Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 110

A major goal of virology research is to understand how protein-protein interactions modulate reproduction cycles and pathogenesis. Consequently, multiple experimental approaches have been devised to identify the entire set of interactions among viral proteins and between viral and cell proteins. The yeast two-hybrid screen, a complementation assay which was designed to discover protein-protein interactions, has been adapted to high-throughput applications. In this assay, a transcriptional regulatory protein is split into two fragments, the DNA-binding domain and the activating domain. The coding sequences of two different proteins are fused with the two domains. If the two proteins interact, when the fusion proteins are produced in cells, transcriptional activation (leading to the transcription of a reporter gene) will take place. For high-throughput applications, libraries of protein-coding DNAs are screened against a single viral protein or all viral proteins. This method was used to describe the virus-host interactome of two herpesviruses.

Other approaches to defining interactomes include coimmunoprecipitation, affinity purification of tagged proteins (Fig. 2.21), and labeling of cell proteins with chemical cross-linkers (used to identify plant proteins that interact with plant virus proteins), followed by mass spectrometry.

While these methods allow definition of virus-cell interactomes, they are not unambiguous. For at least one virus, interactomes determined in different laboratories are very diverse. Most importantly, the observation of a protein-protein interaction does not confirm biological relevance: the roles of such interactions in viral reproduction must be determined by other means (Box 2.12).