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, 77 (11), 6419-29

A New Sendai Virus Vector Deficient in the Matrix Gene Does Not Form Virus Particles and Shows Extensive Cell-To-Cell Spreading

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A New Sendai Virus Vector Deficient in the Matrix Gene Does Not Form Virus Particles and Shows Extensive Cell-To-Cell Spreading

Makoto Inoue et al. J Virol.

Abstract

A new recombinant Sendai virus vector (SeV/DeltaM), in which the gene encoding matrix (M) protein was deleted, was recovered from cDNA and propagated in a packaging cell line expressing M protein by using a Cre/loxP induction system. The titer of SeV/DeltaM carrying the enhanced green fluorescent protein gene in place of the M gene was 7 x 10(7) cell infectious units/ml or more. The new vector showed high levels of infectivity and gene expression, similar to those of wild-type SeV vector, in vitro and in vivo. Virus maturation into a particle was almost completely abolished in cells infected with SeV/DeltaM. Instead, SeV/DeltaM infection brought about a significant increase of syncytium formation under conditions in which the fusion protein was proteolytically cleaved and activated by trypsin-like protease. This shows that SeV/DeltaM spreads markedly to neighboring cells in a cell-to-cell manner, because both hemagglutinin-neuraminidase and active fusion proteins are present at very high levels on the surface of cells infected with SeV/DeltaM. Thus, SeV/DeltaM is a novel type of vector with the characteristic features of loss of virus particle formation and gain of cell-to-cell spreading via a mechanism dependent on the activation of the fusion protein.

Figures

FIG. 1.
FIG. 1.
Construction of an M gene-deficient SeV vector carrying the GFP gene at the M-deficient site and confirmation of the vector's structure. (A) Structures of recombinant SeV genomes. The open reading frame of the GFP gene was inserted with the SeV end and start signals in the respective positions of the deleted gene(s). The positions of the primers for RT-PCR are shown by arrows. (B) The viral genome structure was confirmed by RT-PCR. The DNA fragment of SeV18+/ΔM-GFP from the 5′-terminal of the P gene to the 3′-terminal of the F gene (containing the GFP gene) was amplified from the plasmid (lane PC) and from cDNA prepared in the presence (lane RT+) or absence (lane RT) of reverse transcriptase, and these fragments were compared to the corresponding fragment amplified from the plasmid of SeV18+GFP (lane NC). Amplifications of 1,073- and 1,400-bp DNAs were expected based on the gene structures of SeV18+/ΔM-GFP and SeV18+GFP, respectively. Lane M, markers. (C) Viral proteins were detected by Western blotting. LLC-MK2 cells were infected with SeV18+GFP (lanes Wild), SeV18+/ΔF-GFP (lanes ΔF), or SeV18+/ΔM-GFP (lanes ΔM) at an MOI of 3. The viral proteins in the cells (Cell) and culture supernatants (Sup) prepared 3 days after the infection were detected by Western blotting with anti-M, anti-F, and anti-SeV (which mainly detects NP protein) antibodies. Lane M, markers.
FIG. 2.
FIG. 2.
Virus productivity of SeV18+/ΔM-GFP. LLC-MK2/F7/M62/A cells were infected with SeV18+/ΔM-GFP at an MOI of 0.5 and cultured in serum-free MEM containing 7.5 μg of trypsin per ml at 32°C. The culture supernatants were collected every 24 h, and fresh medium was added immediately to the remaining cells. The infectious ability (GFP-CIU) (bars) and HA activity (HA units [HAU]) (▵) of the recovered supernatants were assayed. The levels of infectious particles were 1 × 103, 3.9 × 104, 9.5 × 105, 2.9 × 107, 7.1 × 107, 2.2 × 107, 3.8 × 106, and 1.3 × 106 GFP-CIU/ml at 1, 2, 3, 4, 5, 6, 7, and 8 days postinfection (p.i.), respectively.
FIG. 3.
FIG. 3.
(Top panels) Gene transfer of the M gene-deficient SeV vector to nondividing cells in vitro. SeV18+/ΔM-GFP was infected into rat primary neuronal cells derived from the rat cerebral cortex at an MOI of 3. GFP expression was detected 36 h after the infection. The cells were also immunostained with anti-MAP2.
FIG. 4.
FIG. 4.
(Bottom panels) Gene transfer of the M gene-deficient SeV vector to gerbil brain in vivo. SeV18+/ΔM-GFP (5 × 106 GFP-CIU) was microinjected into the left lateral ventricle of an adult Mongolian gerbil. GFP expression was detected throughout the brain of the animal sacrificed 3 days following the injection. Fluorescent light micrographs of coronal sections show a positive GFP reaction in the ependymal cells along the bilateral lateral ventricle walls (A) and also in those around the hippocampus in the lateral ventricle (B).
FIG. 5.
FIG. 5.
Semiquantitative comparison of viral protein levels in the culture supernatant of cells infected with SeV18+/ΔF-GFP and SeV18+/ΔM-GFP. LLC-MK2 cells were infected with SeV18+/ΔM-GFP or SeV18+/ΔF-GFP at an MOI of 3 and cultured at 37°C for 3 days. A series of dilutions of the supernatants recovered were assayed by Western blotting with DN-1 antibody, which detects mainly NP protein.
FIG. 6.
FIG. 6.
(Left panels) Comparison of VLP-forming capabilities of SeV18+/ΔF-GFP and SeV18+/ΔM-GFP. (A) Kinetic quantification of VLPs by HA assay. The culture supernatants of LLC-MK2 cells infected with SeV18+/ΔM-GFP or SeV18+/ΔF-GFP at an MOI of 3 were recovered at various times (daily), and fresh MEM was added immediately to the remaining cells. VLPs were quantified by the HA assay. HAU, HA units. (B) Cytopathic effect after SeV infection. LDH released from damaged cells was quantified by assaying the same supernatants as analyzed for panel A. Values are relative (A490/A650). (C) Fluorescence microscopic analysis of the LLC-MK2 cells described above at 5 days after the infection. Magnification, ×100.
FIG. 7.
FIG. 7.
(Right panels) Cationic liposome-mediated transfection of VLP. The culture supernatants of LLC-MK2 cells that had been infected with SeV18+/ΔF-GFP or SeV18+/ΔM-GFP at an MOI of 3 were recovered 5 days after the infection and transfected into newly prepared LLC-MK2 cells by using cationic liposomes (Dosper). The microscopic observation was carried out 2 days after the transfection. Magnification, ×100.
FIG. 8.
FIG. 8.
Electron microscopic ultrastructure of viral particles of SeV/ΔM. The particles were prepared from supernatants of LLC-MK2/F7/M62/A, an M-expressing packaging cell line, or LLC-MK2 after infection of SeV18+/ΔM-GFP at an MOI of 3 and subsequent culturing for 3 days at 37°C. The electron microscopic structures of M gene-deficient and M protein-supplied particles of SeV18+/ΔM-GFP (A) and VLPs of SeV18+/ΔM-GFP lacking M protein (B and C) were observed after negative staining. Bars, 50 nm.
FIG. 9.
FIG. 9.
Quantitative analysis of SeV infection-dependent cytotoxicity. Cytotoxicity was estimated based on the quantity of LDH released into the cell culture medium. CV-1 cells were infected with SeV18+/ΔF-GFP or SeV18+/ΔM-GFP at an MOI of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 and cultured in serum-free MEM. The assay was carried out 3 days after the infection. The values are from three experiments. Error bars indicate standard deviations.
FIG. 10.
FIG. 10.
Spread of M gene-deficient SeV infection in cell-to-cell manner. LLC-MK2 cells were infected with SeV18+/ΔM-GFP at an MOI of 0.2 (A and D), 1 (B), or 5 (C) and cultured in serum-free MEM containing 7.5 μg of trypsin per ml at 37°C. Microscopic observation was carried out 3 days after the infection. Magnifications, ×100 (A to C) and ×600 (D).
FIG. 11.
FIG. 11.
Subcellular localization of F and HN proteins analyzed by confocal laser scanning microscopy. Stereo three-dimensional images of the subcellular localizations of the F (A) and HN (B) proteins observed under a confocal laser microscope are shown. A-10 cells were cultured at 37°C for 24 or 48 h after infection with SeV18+GFP or SeV18+/ΔM-GFP at an MOI of 1. The image was obtained by immunostaining with anti-F and anti-HN antibodies. The nucleus is shown in blue. Magnification, ×600.

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