Biology subjects

Overby, A. K.

Publications and source records attributed to Overby, A. K..

11 recordsLinked to original sources

bioRxiv2025

Investigation of Powassan virus lineage II pathogenesis and neurotropism in mice

Powassan virus (POWV) is an emerging tick-borne flavivirus that causes disease in humans. POWV has considerable genetic and phenotypic diversity, including highly variable replication in vitro and pathogenesis in mice. This study sought to define the extent of variability in pathogenesis within POWV lineage II in mice and investigate possible viral determinants. Relative to other strains, two New York-derived isolates, NY.19.12 and NY.19.32, caused earlier clinical signs and earlier detection of viral RNA (vRNA) in the spleen and brain compared to mice infected with virus derived from a lineage II infectious clone (WI.97.ic). Sequencing revealed these strains share three amino acid substitutions in envelope, NS1, and NS5 compared to other lineage II strains, which were engineered into a mutant infectious clone. At early time points post-infection, clinical signs, vRNA detection in the cerebellum, and viral distribution in the brain were similar between NY.19.12 and the mutant clone, suggesting these mutations may play a role in disease progression and early neuroinvasion. However, NY.19.12 vRNA was detected in the spleen at significantly higher rates compared to both WI.97.ic and the mutant clone, indicating factors other than these mutations are responsible for increased spleen infection. Importantly, this study highlights the complexity of POWV pathogenesis and suggests that POWV lineage II strains have varying disease phenotypes likely driven by multiple genetic differences. ImportanceTick-borne flaviviruses exhibit considerable genetic and phenotypic diversity in nature, influencing their transmission and pathogenesis. Defining the mechanisms of pathogenesis requires understanding how inter-strain variation translates to phenotypic differences in viral dissemination and neuroinvasion. This study demonstrates that even closely related Powassan virus (POWV) lineage II strains display distinct disease phenotypes that are only partially attributable to nonsynonymous consensus mutations within the viral coding sequence. By highlighting the complexity of POWV infection dynamics in mice, these findings provide valuable insights into how POWV lineage II diversity may shape disease progression and severity in humans.

microbiology↗

bioRxiv2024

The influence of the pre-membrane and envelope proteins on structure, pathogenicity and tropism of tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is a neurotropic flavivirus that causes thousands of human infections annually. Viral tropism in the brain is determined by the presence of necessary receptors, entry factors and the ability of the virus to overcome host defenses. The viral structural proteins, pre-membrane (prM) and envelope (E), play an important role in receptor binding, membrane fusion, particle maturation, and antibody neutralization. To understand how these proteins influence virus distribution and tropism in the brain, we generated a chimeric virus harboring the prM and ectodomain of E from TBEV in the background of the low pathogenic Langat virus (LGTV). We solved the atomic structures of both the chimeric virus and LGTV to compare them to the known TBEV structure. We show that this chimeric virus remains low-pathogenic, while being structurally and antigenically similar to TBEV. Using 3D optical whole brain imaging combined with immunohistochemistry, we found that both LGTV and the chimeric virus primarily infect cerebral cortex, with no significant differences in their localization or tropism. In contrast, TBEV shows high infection of the cerebellum and strong preference towards Purkinje cells, indicating that the non-structural proteins are important for determining TBEV tropism in the brain. Together, this provides new insights into the roles of the structural and non-structural proteins of tick-borne flaviviruses.

microbiology↗

bioRxiv2024

Defining short linear motif binding determinants by phage-based multiplexed deep mutational scanning

Deep mutational scanning (DMS) has emerged as a powerful approach for evaluating the effects of mutations on binding or function. Here, we developed a multiplexed DMS by phage display protocol to define the binding determinants of short linear motifs (SLiMs) binding to peptide binding domains. We first designed a benchmarking DMS library to evaluate the performance of the approach on well-known ligands for eleven different peptide binding domains, including the talin-1 PTB domain. Systematic benchmarking against a gold-standard set of motifs from the eukaryotic linear motif (ELM) database confirmed that the DMS by phage analysis correctly identifies known motif binding determinants. The DMS analysis further defined a non-canonical PTB binding motif, with a putative extended conformation. A second DMS library was designed aiming to provide information on the binding determinants for 19 SLiM-based interactions between human and SARS-CoV-2 proteins. The analysis confirmed the affinity determining residues of viral peptides binding to host proteins, and refined the consensus motifs in human peptides binding to five domains from SARS-CoV-2 proteins, including the non-structural protein (NSP) 9. The DMS analysis further pinpointed mutations that increased the affinity of ligands for NSP3 and NSP9. An affinity improved cell-permeable NSP9-binding peptide was found to exert stronger antiviral effects as compared to the initial wild-type peptide. Our study demonstrates that DMS by phage display can efficiently be multiplexed and applied to refine binding determinants, and shows how DMS by phage display can guide peptide-engineering efforts.

biochemistry↗

bioRxiv2024

The flavivirus protein NS4B recruits the cis-Golgi protein ACBD3 to modify ER-Golgi trafficking for virion release

Flavivirus infection involves extensive remodeling of the endoplasmic reticulum (ER), which is key to both the replication of the viral RNA genome as well as the assembly and release of new virions. Yet, little is known about how viral proteins and host factors cooperatively facilitate such a vast transformation of the ER, and how this influences the different steps of the viral life cycle. In this study, we screened for host proteins that interact with the tick-borne encephalitis virus (TBEV) protein NS4B and found that the top candidates were coupled to trafficking between ER exit sites (ERES) and the Golgi. We characterized the role of ACBD3, one of the identified proteins, in flavivirus infection and show that it interacts with NS4B to promote infection across multiple flavivirus species. Using ACBD3 knockout cells, we found that the depletion of ACBD3 inhibited TBEV replication by preventing the trafficking of virions from the cell. We found that ACBD3 promotes flavivirus infection via a different mechanism than its previously described role in picornavirus infection. ACBD3 was enriched at modified ERES-Golgi contact sites to support virus replication. Therefore, we propose that ACBD3 promotes flavivirus replication by modifying the trafficking between the ERES and the Golgi to enable the release of new virions. Author summaryFlaviviruses including dengue virus and tick-borne encephalitis virus are group of viruses that widely affecting the health of human. During infection, flavivirus particles enter host cells and transform the endoplasmic reticulum (ER), which is the main structure for protein synthesis in cells. New flaviviral particles are produced in the transformed ER and then released to the Golgi apparatus, which is the main structure for protein transport in cells. It is unclear how the particles are transported from the ER to Golgi. Here we screened the factors that interact with viral proteins and identified a Golgi protein called ACBD3 as an important factor supporting flavivirus particles release from ER to Golgi. We showed that ACBD3 is recruited by flavivirus to a modified connection between ER and Golgi for viral particles release from the ER. Our work provides new insights into the fine coordination of virus replication and viral particles transport between organelles inside host cells.

cell biology↗

bioRxiv2023

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

microbiology↗

bioRxiv2022

An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy

Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high-resolution optical imaging techniques operating in the mm-cm range, ideally suited for ex vivo 3D whole mouse brain imaging. Although these techniques exhibit high sensitivity and specificity for antibody-labeled targets, the provided anatomical information remains limited. To allow anatomical mapping of fluorescent signal in whole brain, we developed a novel magnetic resonance (MR) - based template with its associated tissue priors and atlas labels, specifically designed for brains subjected to tissue processing protocols required for 3D optical imaging. We investigated the effect of tissue pre-processing and clearing on brain size and morphology and developed optimized templates for BABB/Murrays clear (OCUM) and DBE/iDISCO (iOCUM) cleared brains. By creating optical-(i)OCUM fusion images using our mapping procedure, we localized dopamine transporter and translocator protein expression and tracer innervation from the eye to the lateral geniculate nucleus of thalamus and superior colliculus. These fusion images allowed for precise anatomical identification of fluorescent signal in discrete brain areas. As such, these templates enable applications in a broad range of research areas integrating optical 3D brain imaging by providing an MR template for cleared brains.

neuroscience↗

bioRxiv2022

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.

The infection and replication cycle of all viruses depend on interactions between viral and host proteins. Each of these protein-protein interactions is therefore a potential drug target. These host-virus interactions often involve a disordered protein region on one side of the interface and a folded protein domain on the other. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind peptides from human proteins. Of 281 high/medium confidence peptides, 23 interactions involving eight SARS-CoV-2 protein domains were tested by fluorescence polarization, and binding was observed with affinities spanning the whole micromolar range. The key specificity determinants were established for six of these domains, two based on ProP-PD and four by alanine scanning SPOT arrays. Finally, two cell-penetrating peptides, targeting Nsp9 and Nsp16, respectively, were shown to function as inhibitors of viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously provide information on potential host-virus interactions and identify ligands with antiviral properties.

molecular biology↗

bioRxiv2022

A syntenin inhibitor blocks endosomal entry of SARS-CoV-2 and a panel of RNA viruses

Viruses are dependent on interactions with host factors in order to efficiently establish an infection and replicate. Targeting such interactions provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking, and has previously been shown to be important for HPV infection. Here we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits SARS-CoV-2 infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection, and strongly reduced flavivirus infection, which are completely dependent on receptor mediated endocytosis for their entry. In conclusion, we have identified a novel pan-viral inhibitor that efficiently target a broad range of RNA viruses.

biochemistry↗

bioRxiv2022

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1,712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.

biochemistry↗

bioRxiv2021

Whole-brain fluorescence-MRI coregistration for precise anatomical mapping of virus infection

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combined multimodal imaging (cm-nm scale) with single nuclei RNA- sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection was imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection was limited to grey matter of sensory systems in wildtype mice, but extended into white matter, meninges and choroid plexus in Ifnar-/-mice. Cells in wildtype displayed strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+NK cells, whereas in Ifnar-/-, the absence of this response contributed to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.

microbiology↗

bioRxiv2021

Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, current methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a scalable viral peptide discovery approach covering 229 RNA viruses that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an {Phi}xFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its {Phi}xFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction blocks SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

biochemistry↗