Biology subjects

Belnap, D.

Publications and source records attributed to Belnap, D..

5 recordsLinked to original sources

bioRxiv2025

Cryo-ET Reveals Distinct Gag Lattice Architectures in Virus-like Particles and Immature HIV-1

HIV-1 is released from infected cells as immature virions whose membranes are supported by a Gag lattice. During maturation, this lattice is cleaved by the viral protease to release capsid proteins that assemble into the mature core. The architecture of the Gag lattice is central to this process, and the Gag lattice is targeted by maturation inhibitors that block cleavage. Using cryo-electron tomography, we compared Gag-only virus-like particles (VLPs) with immature HIV-1 virions and found that VLPs assemble denser and more complete lattices, exhibiting a strong correlation between lattice curvature and Gag copy number. In contrast, immature virions incorporate fewer Gag molecules and display weaker coupling between curvature and Gag stoichiometry. These findings show that while Gag alone can form the canonical immature lattice, additional viral components fine-tune lattice organization and curvature, potentially regulating protease accessibility, virion release, and the onset of HIV-1 maturation.

biophysics↗

bioRxiv2024

Enhanced Yield and Gentle Purification of HIV for Cryo-Electron Tomography Analysis of Virion Maturation.

HIV is a lentivirus characterized by the formation of its mature core. Visualization and structural examination of HIV requires purification of virions to high concentrations. The yield and integrity of these virions are crucial for ensuring a uniform representation of all viral particles in subsequent analyses. In this study, we present a method for purification of HIV virions which minimizes forces applied to virions while maximizing the efficiency of collection. This method allows us to capture between 1,000 and 5,000 HIV virions released from individual HEK293 cells after transfection with the NL4.3 HIV backbone, a 10 fold advantage over other methods. We utilized this approach to investigate HIV core formation from several constructs: pNL4-3(RT:D185A&D186A) with an inactive reverse transcriptase, NL4.3(IN: V165A&R166A) with a type-II integrase mutation, and NL4.3(: {Delta}(105-278)&{Delta}(301-332)) featuring an edited packaging signal. Notably, virions from NL4.3(: {Delta}(105-278)&{Delta}(301-332)) displayed a mixed population, comprising immature virions, empty cores, and cores with detectable internal density. Conversely, virions derived from NL4.3(IN: V165A&R166A) exhibited a type II integrase mutant phenotype characterized by empty cores and RNP density localized around the cores, consistent with previous studies. In contrast, virions released from pNL4-3(RT:D185A&D186A) displayed mature cores containing detectable RNP density. We suggest that the purification methods developed in this study can significantly facilitate the characterization of enveloped viruses.

microbiology↗

bioRxiv2023

A weaponized phage suppresses competitors in historical and modern metapopulations of pathogenic bacteria

Bacteriophages, the viruses of bacteria, are proposed to drive bacterial population dynamics, yet direct evidence of their impact on natural populations is limited. Here we identified viral sequences in a metapopulation of wild plant-associated Pseudomonas spp. genomes. We discovered that the most abundant viral cluster does not encode an intact phage but instead encodes a tailocin - a phage-derived element that bacteria use to kill competitors for interbacterial warfare. Each pathogenic Pseudomonas sp. strain carries one of a few distinct tailocin variants, which target variable polysaccharides in the outer membrane of co-occurring pathogenic strains. Analysis of historic herbarium samples from the last 170 years revealed that the same tailocin and receptor variants have persisted in the Pseudomonas populations for at least two centuries, suggesting the continued use of a defined set of tailocin haplotypes and receptors. These results indicate that tailocin genetic diversity can be mined to develop targeted "tailocin cocktails" for microbial control. One-Sentence SummaryBacterial pathogens in a host-associated metapopulation use a repurposed prophage to kill their competitors.

microbiology↗

bioRxiv2020

Minimal system for assembly of SARS-CoV-2 virus like particles

SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 {+/-} 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.

biophysics↗

bioRxiv2017

Membrane Proteins Significantly Restrict Exosome Mobility

Exosomes are membrane nanovesicles that intermediate cell-to-cell signaling through the transfer of their molecular cargo. The exosomes small size facilitates rapid migration through the extracellular matrix and into and out of circulation. Here we report that the mobility of the exosomes is much lower than would be expected from the size of their membrane vesicles. The difference is broadly distributed and caused by surface proteins, which significantly impede exosome migration. The observed wide range in the mobility implies that a subpopulation of hydrodynamically small exosomes is more likely to participate in signaling. The extracellular environment amplifies the size-dependent hindrance to the exosomes migration. The significant contribution of surface proteins to the transport resistance make the exosome mobility a dynamic property that changes with the extracellular environment which affects the membrane protein conformation, glycosylation, specific, and non-specific surface adsorption.

biochemistry↗