Biology subjects

Amarasinghe, G. K.

Publications and source records attributed to Amarasinghe, G. K..

12 recordsLinked to original sources

bioRxiv2026

Phenuivirus glycoprotein Gc domain is a conserved target of cross-functional monoclonal antibodies

Phenuiviruses which include Rift Valley Fever Virus (RVFV), classified by WHO as one of the top priority pathogens with outbreak potential, are emerging arthropod-borne viruses that pose an increasing threat to public health. In line with the prototype pathogen framework advanced by the NIAID, the sandfly-transmitted Toscana virus (TOSV) has been adopted as a prototype to advance understanding of relevant phenuiviruses and inform the development of broadly applicable medical countermeasures. In this study, we sought to isolate and characterize monoclonal antibodies (mAbs) elicited by natural TOSV infection to identify shared antigenic determinants. By employing the conserved envelope Gc glycoprotein as bait for antigen-specific B cell sorting, we recovered more than 1,450 memory B cells and identified 112 Gc-specific neutralizing mAbs. Over one-third of these mAbs exhibited cross-reactivity with RVFV, a genetically distinct phenuivirus. Selected mAbs provided prophylactic protection in a murine model of TOSV infection. Together, our findings establish Gc as an important target of protective neutralizing antibodies and provide insights that may guide the rational design of widely effective interventions, strengthening readiness against future phenuivirus outbreaks.

immunology↗

bioRxiv2025

Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling

Ebola virus (EBOV) is a non-segmented, negative-sense virus (NNSV) with a single-stranded RNA genome. EBOV encodes for a limited number of proteins and thus depends on host factors to facilitate viral replication and pathogenesis. Of the virus-encoded proteins, multifunctional EBOV VP35 (eVP35) is necessary for host immune evasion and viral RNA synthesis. Previous proteomics studies identified an interaction between eVP35 and the host E3 ubiquitin ligase Mindbomb 2 (MIB2). Here, we show how a previously uncharacterized NNLNS motif (residues 201-205) within eVP35 serves as a binding site for MIB2. This motif is critical for eVP35-dependent inhibition of MIB2-mediated IFN induction. It is also important for EBOV RNA synthesis as MIB2 binding to eVP35 inhibited EBOV minigenome activity. Altogether, these findings highlight the importance of the eVP35 protein and the role of host factors in EBOV infection. SIGNIFICANCE STATEMENTThe Ebola virus (EBOV) genome encodes for a limited number of proteins and depends on host factors to facilitate viral replication. Identification and characterization of host-viral interactions are needed to define infection, resolution, and to develop new therapeutics. EBOV VP35 (eVP35) is necessary for mediating host immune evasion and a cofactor for viral RNA synthesis. Here we characterized an interaction between eVP35 and MIB2. We show that the 201NNLNS205 motif in eVP35 is necessary and sufficient for MIB2 binding and inhibition of MIB2-mediated IFN production. Our results also reveal how the eVP35-MIB2 interaction impacts virus infection. These results support the importance of the multifunctional eVP35 to EBOV infection and highlight the significance of host proteins, including E3 ligases, during viral infection.

microbiology↗

bioRxiv2024

Lrp1 facilitates infection of neurons by Jamestown Canyon virus

Jamestown Canyon virus (JCV) is a bunyavirus and arbovirus responsible for neuroinvasive disease in the United States. Little is known about JCV pathogenesis, and no host factors required for cellular infection have been identified. Recently, we identified low-density lipoprotein receptor related protein 1 (Lrp1) as a host entry factor for two other bunyaviruses Rift Valley fever virus (RVFV) and Oropouche virus (OROV). Here, we assessed the role of Lrp1 in mediating JCV cellular infection of neurons. Both neuronal and non-neuronal immortalized cell lines deficient for Lrp1 displayed reduction in infection with JCV, and early stages of infection such as binding and internalization were impacted by lack of Lrp1. In primary rat neurons, Lrp1 was highly expressed, and the neurons were highly permissive for JCV infection. Treatment of primary neurons with recombinant receptor-associated protein (RAP), a high affinity ligand for Lrp1, resulted in reduced infectivity with JCV. In addition, pretreatment of cells with RVFV Gn inhibited JCV infection, suggesting that the two viruses may share overlapping binding sites. These results provide compelling evidence that Lrp1 is an important cellular factor for efficient infection by JCV, and thus multiple bunyaviruses with varying clinical manifestations and tissue tropism are facilitated by the host cell Lrp1. Reliance of multiple bunyaviruses on Lrp1 makes it a promising target for pan-bunyaviral antivirals and therapeutics.

microbiology↗

bioRxiv2024

Characterization of neural infection by Oropouche orthobunyavirus

Oropouche fever is a re-emerging global viral threat caused by infection with Oropouche orthobunyavirus (OROV). While disease is generally self-limiting, historical and recent reports of neurologic involvement highlight the importance of understanding the neuropathogenesis of OROV. In this study, we characterize viral replication kinetics in neurons and microglia derived from immortalized, primary, and induced pluripotent stem cell-derived cells, which are all permissive to infection. We demonstrate that ex vivo rat brain slice cultures can be infected by OROV and produce antiviral cytokines and chemokines, including IL-6, TNF- and IFN-{beta}, which introduces an additional model to study viral kinetics in the central nervous system. These findings provide additional insight into OROV neuropathogenesis and in vitro modeling strategies for a newly re-emerging arbovirus.

microbiology↗

bioRxiv2024

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 M. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.

microbiology↗

bioRxiv2021

Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants

The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used naive phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

synthetic biology↗

bioRxiv2021

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein are approved for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb was purified in high yield, and it exhibited biophysical characteristics that were comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM), and it exhibited extremely high potencies against a broad array of pseudoviruses, chimeric viruses, and authentic virus variants. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

bioengineering↗

bioRxiv2021

Intrinsically disordered pathogen effector alters the STAT1 dimer to prevent recruitment of co-transcriptional activators CBP/p300

Signal transducer and activator of transcription (STATs) proteins signal from cell-surface receptors to drive transcription of immune response genes. The parasite Toxoplasma gondii blocks STAT1-mediated gene expression by secreting the intrinsically disordered protein TgIST that traffics to the host nucleus, binds phosphorylated STAT1 dimers, and occupies nascent transcription sites that unexpectantly remain silenced. Here we define a core repeat region within internal repeats of TgIST that is necessary and sufficient to block STAT1-mediated gene expression. Cellular, biochemical, mutational, and structural studies demonstrate that the repeat region of TgIST adopts a helical conformation upon binding to STAT1 dimers. The binding interface is defined by a groove formed from two loops in the STAT1 SH2 domains that reorient during dimerization. TgIST binding to this newly exposed site at the STAT1 dimer interface altered its conformation and prevented recruitment of co-transcriptional activators, thus defining the mechanism of blocked transcription.

microbiology↗

bioRxiv2020

Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

biochemistry↗

bioRxiv2020

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs. In these interactions the largely alpha-helical carboxy-terminal domain of the EBOV VP30 engages with the motif such that the prolines adopt non-canonical orientations, as compared to other proline-rich motifs. Affinity tag-purification mass spectrometry identified additional PPxPxY-containing host proteins, including hnRNP L, hnRNPUL1 and PEG10, as VP30 interactors. Of these, hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV replication, whereas hnRNPUL1 enhances. Further, double knockdown studies support additive effects of RBBP6 and hnRNP L. Binding studies demonstrate variable capacity of PPxPxY motifs to bind VP30 and the extended motif PxPPPPxY is demonstrated to confer optimal binding and to inhibit RNA synthesis, with the fifth proline and the tyrosine being most critical. Competition binding and hydrogen-deuterium exchange studies demonstrate that each protein binds a similar interface on VP30 and impacts VP30 phosphorylation. VP30 therefore represents a novel proline recognition domain that allows multiple host proteins to target a single viral protein-protein interface to modulate viral transcription.

microbiology↗

bioRxiv2020

Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2.

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune {gamma}-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.

microbiology↗

bioRxiv2020

Discovery of a cryptic allosteric site in Ebola's 'undruggable' VP35 protein using simulations and experiments

Protein-protein and protein-nucleic acid interactions are often considered difficult drug targets because the surfaces involved lack obvious druggable pockets. Cryptic pockets could present opportunities for targeting these interactions, but identifying and exploiting these pockets remains challenging. Here, we apply a general pipeline for identifying cryptic pockets to the interferon inhibitory domain (IID) of Ebola viral protein 35 (VP35). VP35 plays multiple essential roles in Ebolas replication cycle but lacks pockets that present obvious utility for drug design. Using adaptive sampling simulations and machine learning algorithms, we predict VP35 harbors a cryptic pocket that is allosterically coupled to a key dsRNA-binding interface. Thiol labeling experiments corroborate the predicted pocket and mutating the predicted allosteric network supports our model of allostery. Finally, covalent modifications that mimic drug binding allosterically disrupt dsRNA binding that is essential for immune evasion. Based on these successes, we expect our pipeline is equally applicable to other proteins.

biochemistry↗