Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain.
The cytotoxic T-lymphocyte (CTL) response to rabies virus glycoprotein has been studied. A primary in vivo CTL response was obtained following inoculation of A/J mice with 10 micrograms of glycoprotein, but only when in the form of reconstituted glycoprotein-lipid vesicles. These glycoprotein-lipid vesicles were prepared with lipids from BHK-21 cells, and did not incorporate mouse major histocompatibility complex (MHC) antigens. Secondary in vitro stimulation of rabies virus-specific CTL was obtained with inactivated virus and with larger quantities of glycoprotein. This response, but not that induced by rabies virus-infected stimulator cells, was dependent on the presence of radiation-resistant accessory cells which could not be replaced with T-cell growth factors. An analysis of the molecular requirements for stimulation of CTL by glycoprotein revealed that cleavage by cyanogen bromide (CNBr) or limited tryptic digestion actually enhanced stimulation of CTL. In contrast, reduction and alkylation destroyed activity. Following separation of CNBr or tryptic fragments by gel electrophoresis or high-pressure liquid chromatography (HPLC) (under nonreducing conditions), a nominal determinant of glycoprotein was identified. One CNBr peptide (residues 103-178) and one peak of tryptic peptides were found to stimulate rabies virus-specific CTL. The tryptic peak was further analyzed by Edman degradation-sequencing, and found to consist of three peptides with amino terminals at residues 130, 251 and 279. This evidence suggests that a nominal determinant of glycoprotein responsible for stimulating rabies virus-specific CTL is located between residues 130-178 of the glycoprotein, and incorporates a single disulfide loop (159-169) which is necessary for biologic activity.
D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology*; D003488:Cyanogen Bromide / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D005260:Female; D006023:Glycoproteins / Q000276:immunology*; D051379:Mice; D008815:Mice, Inbred Strains; D011820:Rabies virus / Q000276:immunology*; D013154:Spleen / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014357:Trypsin / Q000494:pharmacology
D000956:Antigens, Viral; D006023:Glycoproteins; D014357:Trypsin; D003488:Cyanogen Bromide