Biology subjects

McFadden, W. M.

Publications and source records attributed to McFadden, W. M..

6 recordsLinked to original sources

bioRxiv2026

Structural Basis of Polypurine Track Strand Displacement by HIV-1 Reverse Transcriptase

To complete reverse transcription, HIV-1 reverse transcriptase (RT) must displace the RNase H-resistant polypurine tract (PPT) primers. This enables synthesis of the long terminal repeats and formation of the central cDNA flap. However, the molecular mechanism of this PPT strand displacement (SD) has remained unknown, and no structural data exist on how a retroviral polymerase execute these reactions. We report the first cryo-EM structures of HIV-1 RT bound to nucleic acid substrates containing either a PPTRNA or PPTDNA displacement strand, with incoming dATP positioned at the polymerase catalytic site. These structures reveal key features of the PPT displacement mechanism by RT. Specifically, we observed a binding mode where the template nucleotide (T1) base-paired to the first displacement nucleotide (D1) undergoes a 90{degrees} rotation relative to the preceding template base (T0). This sharp template flip positions D1 [~]30 [A] away from the primers 3-end and is coordinated by RTp66 residues at the SD interface: F61 and R78 contact T1/T0 to drive template translocation, while W24 engages both T1 and D1 to stabilize the displacement strand. Biochemical and virological mutagenesis experiments confirm that interactions with F61 and R78 are essential for both canonical cDNA polymerization and SD, whereas the W24-nucleotide interactions are required exclusively for SD but are dispensable for standard cDNA synthesis. These results contribute to the structural and functional understanding of PPT strand displacement by HIV-1 RT and reveal a distinct mechanistic vulnerability for the design of next-generation antiretrovirals.

biochemistry↗

bioRxiv2026

Damaging the conical morphology of HIV-1 capsid by targeting the FG-binding pocket and disfavoring pentameric subunits needed for core closure

The HIV-1 capsid is an essential viral component, targeted by the long-acting antiretroviral Lenacapavir (LEN). LEN binds to the HIV-1 capsid protein (CA) at the phenylalanine-glycine (FG) binding pocket (FGBP), a site for multiple host-factor and antiviral interactions in CA hexamers (CAHEX). Previously, we generated a chemical library to investigate the FGBP; ZW-1261, a lead compound, exhibits potent antiviral activity and strong inter-subunit interactions within CAHEX. Here, we report the molecular mechanism by which ZW-1261 affects the morphology and integrity of capsid lattice. ZW-1261 alone rapidly induces tubular CA assemblies; simultaneous addition of ZW-1261 with the assembly cofactor inositol hexaphosphate (IP6) forms morphologically distinct tubes. In mature virions, IP6 is required for the assembly of both CAHEX and CA pentamers (CAPENT). Cryogenic-electron microscopy analysis of in vitro assembled capsid-like particles (CLPs) with IP6 suggests that ZW-1261 leads to the absence of CAPENT and damages the pre-formed conical lattice. To elucidate how this FGBP-targeting antiviral impacts CAPENT, we further solved structures of CAPENT-only icosahedral assemblies (T = 1), formed by reported mutations, that were treated with ZW-1261. We find that ZW-1261 binding in these constrained T = 1 assemblies converts CAPENT to a CAHEX-like conformation. Collectively, this suggests a mechanism by which addition of FGBP-binding inhibitor to native cores leads to the absence of CAPENT, impacting capsid closure and core integrity.

molecular biology↗

bioRxiv2025

Structural, biophysical, and virological mechanistic characterization of HIV-1 capsid-targeting antivirals

Due to its significant role in virus replication, the HIV capsid is an attractive antiviral target. This is validated by the recent clinical approval of lenacapavir for both treatment and pre-exposure prophylaxis (PrEP). PF74 is a well-characterized capsid-targeting antiviral that was discontinued in further study due to potency and metabolic issues. We hypothesized that making chemical modifications at certain sites of PF74 could result in capsid-targeting antivirals with improved potency and bioavailability. Our cumulative studies show that making changes at the R1 and R3 positions of PF74 results in compounds with increased antiviral potency, increased stability of wild-type HIV capsid hexamers and virions, tighter binding to wild-type HIV capsid hexamer compared to PF74, and different interactions at the "FG" binding site of capsid compared to PF74. These data provide insights into the design of future capsid-targeting antivirals relevant for clinical use.

biochemistry↗

bioRxiv2025

Characterization of antiviral compounds using Bio-Layer Interferometry

Small molecule-protein interactions underpin many biological functions and play an integral role in the treatment and prevention of several human diseases. These interactions can be key to understanding the mechanism of action of these compounds. Previous methods of determining protein-protein or protein-antibody interactions have been well established; however, the use of BLI in antiviral discovery is a promising and relatively new avenue. The high-throughput nature of this method in tandem with its pM sensitivity allows for quick and seamless identification of hit compounds. Here we discuss ways to overcome common pitfalls that can occur while using BLI such as nonspecific binding (NSB) and ligand drift while offering possible solutions. Characterizing small molecule-protein interactions is not trivial and optimizing the experimental conditions is imperative. To address this gap in knowledge, we present optimized BLI protocols for the study of three cases of protein-small molecule interactions: PF74 or Lenacapavir (LEN) with HIV-1 capsid protein (CA), and Nirmatrelvir (NIR) with SARS-CoV-2 Mpro. LEN and NIR are of particular interest because they are clinically relevant, and PF74, a well-studied control, was the first compound reported to target the LEN binding site. We demonstrate that BLI can be a powerful and effective tool in calculating the binding affinities between a protein and small molecule. These newly designed methods enabled calculation of KD values, the affinity between ligand and analyte, ranging from the micro to the sub-nanomolar range for CA binding events and confirmed the covalent interaction between NIR and Mpro. These protocols will facilitate efficient testing of new antivirals or derivatives in a high- throughput format. SummaryBio-Layer Interferometry (BLI) is a multifunctional technology that is used to determine valuable information on real-time kinetics including association and dissociation. Optimizing experimental conditions to acquire data about protein-ligand interactions can be challenging. We provide three example methods of collecting binding data that characterize how viral proteins interact with antivirals.

biochemistry↗

bioRxiv2024

Disruption of LEDGF/p75-directed integration derepresses antisensetranscription of the HIV-1 genome

AbstractDisruption of HIV-1 Integrase (IN) interactions with the host-factor Lens Epithelium-Derived Growth Factor (LEDGF)/p75 leads to decreased, random integration, increased latent infection, and described here, accumulation of HIV-1 antisense RNA (asRNA). asRNA increase was observed following interruptions of IN-LEDGF/p75 interactions either through pharmacologic perturbations of IN-LEDGF/p75 by treatment with allosteric HIV-1 integrase inhibitors (ALLINIs) or in cell lines with LEDGF genetic knockout. Additionally, by impairing Tat-dependent HIV transcription, asRNA abundance markedly increases. Illumina sequencing characterization of asRNA transcripts in primary T cells infected in the presence of ALLINIs showed that most initiate from within the HIV-1. Overall, loss of IN-LEDGF/p75 interactions increase asRNA abundance. Understanding the relationship between ALLINIs, integration sites, asRNA, and latency could aid in future therapeutic strategies.

microbiology↗

bioRxiv2023

Use of TSAR, Thermal Shift Analysis in R, to identify Folic Acid as a Molecule that Interacts with HIV-1 Capsid

The thermal shift assay (TSA) is a versatile biophysical technique for studying protein-ligand interactions in vitro. Here, we report a free, open-source software tool, TSAR ("Thermal Shift Analysis in R"), to expedite the analysis of TSA data. The TSAR package incorporates multiple workflows that facilitate TSA analyses, returns publication-ready graphics, and includes an optional graphic user interface. The package is available at https://bioconductor.org/packages/TSAR/. Applying TSAR, we screened two chemical libraries and found multiple molecules that potentially interact in vitro with the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1). First, a library of vitamins exemplifies the different graphic outputs of TSAR, and we report a change in the 50% melting temperature ({Delta}Tm) for folic acid-treated CA hexamers (CAHEX). Since HIV-1 CAHEX interacts with host-derived acids like inositol hexaphosphate (IP6) or dNTPs, a second library was screened containing 96 organic, acidic metabolites; multiple anionic ligands caused a {Delta}Tm for CAHEX. Subsequent investigation of these interactions includes biolayer interferometry, antiviral activity against pseudotyped HIV-1, and endogenous reverse transcriptase assays that were used to validate and investigate the biological impact of these native ligands that thermally-stabilize CAHEX. One compound hit, gallic acid, exhibited anti-HIV-1 activity as previously reported, and we show interacts with CAHEX as a potentially novel mechanism. Overall, the TSAR package facilitated quick analysis of TSA data from multiple libraries to help identify a biologically relevant hit, gallic acid, as a molecule that can inhibit HIV-1 replication and targets CAHEX. ImportanceThe TSAR package is freely available (AGPL-3) and is designed for both experienced or new R users, having command-line code for handling large and challenging datasets while also including an optional GUI that enables easy use by non-programmers. This is the first TSA analysis program written in R, a free and open-source language; TSAR simplifies TSA analysis while maintaining diverse visualization options for small-to-large libraries and multidimensional analysis. Additionally, we report multiple endogenous metabolites that potentially interact with the HIV-1 capsid protein hexamers (CAHEX) in vitro, including folic acid, gallic acid, and multiple others, primarily from the tricarboxylic acid (TCA) cycle. Various methods validate gallic acid interactions with CAHEX, leading to a novel suggested mechanism of action, and in-line with previous reports, this metabolite has potential for natural-based treatments of HIV-1. Further, we advance the understanding of the potential mechanism(s) for HIV-1 inhibition by gallic acid treatment.

biochemistry↗