Pseudotyped lentivirus vectors derived from simian immunodeficiency virus SIVagm with envelope glycoproteins from paramyxovirus

Abstract

We describe the development of novel lentivirus vectors based on simian immunodeficiency virus from African green monkey (SIVagm) pseudotyped with Sendai virus (SeV) envelope glycoproteins. SeV fusion (F) and hemagglutinin-neuraminidase (HN) proteins were successfully incorporated into the SIVagm-based vector by truncation of the cytoplasmic tail of the F protein and by addition of the cytoplasmic tail of SIVagm transmembrane envelope protein to the N terminus of the HN protein. As with the vesicular stomatitis virus G glycoprotein-pseudotyped vector, the mutant SeV F- and HN-pseudotyped SIVagm vector was able to transduce various types of animal and human cell lines. Furthermore, the vector was able to transduce an enhanced green fluorescent protein reporter gene into polarized epithelial cells of rat trachea from the apical and basolateral sides. Therefore, SeV F- and HN-pseudotyped SIVagm vectors have considerable potential for effective use in gene therapy for various therapies, including respiratory diseases.

FIG. 1.

Organization of SeV wild-type (wt) and mutant F and HN proteins. (A) HN expression vectors. Schematic representation of HN protein and the amino acid sequence of the cytoplasmic tail (CT) of SIVagm TMP are shown. The numbers represent amino acids derived from SIVagm TMP. term, terminus. (B) F expression vectors. The amino acid sequences of part of the transmembrane domain and the cytoplasmic tail are shown. The plasmid names (Fct4, Fct14, and Fct27) indicate the number of residues in the cytoplasmic tail of the F protein.

FIG. 2.

Flow cytometric analysis of cell surface expression of SeV wild-type (wt) and mutant F or HN protein. 293T cells were transfected as described in Materials and Methods. At 24 h after transfection, cells were immunostained with a specific monoclonal antibody against F or HN protein and reacted with fluorescein-labeled anti-mouse immunoglobulin G. The solid area of each histogram indicates positive cells.

FIG. 3.

Western blot analysis of incorporation of SeV wild-type and mutant F and HN proteins into SIVagm vectors. Virion lysates of SeV wild-type (wt) and mutant F- and HN-pseudotyped vectors were prepared from pellets of supernatants of producer cells by ultracentrifugation. Samples were transferred to a polyvinylidene difluoride membrane and blotted with the anti-HN antibody HN-2 (A) or the anti-F1 antibody (B). SeV virion lysates were used as the positive control for each protein.

FIG. 4.

Western blot analysis of incorporation of a SeV mutant F or HN protein into SIVagm vectors. 293T cells were cotransfected with the Fct4 or SIVct+HN expression plasmid in combination with gene transfer and packaging vectors. Preparation of pseudotyped virion lysates and Western blot analysis with the anti-F1 antibody (lanes 1 to 4) or the anti-HN antibody HN-2 (lanes 5 to 8) was performed as described for Fig. 3. SeV virion lysates were used as the positive control for each protein. Lanes: 1 and 5, SeV; 2 and 6, Fct4; 3 and 7, SIVct+HN; and 4 and 8, Fct4 and SIVct+HN.

FIG. 5.

Inhibition of transduction by SeV mutant F- and HN-pseudotyped SIVagm vector with a neutralizing antibody specific to SeV. The SeV Fct4- and SIVct+HN-pseudotyped vector at 0.8 × 10⁴ T.U. or the VSV-G-pseudotyped vector at 1.2 × 10⁶ T.U. was incubated with the immunoglobulin G fraction of a neutralizing antibody specific to SeV (anti-HN antibody HN-2) or VSV (anti-VSV-G antibody I-1) at 4°C for 30 min, followed by transduction of 293T cells. Seventy-two hours after the transduction, EGFP-expressing cells were detected by fluorescence microscopy.

FIG. 6.

Cell tropism of SeV mutant F and HN vector. Cells were replated the day before the Fct4- and SIVct+HN- or VSV-G-pseudotyped SIVagm vector infection. About 10⁶ cells in six-well plates were infected with 1 ml of the diluted vector solution. The number of EGFP-expressing cells was counted 72 h after infection. Titers of the vector solution measured in 293T cells were 1 × 10⁵ T.U./ml in the Fct4- and SIVct+HN-pseudotyped vector and 2 × 10⁶ T.U./ml in the VSV-G-pseudotyped vector.

FIG.7.

Transduction of RTE cells with pseudotyped SIVagm vectors. Fluorescent images of EGFP expression on day 3 and phase contrast microscopy of RTE cells. RTE cells were exposed to Fct4- and SIVct+HN-pseudotyped SIVagm vectors at the apical or basolateral side. The titers of the VSV-G-pseudotyped SIVagm vector were (a) 4.9 × 10⁵ T.U./0.1 ml and (b) 1.2 × 10⁸ T.U./0.1 ml; the titer of the SeV Fct4- and SIVct+HN-pseudotyped SIVagm vector was 1.2 × 10⁴ T.U./0.1 ml; and the titer of the SeV Fct4- and SIVct+HN-pseudotyped Moloney murine leukemia virus vector was 1.7 × 10⁴ T.U./0.1 ml.