Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

Abstract

Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCEThe exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy. P hloem-limited viruses, such as poleroviruses, are among the most devastating, but least understood pathogens infecting plants due to the technical challenges encountered when studying pathogens that naturally replicate and move within intractable cell types (1). Mutagenesis studies performed with infectious clones have greatly enhanced our understanding of the domains within viral proteins that are re...

Plant Genomics

“…bacterial lacZ gene and the yeast HIS3 gene, which are both controlled by the LexA operators [29,30]. Another novel reporter system that has been created for Y2H assays utilizes an enhanced green fluorescent protein (EGFP)-based reporter in order to characterize interactions.…”

“…1). Modifying protein structures through mutation or domain deletion can reveal functionally significant PPIs and increase understanding of the structure function relationships of your POIs [29,30]. Mechanism-based trapping of interactors by protein engineering can increase binding affinity and has been applied to kinases, phosphatases, proteases, E3 ligases, and many other protein types [29,[31][32][33].…”

Proteomics Tracing the Footsteps of Infectious Disease

“…Although this technology has not yet been applied in human infections, the study found several instances of interaction topologies that could reflect conserved functions in unrelated human viruses. For example, a PLRV-host interaction was identified that was analogous to the known structural bind-ing of the animal zinc binding protein BS69 to a conserved PXLXP motif in viral proteins from the Heresviridae and Adenoviridae families (84). Overall, this technology has enormous potential in pathogen proteomics, with the ability to identify in a single experiment both the interacting protein pairs and the higher order structures that mediate the interaction.…”

“…Toward this goal, the continued development of in vivo chemical labeling techniques paired with mass spectrometry will increase the specificity of identifying interactions in infected cells and provide the direct points of hostpathogen contact. For instance, an in vivo chemical crosslinking strategy paired with protein interaction reporter technology and mass spectrometry analysis has been used to identify protein interaction interfaces in cultured cells (83), viruses (84), and bacteria (85). The first application of this technology to viral infection characterized the protein interaction topologies of the plant pathogen Potato leafroll virus (PLRV) (84).…”

“…For instance, an in vivo chemical crosslinking strategy paired with protein interaction reporter technology and mass spectrometry analysis has been used to identify protein interaction interfaces in cultured cells (83), viruses (84), and bacteria (85). The first application of this technology to viral infection characterized the protein interaction topologies of the plant pathogen Potato leafroll virus (PLRV) (84). Although this technology has not yet been applied in human infections, the study found several instances of interaction topologies that could reflect conserved functions in unrelated human viruses.…”