Biology subjects

Barklis, E.

Publications and source records attributed to Barklis, E..

6 recordsLinked to original sources

bioRxiv2026

Mapping SARS-CoV-2 Nucleocapsid Function with Nanobodies

The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the proteins structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential. ImportanceSARS-CoV-2 and emerging coronaviruses remain a major global health threat, yet critical gaps persist in our understanding of their molecular pathogenesis. The nucleocapsid (N) protein, the most abundantly expressed SARS-CoV-2 antigen, plays essential roles beyond genome packaging, including immune evasion and intracellular organization. Here, we generate and characterize a panel of domain-specific nanobodies (VHHs) that enable precise dissection of Ns functional architecture. Using integrated biochemical, structural, and virological approaches, we uncover distinct mechanisms of viral inhibition, including disruption of phase condensation through a conserved linker region. These findings address long-standing knowledge gaps about a multifunctional viral protein and establish VHHs as powerful, modular tools for probing coronavirus biology, with broad potential for therapeutic, diagnostic, and cell biology applications.

microbiology↗

bioRxiv2025

Analysis of factors that regulate HIV-1 fusion in reverse

Based on observations that HIV-1 envelope (Env) proteins on the surfaces of cells have the capacity to fuse with neighboring cells or enveloped viruses that express CD4 receptors and CXCR4 co-receptors, we tested factors that affect the capacities of lentiviral vectors pseudotyped with CD4 and CXCR4 variants to infect Env-expressing cells. The process, which we refer to as fusion in reverse, involves the binding and activation of cellular Env proteins to fuse membranes with lentiviruses carrying CD4 and CXCR4 proteins. We have found that infection via fusion in reverse depends on cell surface Env levels, is inhibitable by an HIV-1-specific fusion inhibitor, and preferentially requires lentiviral pseudotyping with a glycosylphosphatidylinositol (GPI) anchored CD4 variant, and a cytoplasmic tail-truncated CXCR4 protein. We have demonstrated that latently HIV-1-infected cells can be specifically infected using this mechanism, and that activation of latently infected cells increases infection efficiency. The fusion in reverse approach allowed us to characterize how alteration of CD4 plus CXCR4 lipid membranes affected Env protein activities. In particular, we found that perturbation of membrane cholesterol levels did not affect Env activity. In contrast, viruses assembled in cells deficient for long chain sphingolipids showed increased infectivities, while viruses that incorporated a lipid scramblase were non-infectious. Our results yield new insights as to factors that influence envelope protein functions.

molecular biology↗

bioRxiv2024

A Nanobody Interaction with SARS-CoV-2 Spike Allows the Versatile Targeting of Lentivirus Vectors

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor binding domain to direct lentivirus infection of Spike-expressing cells. Using three different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. The targeting is dependent on the fusion function of Spike, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion, and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections. IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein, and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion, and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

molecular biology↗

bioRxiv2023

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

biochemistry↗

bioRxiv2022

A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants

The spike glycoprotein of SARS-CoV-2 engages with human angiotensin-converting enzyme 2 (ACE2) to facilitate infection. Here, we describe an alpaca-derived heavy chain antibody fragment (VHH), saRBD-1, that disrupts this interaction by competitively binding to the spike protein receptor-binding domain. We further generated an engineered bivalent nanobody construct engineered by a flexible linker, and a dimeric Fc conjugated nanobody construct. Both multivalent nanobodies blocked infection at picomolar concentrations and demonstrated no loss of potency against emerging variants of concern including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Epsilon (B.1.427/429), and Delta (B.1.617.2). saRBD-1 tolerates elevated temperature, freeze-drying, and nebulization, making it an excellent candidate for further development into a therapeutic approach for COVID-19.

immunology↗

bioRxiv2020

A global lipid map defines a network essential for Zika virus replication

Zika virus (ZIKV), an arbovirus of global concern, remodels intracellular membranes to form replication sites. How ZIKV dysregulates lipid networks to allow this, and consequences for disease, is poorly understood. Here, we performed comprehensive lipidomics to create a lipid network map during ZIKV infection. We found that ZIKV significantly alters host lipid composition, with the most striking changes seen within subclasses of sphingolipids. Ectopic expression of ZIKV NS4B protein resulted in similar changes, demonstrating a role for NS4B in modulating sphingolipid pathways. Disruption of sphingolipid biosynthesis in various cell types, including human neural progenitor cells, blocked ZIKV infection. Additionally, the sphingolipid ceramide redistributes to ZIKV replication sites and increasing ceramide levels by multiple pathways sensitizes cells to ZIKV infection. Thus, we identify a sphingolipid metabolic network with a critical role in ZIKV replication and show that ceramide flux is a key mediator of ZIKV infection.

microbiology↗