Biology subjects

Malonis, R. J.

Publications and source records attributed to Malonis, R. J..

5 recordsLinked to original sources

bioRxiv2026

Expandable DNA-origami rings as a nanomechanical platform for studying disordered nucleoporins

Phe-Gly-rich nucleoporins (FG-Nups) within the nuclear pore complex (NPC) form a diffusion barrier that controls the exchange of macromolecules between the nucleus and cytoplasm. The behaviors of these intrinsically disordered FG-Nups are fundamental to the NPC function but remain difficult to probe within the dynamic, native nuclear pore. To bridge this knowledge gap, here we build expandable DNA-origami rings with tunable elasticity and study FG-Nups site-specifically tethered in such mechanically responsive NPC mimics. The elastic deformation of DNA rings, measured by single-particle TEM, reveals the type (attraction or repulsion) and strength of FG-Nup interactions that shape the collective morphology of these unstructured proteins. Analyzing homotypic interactions of five FG-Nup species finds diverse FG-Nup behaviors that can be classified as repulsive/extended (Nsp1), moderately cohesive/extended (Nup62, Nup153, and Nup214), and strongly cohesive/compact (Nup98). Furthermore, we examine how FG-Nups' primary sequence, O-glycosylation, grafting density, and binding to nuclear transport receptors (NTRs) impact their biophysical state. For example, O-GlcNAcylation reduced FG-Nup cohesiveness, switching Nup98 from a collapsed state to more extended conformations. Our work thus elucidates spatially confined Nup-Nup and Nup-NTR interactions within an NPC-like nanopore, suggests a previously underappreciated role of FG-Nups in modulating NPC dilation, and establishes a generalizable method for studying multivalent interactions among disordered proteins.

biophysics↗

bioRxiv2025

Dissecting the unique self-assembly landscape of the HIV-2 capsid protein

Human immunodeficiency virus type 2 (HIV-2) is a lentivirus closely related to HIV-1 but exhibits distinct molecular and clinical features that influence viral infectivity and efficacy of antiretroviral therapy. The HIV capsid is a critical structural component with multifaceted roles during infection and mediates some of the observed divergence between HIV-1 and HIV-2. Unlike HIV-1, study of the HIV-2 capsid is limited and standard protocols for the in vitro assembly of HIV-1 capsid protein (CA) lattice structures have not been successfully translated to the HIV-2 context. This work identifies effective approaches for the assembly of the HIV-2 CA lattice and leverages this to biochemically characterize HIV-2 CA assemblies and mutant phenotypes. Our findings elaborate on the sensitivity of HIV-2 CA to chemical conditions and reveal that it assembles into a more varied spectrum of particle morphologies compared to HIV-1. Utilizing these assemblies, we tested the hypothesis that HIV-1 and HIV-2 employ divergent mechanisms to stabilize CA oligomer forms and investigate the effects of non-conserved substitutions at the CA inter-protomer interfaces. This work advances our understanding of the key biochemical determinants of HIV-2 CA assembly that are distinct from HIV-1 and may contribute to their divergent virological properties.

biochemistry↗

bioRxiv2021

A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. ImportanceThe US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.

microbiology↗

bioRxiv2020

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

biochemistry↗

bioRxiv2020

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

microbiology↗