ABSTRACT
The formation of nontransmissible virus-like particles (NTVLP) by cells infected with F-deficient Sendai virus (SeV/ΔF) 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/ΔF to generate SeV/MtsHNtsΔF. 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/MtsHNtsΔF at 37°C was decreased to about 1/10 of that by SeV/ΔF, 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/ΔF at an incompatible temperature, 38°C, or in those infected by SeV/MtsHNtsΔF at 37 or 38°C. The absence of F protein in SeV/ΔF and the additional mutations in M and HN in SeV/MtsHNtsΔF probably weaken the ability to transport M protein to the plasma membrane, leading to the diminished formation of NTVLP.
Gene Expression Profiling Related to Hyphal Growth in a Temperature-Sensitive Mutant of Magnaporthe oryzae
