Webb, N. | Search bioRxiv

bioRxiv2026

An ex vivo gut mucosal explant assay to compare HIV tissue susceptibility shows increased HIV susceptibility with methamphetamine exposure

BackgroundExisting ex vivo mucosal explant models for HIV assess viral replication but do not fully capture the ability of tissue to support productive infection and transfer to target cells, an important indicator of tissue susceptibility. Additional tools to investigate relative mucosal susceptibility to HIV are necessary to better understand risk factors, such as substance use, or to evaluate therapeutics. ResultsWe developed an ex vivo gut mucosal explant assay incorporating a CCR5/CXCR4-expressing GFP-reporter cell co-culture to quantify both viral replication and transfer. Susceptibility was quantified using a composite metric based on time to infection endpoint across viral doses. Modulators of susceptibility, including anti-CD3 stimulation, tenofovir, and methamphetamine, were evaluated. The assay reliably detected differences in tissue susceptibility under various experimental conditions. We detected enhanced susceptibility following anti-CD3 stimulation, while tenofovir pretreatment reduced susceptibility in a dose-dependent manner. Methamphetamine exposure resulted in a modest but significant increase in gut tissue susceptibility to HIV. ConclusionsThis novel ex vivo explant susceptibility assay advances HIV mucosal transmission research by measuring both viral production and transfer. It can detect subtle changes in susceptibility, providing a versatile platform for evaluating prevention therapeutics, host and microbial factors, and substance use effects.

microbiology↗

bioRxiv2024

Natural Variation in HIV-1 Entry Kinetics Map to Specific Residues and Reveal an Interdependence Between Attachment and Fusion

HIV-1 entry kinetics reflect the fluid motion of the HIV envelope glycoprotein through at least three major structural configurations that drive virus-cell membrane fusion. The lifetime of each state is an important component of potency for inhibitors that target them. We used the time-of-addition inhibitor assay and a novel analytical strategy to define the kinetics of pre-hairpin exposure (using T20) and co-receptor engagement (via. maraviroc), through a characteristic delay metric, across a variety of naturally occurring HIV Env isolates. Among 257 distinct HIV-1 envelope isolates we found a remarkable breadth of T20 and maraviroc delays ranging from as early as 30 seconds to as late as 60 minutes. The most extreme delays were observed among transmission-linked clade C isolates. We identified four single-residue determinants of late T20 and maraviroc delays that are associated with either receptor engagement or gp41 function. Comparison of these delays with T20 sensitivity suggest co-receptor engagement and fusogenic activity in gp41 act cooperatively but sequentially to drive entry. Our findings support current models of entry where co-receptor engagement drives gp41 eclipse and have strong implications for the design of entry inhibitors and antibodies that target transient entry states. Author Summary.The first step of HIV-1 infection is entry, where virus-cell membrane fusion is driven by the HIV-1 envelope glycoprotein through a series of conformational changes. Some of the most broadly active entry inhibitors work by binding conformations that exist only transiently during entry. The lifetimes of these states and the kinetics of entry are important elements of inhibitor activity for which little is known. We demonstrate a remarkable range of kinetics among 257 diverse HIV-1 isolates and find that this phenotype is highly flexible, with multiple single-residue determinants. Examination of the kinetics of two conformational landmarks shed light on novel kinetic features that offer new details about the role of co-receptor engagement and provide a framework to explain entry inhibitor synergy.

molecular biology↗