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The formation of nontransmissible virus-like particles (NTVLP) by cells infected with F-deficient Sendai virus (SeV/deltaF) was found to be temperature sensitive. Analysis by hemagglutination assays and Western blotting demonstrated that the formation of NTVLP at 38 degrees C was about 1/100 of that at 32 degrees 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/deltaF to generate SeV/M(ts)HN(ts)deltaF. The use of these mutations allows vector production at low temperature (32 degrees C) and therapeutic use at body temperature (37 degrees C) with diminished NTVLP formation. As expected, the formation of NTVLP by SeV/M(ts)HN(ts)deltaF at 37 degrees C was decreased to about 1/10 of that by SeV/deltaF, 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/deltaF at an incompatible temperature, 38 degrees C, or in those infected by SeV/M(ts)HN(ts)deltaF at 37 or 38 degrees C. The absence of F protein in SeV/deltaF and the additional mutations in M and HN in SeV/M(ts)HN(ts)deltaF probably weaken the ability to transport M protein to the plasma membrane, leading to the diminished formation of NTVLP.

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