LaCount, D. J. | bioRxiv Science

bioRxiv2026

A Yeast Two-Hybrid Protein Domain Screening Approach for Ebola Virus-Human Protein Interactions Identifies PABPC1 as a Host Factor Required for Replication

Ebola virus (EBOV) causes severe hemorrhagic fever and remains a global health threat despite advances in vaccines and antibody-based therapeutics. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet the full spectrum of these interactions is unknown. To systematically identify EBOV-human protein-protein interactions, we performed more than 200 yeast two-hybrid (Y2H) screens using full-length EBOV proteins and domain fragments as baits and cDNA libraries derived from human liver tissue, macrophages, and interferon-stimulated macrophages. This approach revealed 521 unique interactions supported by confidence scoring based on retest assays and library screen data. The interactome includes proteins involved in RNA metabolism, transcriptional regulation, and post-translational modification, with enrichment for RNA-binding proteins among partners of NP, VP30, and L. By employing multiple constructs for each EBOV gene, interaction interfaces could be localized to specific viral domains. This approach identified a likely MYND domain binding site in the intrinsically ordered region of NP. Similarly, overlapping cDNA fragments from Y2H screens revealed potential binding sites within human proteins, including putative VP30 binding sites on NUFIP2 and PABPC1 that lacked canonical PPPPxY motifs. PABPC1 interacted with VP30 in uninfected human cells and accumulated in inclusion bodies in infected cells that resembled respiratory syncytial virus inclusion body associated granules (IBAGs). Inhibiting PABPC1 expression reduced EBOV replication early in infection. These findings expand the EBOV-host interaction network, identify candidate regulators of viral RNA synthesis, and provide a resource for mechanistic studies and antiviral target discovery. ImportanceEbola virus (EBOV) remains a major global health threat due to its high mortality rate and limited treatment options. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet these interactions are incompletely understood. This study uses a large-scale yeast two-hybrid approach to systematically map EBOV-human protein-protein interactions, revealing hundreds of previously unreported partners and identifying cellular pathways that may be exploited by the virus. By integrating domain mapping, confidence scoring, and functional validation, we provide a resource that advances understanding of EBOV biology and may be used to discover candidates for antiviral development.

microbiology↗

bioRxiv2022

Shifting a Cellular Metabolic Landscape Identifies a Refractory Environment for Flavivirus Replication

Host-targeted therapeutics to control viral infection are gaining prominence given the vulnerability of viral replication at select host-interaction points and the limited possibility of developing drug resistant mutants. Nevertheless, the chemical and biological impact of many host-targeted therapeutics on both the cell and virus has not been elucidated and remains a key complication. Previously, it has been demonstrated that inhibition of fatty acid metabolism has significant antiviral potential. Here, we use a multidisciplinary approach to demonstrate how inhibition of fatty acid biosynthesis creates a metabolically refractory environment that drives viral dependence on alternate metabolic pathways for survival. By profiling the global metabolic landscape following inhibition of fatty acid biosynthesis, we identified additional biochemical pathways that, when inhibited in combination with fatty acid biosynthesis, displayed increased antiviral potential. Our studies also demonstrated that there was a direct link between changes in cellular chemical composition and the ultrastructural membrane architecture induced by viral gene products. Utilizing inhibitors to change these metabolic environments significantly impacted early viral replication and disrupted the membrane architecture critical for the viral life cycle. Here, we have defined at a molecular level how shifting metabolic landscapes can be exploited to identify combinations of therapeutics that have a greater antiviral effect. Author SummaryDengue viruses are transmitted by Aedes aegypti mosquitoes which are prevalent in the tropical and subtropical regions of the world. These viruses cause over 350 million infections annually. There are no antivirals to combat infection and the only vaccine available is suboptimal. Since these viruses are obligate pathogens, they hijack lipid metabolic pathways in host cells to drive new lipid synthesis critically required for their replication. Mechanisms of how lipid synthesis impacts viral replication is unknown. These viruses also rearrange cellular membranes to form platforms for assembly of viral replication complexes. Here, for the first time, we show that virus-hijacking of de novo fatty acid biosynthesis pathways is required for the formation of membranous replication platforms and if inhibited disrupted synthesis of replicative form viral RNA. Importantly, these inhibitors drastically rearranged the metabolic landscape of the cell resulting in an activation of compensatory nucleotide synthesis pathways that allowed the virus to survive at a low level through the inhibition. However, if both pathways were inhibited in combination, infectious virus release was reduced to below detection limits. The study demonstrates how understanding the metabolic landscape altered by specific inhibitors can lead to the discovery of compensatory metabolic pathways and targets that in combination can enhance intervention efficacy.

microbiology↗