Biology subjects

Stubbs, S. H.

Publications and source records attributed to Stubbs, S. H..

3 recordsLinked to original sources

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↗

bioRxiv2021

Vesicular stomatitis virus chimeras expressing the Oropouche virus glycoproteins elicit protective immune responses in mice.

Oropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections. Like other members of the family Peribunyaviridae, the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins Gn and Gc responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized with VSV-OROV specifically neutralized wild type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild type OROV shows that the VSV-OROV chimeras reduce wild type viral infection and suggest that antibodies that recognize the variable N-terminus of Gc afford less protection than those that target more conserved regions of Gc. ImportanceOropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wildtype virus. This data provides the first evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

microbiology↗

bioRxiv2018

Reconstruction of the cell entry pathway of an extinct virus

Endogenous retroviruses (ERVs), remnants of ancient germline infections, comprise 8% of the human genome. The most recently integrated includes human ERV-K (HERV-K) where several envelope (env) sequences remain intact. Viral pseudotypes decorated with one of those Envs are infectious. Using a recombinant vesicular stomatitis virus encoding HERV-K Env as its sole attachment and fusion protein (VSV-HERVK) we conducted a genome-wide haploid genetic screen to interrogate the host requirements for infection. This screen identified 11 genes involved in heparan sulfate biosynthesis. Genetic inhibition or chemical removal of heparan sulfate and addition of excess soluble heparan sulfate inhibit infection. Direct binding of heparin to soluble HERVK Env and purified VSV-HERVK defines it as critical for viral attachment. Cell surface bound VSV-HERVK particles are triggered to infect on exposure to acidic-pH, whereas acid pH pretreatment of virions blocks infection. Testing of additional endogenous HERV-K env sequences reveals they bind heparin and mediate acid pH triggered fusion. This work reconstructs and defines key steps in the infectious entry pathway of an extinct virus.\n\nAuthor SummaryThe genomes of all vertebrates are littered with the remains of once exogenous retroviruses. The properties of these ancient viruses that fostered germline colonization and their subsequent inheritance as genetic elements are largely unknown. The viral envelope protein (Env) dictates the cell entry pathway. Here we define host factors involved in the cell-entry of the youngest human ERV, HERV-K. Using a forward genetic screen, we identified heparan sulfate as a critical mediator of productive cell-entry. The abundance of this carbohydrate on almost all cells in the body suggests that HERV-K endogenization was a consequence of a broad tropism and not a specific targeting of germ cells. We demonstrate that multiple HERV-K Env protein encoded in the genome bind heparin. As HERV-K Envs are expressed in some transformed and virus-infected cells as well as during inflammation, it is tempting to speculate that this heparan sulfate binding property could be physiologically relevant during disease.

microbiology↗