Technologies for Proteome-Wide Discovery of Extracellular Host-Pathogen Interactions

Martinez-Martin, Nadia

Related to the NAPPA technology, Glick and colleagues recently built a miniaturized platform focused on human membrane proteins. By integrating the microfluidics technology, protein microarrays, and an IVTT system, this group built a new device named microfluidic-based comprehensive human membrane protein array (MPA) [32] . A notable improvement introduced by these investigators was the addition of microsomal membranes to the IVTT system to allow for improved folding and posttranslational modifications in plasma membrane proteins, both common limitations of IVTT systems. In this work, a library of 2,700 human genes encoding for membrane proteins was built and subsequently utilized to screen the large-form delta antigen (L-HDAg) encoded by the hepatitis delta virus (HDA) and whole viral particles of the simian virus 40 (SV40), a nonenveloped human pathogen. Proof-of-concept assays showed encouraging results, with over 75% true-positive rate within a small set of proteins with known interactors and, more importantly, indicated the feasibility of this approach for expression of multitransmembrane-containing proteins, a protein type that has proven challenging given their high hydrophobicity. The MPA screens identified 99 and over 150 interactions for SV40 particles and L-HDAg viral protein, respectively, and around 35 interactions were validated by coimmunoprecipitation or protein-fragment complementation assays [32] . To our knowledge, this is the first study to assess ePPIs using a comprehensive human protein library and whole viral particles (SV40) as baits, a valuable approach that may provide important insights into pathogen tropism, alongside a molecular explanation for the cell surface receptors engaged by the pathogen. Further utilization of this platform followed by a more systematic analysis of the candidate hits, including nonspecific binder determination, will be needed to assess the overall performance of the MPA technology. Regardless, this platform provides an extended version of the NAPPA approach that focuses on mammalian ePPIs and may therefore provide relevant insights into extracellular hostpathogen interactions.