EVALUATION OF MHC CLASS I PEPTIDE-SOURCE PROTEINS USING WESTERN BLOTS AND REAL-TIME PCR.

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EVALUATION OF MHC CLASS I PEPTIDE-SOURCE PROTEINS USING WESTERN BLOTS AND REAL-TIME PCR.
Angela D. Luis, B.S. , Heather D. Hickman, M.S. , Wilfried Bardet M.S. and William H. Hildebrand, Ph.D. . Oklahoma City OK, University of Oklahoma Health Sciences Center, 73104, Microbiology and Immunology .

Cellular infection by pathogens typically involves utilization of host cellular protein or pathways. As a consequence of this, MHC class I molecules bind and present peptides unique to the infected cell. The foremost explanations for presentation of peptides are altered RNA or protein levels. In order to validate peptide changes during infection through an understanding of their derivation, we have adapted experiments to determine the contribution of transcriptional activation and/or protein expression on peptide presentation. After peptide source protein identification, uninfected and infected cells are lysed and protein and total RNA extracted. Peptide presentation due to transcriptional up-regulation is examined by using real-time PCR on cDNA. Likewise, unique peptide presentation resulting from altered protein levels of the peptide’s source protein is examined by performing Western blots on the cell lysates. Application of this technique to HIV-infected cells allows determination of causes for peptide up-regulation. For example, infection with HIV results in the unique presentation of an HLA-B*0702 peptide derived from eukaryotic translation initiation factor 4GI (eIF4GI). When infected cells were examined, no change in RNA levels was detected by real-time PCR between HIV infected and uninfected cells. However, Western blotting with a monoclonal antibody against eIF4GI showed a dramatic decrease in protein level by day 3 post infection. Therefore, the up-regulation of this HLA-bound peptide was a result of increased degradation of the source protein. In conclusion, we found that real-time PCR and Western blotting are valid methods of evaluating source proteins during pathogenic infection while providing additional information concerning host-pathogen interactions.