Biochemical characterization of temperature-sensitive rabies virus mutants

Abstract

Biochemical characterization of 70 temperature-sensitive (ts) mutants of rabies virus was done by following the appearance of viral proteins and RNA molecules in infected [baby hamster kidney BHK-21] cells at both permissive and nonpermissive temperature. The presence or absence of the nucleocapsid protein (N) was demonstrated by treating infected cells with anti-N fluorescent antibodies. At 33.degree. C, all the mutants induced a fluorescence comparable to the wild type. At 39.6.degree. C, the mutants can be classified into 3 groups. Three mutants induced a fluorescence comparable to the wild type (F+ mutants); 54 mutants induced a faint fluorescence which was proportional to the multiplicity of infection and increased with time (F+- mutants). No fluorescence could be detected for the 13 remaining mutants (F- mutants). The synthesis of all viral proteins was normal for F+ mutants, indicating that transcription and replication of the virus were normal and that the ts lesion was located in a protein which is not directly required for those functions. The synthesis of all viral proteins was similarly decreased for F+- mutants and undetectable for the F- mutants. This suggests that the ts lesion affects the transcription and/or replication of the virus. Annealing techniques demonstrated that the F+- mutants were able to perform some amount of secondary transcription at nonpermissive temperature. No secondary transcription occurred with F- mutants. When detectable (i.e., at higher multiplicity of infection), primary transcription of F- mutants was normal.