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Journal of Virology, March 2003, p. 3238-3246, Vol. 77, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.5.3238-3246.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Nontransmissible Virus-Like Particle Formation by F-Deficient Sendai Virus Is Temperature Sensitive and Reduced by Mutations in M and HN Proteins

DNAVEC Research Inc., Tsukuba-shi, Ibaraki 305-0856,¹ Toyama Institute of Health, Kosugi-machi, Imizu-gun, Toyama 939-0363, Japan²

Received 13 September 2002/ Accepted 28 November 2002

The formation of nontransmissible virus-like particles (NTVLP) by cells infected with F-deficient Sendai virus (SeV/F) was found to be temperature sensitive. Analysis by hemagglutination assays and Western blotting demonstrated that the formation of NTVLP at 38°C was about 1/100 of that at 32°C, whereas this temperature-sensitive difference was only moderate in the case of F-possessing wild-type SeV. In order to reduce the NTVLP formation with the aim of improving SeV for use as a vector for gene therapy, amino acid substitutions found in temperature-sensitive mutant SeVs were introduced into the M (G69E, T116A, and A183S) and HN (A262T, G264R, and K461G) proteins of SeV/F to generate SeV/M^tsHN^tsF. The use of these mutations allows vector production at low temperature (32°C) and therapeutic use at body temperature (37°C) with diminished NTVLP formation. As expected, the formation of NTVLP by SeV/M^tsHN^tsF at 37°C was decreased to about 1/10 of that by SeV/F, whereas the suppression of NTVLP formation did not cause either enhanced cytotoxicity or reduced gene expression of the vector. The vectors showed differences with respect to the subcellular distribution of M protein in the infected cells. Clear and accumulated immunocytochemical signals of M protein on the cell surface were not observed in cells infected by SeV/F at an incompatible temperature, 38°C, or in those infected by SeV/M^tsHN^tsF at 37 or 38°C. The absence of F protein in SeV/F and the additional mutations in M and HN in SeV/M^tsHN^tsF probably weaken the ability to transport M protein to the plasma membrane, leading to the diminished formation of NTVLP.

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