Application of in vitro transcribed and translated HIV-1 reverse transcriptase and integrase in protein-protein interaction studies
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Replication of the human immunodeficiency virus (HIV) necessitates the two viral enzymes reverse transcriptase (RT) and integrase (IN). These two proteins are found to associate in a large nucleoprotein complex called the preintegration complex (PIC) together with viral nucleic acids and other viral and host proteins. Since the two proteins are localised together in the host cell, a direct interaction between them can exist, although it has been difficult to demonstrate such interaction. In this study, RT and IN were produced by in vitro transcription and translation and the proteins were used to study possible RT-IN interactions by ELISA. The RT and IN coding regions were amplified by PCR from a full length HIV-1 HxB2 clone and ligated in-frame into the in vitro translation vector pCITE-5b(+). The two plasmids generated, pCITE-RT and pCITE-IN were constructed and verified by sequencing and restriction enzyme analysis. A coupled in vitro transcription and translation kit based on rabbit reticulocyte lysate was utilised together with the two plasmids in order to obtain RT and IN. The proteins were produced unmarked since marked amino acids possibly could obscure an eventual interaction. Verification of the protein products was done by Western blotting having specific monoclonal antibodies. The proteins produced were not purified from the reticulocyte lysate but used directly in an ELISA-format to try to detect any interactions between RT and IN. With the ELISA-format, no interactions could be demonstrated. The signal was the same for all dilutions of the coating protein, as well as for the negative control. The most likely explanation for this is that there are some proteins in the reticulocyte lysate that interacts with both RT and IN. If in vitro translated proteins are going to be used to investigate protein-protein interactions by this ELISA method they need to be purified first. An other possibility is to cotranslate the two proteins in vitro. It is not sure that an eventual direct interaction between RT and IN can be detected by ELISA. Other components of the PIC may need to be present for a direct interaction to occur.
PublisherThe University of Bergen
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