Plasmodium falciparum enolase (Pfeno) localizes to the cytosol, nucleus, cell membrane and cytoskeletal elements, suggesting multiple non-glycolytic functions for this protein. Our recent observation of association of enolase with the food vacuole (FV) in immuno-gold electron microscopic images of P. falciparum raised the possibility for yet another moonlighting function for this protein. Here we provide additional support for this localization by demonstrating the presence of Pfeno in purified FVs by immunoblotting. To examine the potential functional role of FV-associated Pfeno, we assessed the ability of Pfeno to complement a mutant Saccharomyces cervisiae strain deficient in enolase activity. In this strain (Tetr-Eno2), the enolase 1 gene is deleted and expression of the enolase 2 gene is under the control of a tetracycline repressible promoter. Enolase deficiency in this strain was previously shown to cause growth retardation, vacuolar fragmentation and altered expression of certain vacuolar proteins. Expression of Pfeno in the enolase-deficient yeast strain restored all three phenotypic effects. However, transformation of Tetr-eno2 with an enzymatically active, monomeric mutant form of Pfeno (Δ(5)Pfeno) fully restored cell growth, but only partially rescued the fragmented vacuolar phenotype, suggesting that the dimeric structure of Pfeno is required for the optimal vacuolar functions. Bioinformatic searches revealed the presence of Plasmodium orthologs of several yeast vacuolar proteins that are predicted to form complexes with Pfeno. Together, these observations raise the possibility that association of Pfeno with food vacuole in Plasmodium may have physiological function(s).

Zika virus (ZIKV) nonstructural protein 5 (NS5) plays a critical role in viral RNA replication and mediates key virus-host cell interactions. As with other flavivirus NS5 proteins, ZIKV NS5 is primarily found in the nucleus. We previously reported that the NS5 protein of dengue virus, another flavivirus, localized to centrosomes during cell division. Here we show that ZIKV NS5 also relocalizes from the nucleus to centrosomes during mitosis. In infected cells with supernumerary centrosomes, NS5 was present at all centrosomes. Transient expression of NS5 in uninfected cells confirmed that centrosomal localization was independent of other viral proteins. Live-cell imaging demonstrated that NS5-GFP accumulated at centrosomes shortly after break down of nuclear membrane and remained there through mitosis. Cells expressing NS5-GFP took longer to complete mitosis than control cells. Finally, an analysis of ZIKV NS5 binding partners revealed several centrosomal proteins, providing potential direct links between NS5 and centrosomes.

The replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors involved in actin nucleation, such as Rac1 and Arp2/3, were shown important in both steps. However, downstream of nucleation, many other cell factors are needed to control actin dynamics. How these regulate EBOV infection remains largely unclear. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown of CAPG specifically inhibited viral infectivity and yield of infectious particles. Cell-based mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split-green fluorescent protein reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells.

In order to establish a successful infection in the human host, the malaria parasite Plasmodium falciparum must establish interactions with a variety of human proteins on the surface of different cell types, as well as with proteins inside the host cells. To better understand this aspect of malaria pathogenesis, a study was conducted with the goal of identifying interactions between proteins of the parasite and those of its human host.

Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs. Yeast two-hybrid screens with ANK1 and 4.1R identified eight interactions with P. falciparum exported proteins, including an interaction between 4.1R and PF3D7_0402000 (PFD0090c). This interaction was first identified in a large-scale screen (Vignali et al., Malaria J, 7:211, 2008), which also reported an interaction between PF3D7_0402000 and ANK1. We confirmed the interactions of PF3D7_0402000 with 4.1R and ANK1 in pair-wise yeast two-hybrid and co-precipitation assays. In both cases, an intact PHIST domain in PF3D7_0402000 was required for binding. Complex purification followed by mass spectrometry analysis provided additional support for the interaction of PF3D7_0402000 with ANK1 and 4.1R. RBC ghost cells loaded with maltose-binding protein (MBP)-PF3D7_0402000 passed through a metal microsphere column less efficiently than mock- or MBP-loaded controls, consistent with an effect of PF3D7_0402000 on RBC rigidity or membrane stability. This study confirmed the interaction of PF3D7_0402000 with 4.1R in multiple independent assays, provided the first evidence that PF3D7_0402000 also binds to ANK1, and suggested that PF3D7_0402000 affects deformability or membrane stability of uninfected RBC ghosts.

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Proximity biotinylation was developed to detect physiologically relevant protein-protein interactions in living cells. In this method, the protein of interest is tagged with a promiscuous biotin ligase, such as BioID or BioID2, which produces activated biotin that reacts with nearby proteins; these proteins can subsequently be purified and identified by mass spectrometry. Here we report a novel modification of this technique by combining it with a self-associating split-GFP system in which we exploit the high-affinity interaction between GFP1-10 and GFP11 to recruit BioID2 to the protein of interest. As a test case, we fused GFP11 to clathrin light chain (CLTB) and BioID2 to GFP1-10. Co-expression of GFP11-CLTB and BioID2-GFP1-10 yielded a green fluorescent complex that co-localized with clathrin heavy chain. To facilitate removal of non-specifically biotinylated proteins, we generated an inducible cell line expressing BioID2-GFP1-10. Proximity biotinylation in this cell line with GFP11-CLTB yielded a higher percentage of biologically relevant interactions than direct fusion of BioID2 to CLTB. Thus, this system can be used to monitor expression and localization of BioID bait proteins and to identify protein-protein interactions.