Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation.
Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of approximately 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.
Hahn
C S
CS
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110-1093.
Hahn
Y S
YS
Braciale
T J
TJ
Rice
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology*; D000941:Antigens / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D004274:DNA, Recombinant; D000939:Epitopes; D005822:Genetic Vectors; D006183:H-2 Antigens / Q000276:immunology; D006389:Hemagglutinins, Viral / Q000276:immunology; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D012845:Sindbis Virus / Q000235:genetics* / Q000254:growth & development; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014779:Virus Replication
D000941:Antigens; D004274:DNA, Recombinant; D000939:Epitopes; D006183:H-2 Antigens; D006389:Hemagglutinins, Viral