat brain. GFP expression was observed 4 days after vector injection. Fluorescent photomicrographs at lower (A and B) and higher (C and D) magnifications of pyramidal cells of the CA1 region in the hippocampus and ependymal cells of the lateral ventricle.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The development of a reverse genetic system has enabled the genetic engineering of negative-strand RNA viruses. This system has been used to analyze the function of viral genes and to construct recombinant viruses which express foreign proteins. In this study, we made an improvement to this system by devising a new method to generate the F-defective SeV vector from a cloned cDNA of a defective RNA genome. This is the first report on constructing a replicon-based RNA vector in the family Paramyxoviridae which replicates in infected cells but does not infect neighboring cells. The improvements achieved in this study are (i) optimization of RNP recovery efficiency by using a UV-inactivated recombinant vaccinia virus expressing T7 RNA polymerase, (ii) construction of an inducible F-expressing packaging LLC-MK2 cell line supplemented with the F protein in trans, and (iii) development of a transfection process for RNP recovered from LLC-MK2 cells. An attempt to recover the F-defective SeV vector directly in the F-expressing packaging cell line by transfecting F-defective cDNA together with three plasmids expressing NP, P, and L proteins was unsuccessful. Our observation on the gross reduction in F protein expression after vaccinia virus infection of packaging cells suggests that this protein was depleted during this approach (data not shown). The fact that the F-defective SeV vector cannot spread to F-nonexpressing cells indicates that F protein is indispensable for viral infection. Since this system requires the NP, P, and L genes for self-replication and transcription of RNP, a variety of similar self-replicating SeV vectors defective in M, HN, and/or a combination of M, HN, and F genes could be designed if proper complementing cell lines are constructed. Further, we speculate that the strategy developed in this study for rescuing defective viruses is applicable to other negative-strand RNA viruses and represents an innovative method for generation of novel types of vectors.

As to paramyxoviruses carrying defective genome, measles virus defective in M gene were isolated from the brains of subacute sclerosing panencephalitis patients and generated by reverse genetic techniques (4). These viruses were not able to generate progeny viral particles because of the defect in viral envelope assembly but did spread by cell-to-cell fusion. Defective interfering particles of negative-strand RNA viruses which are defective in several viral genes and interfere with the replication of nondefective virus are generated in nature (35). Furthermore, minigenomes in which the entire coding region was replaced with a reporter gene were constructed by genetic engineering in negative-strand RNA viruses (5, 25, 31). Defective interfering particles and minigenomes require helper virus for their replication and virion assembly. The F-defective SeV vector reported in this study is independent of helper virus for its reproduction and is able to self-replicate in infected cells. In the family Rhabdoviridae, generation of G-gene-deficient viruses which carry human immunodeficiency virus (HIV) receptor and coreceptor genes has been performed in the vesicular stomatitis virus and rabies virus groups (19, 29). These pseudotyped rhabdoviruses were constructed specifically for targeting to cells infected with HIV-1. Vesicular stomatitis virus has also been used as a vaccine vector (27).

The F-defective SeV vector has several advantages over existing vectors as a gene delivery system for human treatments. (i) SeV is a murine parainfluenza virus, and pathogenicity to humans has not been reported. (ii) This vector replicates exclusively in the cytoplasm of infected cells and does not go through a DNA phase; therefore, there is no concern about unwanted integration of foreign sequences into chromosomal DNA. (iii) This vector has shown a high efficiency of gene transfer and expression of a foreign reporter gene to a wide spectrum of cells and tissues, which is comparable to SeV vectors derived from the wild-type genome. The highest level of expression in mammalian cells has been found in a recombinant SeV expressing HIV-1 envelope glycoprotein gp120 (36). For expression of foreign genes in recombinant F-defective SeV vectors, the genes can be designed as the 3' proximal first gene of the viruses. A vector with a 3.2-kb foreign gene has been successfully recovered (data not shown). (iv) This vector is not likely to generate wild-type virus in a packaging cell line, since homologous recombination between RNA genomes has not been observed in nonsegmented negative-strand RNA viruses (33). The following studies have confirmed this idea. The F-defective SeV vector was inoculated into embryonated hen eggs or into non-F-expressing LLC-MK2 cells. The allantoic fluids or the culture supernatants were harvested several days after the vector infection and reinoculated into LLC-MK2 cells. The presence of infectious viruses in infected cells was examined by GFP expression or immunostaining with an anti-SeV serum. Repeated studies have detected no infectious particles.

Replicon-based vectors derived from positive-strand RNA viruses such as Sindbis virus and Semliki Forest virus expressed foreign genes with high efficiency, but foreign genes were rapidly lost on passaging of infected supernatant. Also, these vectors had severe cytopathic effects on infected cells (8, 17). The F-defective SeV vector developed in this study is likely to overcome these disadvantages of positive-strand RNA vectors.

One application of this vector is for human gene therapy. The high-level expression of therapeutic genes in wide varieties of cell types, including nondividing types, and the potential safety to humans suggest that this novel vector has great potential for use in transient gene therapy at least (6). Another potential application is in the development of vaccines. This vector resembles DNA vaccines because of its ability to express epitopes of foreign proteins without generating infectious viruses. Therefore, this vector is useful for the design of improved attenuated vaccines. The applications to the treatment of human diseases are now in progress.


    ACKNOWLEDGMENTS

We acknowledge B. Moss for supplying vTF7-3; D. Kolakofsky for supplying pGEM-N, pGEM-P, and pGEM-L; H. Taira for supplying anti-F antibody and for helpful discussions; I. Saito for supplying AxCANCre; H. Iba for supplying pCALNdlw; N. Miura for supplying anti-HN antibody; and Y. Ito and M. Okayama for helpful discussions. We extend our thanks to T. Fujikawa, H. Hosonuma, K. Washizawa, and S. Komaba for excellent technical assistance.


    FOOTNOTES

* Corresponding author. Mailing address: DNAVEC Research Inc., 1-25-11 Kannondai, Tsukuba-shi, Ibaraki 305-0856, Japan. Phone: 81-298-38-0540. Fax: 81-298-39-1123. E-mail: iida{at}dnavec.co.jp.

dagger Present address: Department of Viral Diseases and Vaccine Control, National Institute of Infectious Diseases, Tokyo 208-0011, Japan.

Dagger Present address: AIDS Research Center, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Arai, T., K. Matsumoto, K. Saitoh, M. Ui, T. Ito, M. Murakami, Y. Kanegae, I. Saito, F.-L. Cosset, Y. Takeuchi, and H. Iba. 1998. A new system for stringent, high-titer vescular stomatitis virus G protein-pseudotyped retrovirus vector induction by introduction of Cre recombinase into stable prepackaging cell lines. J. Virol. 72:1115-1121[Abstract/Free Full Text].
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Journal of Virology, July 2000, p. 6564-6569, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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