Biology subjects

Preece, B.

Publications and source records attributed to Preece, B..

7 recordsLinked to original sources

bioRxiv2026

A mechanism-aware stoichiometry platform resolves functional viral thresholds and induces antiviral hypersensitivity

Antiviral discovery is frequently hindered by a stoichiometric blind spot--a lack of resolution regarding the functional density of viral enzymes required for replication. Traditional screening pipelines rely on target-based or phenotypic assays that cannot distinguish between simple molecular binding and the crossing of a functional stoichiometric cliff. Here, we present a mechanism-aware platform that resolves these enzymatic requirements with single-virion precision. By integrating quantitative cryo-electron microscopy and genomic validation with Monte Carlo modeling, we map the stoichiometric landscapes of HIV-1 Protease (PR) and Reverse Transcriptase (RT). We uncover a striking disparity in enzymatic demand: a high-redundancy buffering capacity for PR ([~]40 monomers) contrasted with a high-threshold requirement for RT ([~]95 subunits). We demonstrate that by systematically de-buffering the virion, our platform induces a state of antiviral hypersensitivity, enabling the detection of therapeutic activity in novel and clinical inhibitors that remain invisible to traditional workflows. Furthermore, this multiplexed profiling enables de novo target identification, as inhibitors trigger failure exclusively on their respective stoichiometric arms. This platform provides a deterministic roadmap for de-risking drug discovery and identifying viral sub-stoichiometric vulnerabilities.

biophysics↗

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↗

bioRxiv2024

Competitive assembly resolves the stoichiometry of essential proteins in infectious HIV-1 virions.

During assembly on the plasma membrane, HIV-1 virions incorporate Gag-Pol as well as gp120/gp41 trimers. The Pol region consists of protease, reverse transcriptase and integrase precursors which are essential enzymes required for maturation, reverse transcription, and integration of the viral genome in the next host. gp120/gp41 trimers catalyze the fusion of the virion with its next host. Only a fraction of released virions are infectious. The stoichiometry of gp120/gp41 and Gag-Pol proteins in HIV virions was previously measured using cryotomography and ratiometric protein analysis, but what is the stoichiometry of these proteins in infectious virions remained to be determined. Here we developed a method based on competition between infectious HIV backbones with noninfectious mutants and measured 100 {+/-} 10 Gag-Pol and 15 {+/-} 3 gp120/gp41 proteins incorporated in infectious virions assembled in HEK293 cells from NL4.3 HIV-1 backbone. Our measurements are in broad agreement with cryotomography and ratiometric protein analysis and therefore stoichiometry of gp120/gp41 and Gag-Pol in infectious virions is the same as all released virions. With the development of appropriate mutants and infectivity assays, our method is applicable to other infectious viruses. Statement of significanceThere are 30 million people who have succumbed to the AIDS pandemic with 600,000 additional deaths per year. HIV has an accelerated rate of mutational accumulation with the virus mutating out of neutralizing antibodies within the same patient making development of vaccines challenging. Like most enveloped viruses, only a fraction of released virions are infectious and the question of what selects these virions has remained a mystery. The method developed in this article will allow stoichiometric measurements on infectious virions and therefore allows further studies of causes of infectivity.

biophysics↗

bioRxiv2022

Competitive HIV budding suggests that a self-packaging gRNA:Gag-Pol complex directs HIV assembly and enforces infectivity.

To resolve the assembly mechanism of infectious HIV virions, we tested the ability of HIV to assemble infectious virions in the presence of a titrated mix of infectious/ non-infectious proviral genomes. The analysis of our assembly competitions shows that during translation, 15 {+/-} 5 Gag-Pols bind back to their parental gRNA creating a gRNA1: Gag-Pol15 complex. This complex initiates the infectious virion assembly through interactions mediated by cis packaged Gag/Gag-pols and the plasma membrane. Our analysis also shows the number of Gag-Pol and Env proteins packaged in an infectious HIV virion and the minimum functional units of these proteins required for viral infectivity. We suggest that aside from orchestrating the infectious virion assembly the gRNA1: Gag-Pol15 complex plays a major role in HIV evolution and likely hampers effectiveness of antiviral therapies. One-Sentence SummaryCompetitive HIV budding reveals gRNA1: Gag-Pol15 complex orchestrating viral assembly.

microbiology↗

bioRxiv2020

Structural stability of SARS-CoV-2 degrades with temperature

SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter. Statement of Scientific SignificanceThe economic and public health impact of the COVID-19 pandemic are very significant. However scientific information needed to underpin policy decisions are limited partly due to novelty of the SARS-CoV-2 pathogen. There is therefore an urgent need for mechanistic studies of both COVID-19 disease and the SARS-CoV-2 virus. We show that individual virus particles suffer structural destabilization at relatively mild but elevated temperatures. Our nanoscale results are consistent with recent observations at larger scales. Our work strengthens the case for COVID-19 resurgence in winter.

biophysics↗

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↗