Biology subjects

Musier-Forsyth, K.

Publications and source records attributed to Musier-Forsyth, K..

7 recordsLinked to original sources

bioRxiv2026

Psi RNA-specific Binding Promotes HIV-1 Gag Conformational Change Critical for Immature Viral Particle Assembly

The immature HIV-1 virion is assembled by the Gag polyprotein using inositol hexakisphosphate (IP6) as an essential assembly co-factor. Gag binds the genomic RNA Psi packaging signal via the nucleocapsid (NC) domain and associates with the plasma membrane via the matrix (MA) domain. Previous studies revealed that Gag exists in both compact (C) and extended (E) conformational states in solution. Only E-Gag formed virus-like particles with the correct size and IP6 shifted the equilibrium of DNA-bound Gag to the E state. The influence of specific RNA elements on this conformational change is unknown. In this work, a dual dye-labeled Gag was prepared for probing the effect of RNA binding on Gag conformation using Forster resonance energy transfer (FRET). In low salt and in the absence of other factors, Gag was primarily in the C state. Psi RNA binding induced a more significant FRET decrease than binding to non-Psi RNAs, consistent with a shift to E-Gag. IP6 alone also promoted the E-Gag state in the absence and presence of RNA. Atomistic molecular dynamics simulations are consistent with and provide detail into the role of NC-Psi RNA binding in the conformational switch of C-Gag to assembly-competent E-Gag. Simulations also showed that this switch is driven by capsid (CA) linker domain orientational flexibility and MA-CA unbinding dynamics. Thus, the highly flexible multi-domain Gag polyprotein leverages both viral and host cell factors to sample and stabilize distinct conformations, thereby orchestrating the viral assembly process.

biophysics↗

bioRxiv2025

Mechanism of SARS-CoV-2 Nucleocapsid Protein Phosphorylation-induced Functional Switch

The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed WT Np formed discrete complexes on viral 5' UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1-4. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs. WT Np also compacted and irreversibly bound single-stranded DNA; this activity was significantly reduced by phosphorylation. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants.

biochemistry↗

bioRxiv2025

Human RPL7 and DDX21 interact with HTLV-1 Gag and enhance tRNAPro primer annealing to genomic RNA

Human T-cell leukemia virus type 1 (HTLV-1), an oncogenic retrovirus, uses human tRNAPro to prime reverse transcription (RT). However, how tRNAPro is annealed to the primer binding site (PBS), which is embedded in a highly structured hairpin in the genomic RNA (gRNA), remains unclear. We hypothesize that HTLV-1 Gag may have more robust chaperone activity than mature HTLV-1 nucleocapsid (NC), which in contrast to HIV-1 NC, displays relatively weak chaperone function, and that a cellular co-factor may be required to facilitate primer tRNA annealing. Recombinant HTLV-1 Gag was successfully purified for the first time and used to perform primer-annealing assays. Relative to mature NC and matrix (MA) domains, HTLV-1 Gag is only slightly more effective at chaperoning the annealing of tRNAPro to the PBS. To identify potential HTLV-1 Gag interacting co-chaperones of tRNA annealing in cells, we performed affinity tagging/purification-mass spectrometry (AP-MS). Two significant AP-MS hits, RPL7 and DDX21, were further validated by reciprocal co-IP studies in both HEK293T and chronically HTLV-1-infected MT-2 cells. Domain mapping studies revealed that HTLV-1 Gag interacts with RPL7 and DDX21 through the zinc fingers in the NC domain independent of the presence of RNA. In addition, we showed that both RPL7 and DDX21 are packaged into virions. RPL7 or DDX21 alone was more effective than HTLV-1 Gag at annealing tRNAPro to the PBS. Synergistic effects of the Gag/RPL7/DDX21 combination in facilitating tRNAPro annealing to the PBS were found. Taken together, the mechanistic insights gained from these studies could be exploited for the development of new therapeutic strategies aimed at targeting HTLV-1 RT. HighlightsO_LIMechanism for HTLV-1 reverse transcription primer annealing to the primer binding site in viral RNA is proposed. C_LIO_LIRecombinant HTLV-1 Gag was purified successfully from E. coli for the first time. C_LIO_LIHTLV-1 Gag interacts directly with human RPL7 and DDX21 in cells. C_LIO_LIRPL7 and DDX21 enhance HTLV-1 Gags tRNAPro primer annealing activity. C_LIO_LItRNAPro annealing activity: DDX21 + RPL7 + Gag > DDX21 + RPL7 > DDX21 > RPL7 > Gag. C_LI

biochemistry↗

bioRxiv2024

Structural basis for aminoacylation of cellular modified tRNALys3 by human lysyl-tRNA synthetase

The average eukaryotic tRNA contains 13 posttranscriptional modifications; however, their functional impact is largely unknown. Our understanding of the complex tRNA aminoacylation machinery in metazoans also remains limited. Herein, using a series of high-resolution cryo-electron microscopy (cryo-EM) structures, we provide the mechanistic basis for recognition and aminoacylation of fully-modified cellular tRNALys3 by human lysyl-tRNA synthetase (h-LysRS). The tRNALys3 anticodon loop modifications S34 (mcm5s2U) and R37 (ms2t6A) play an integral role in recognition by h-LysRS. Modifications in the T-, variable-, and D-loops of tRNALys3 are critical for ordering the metazoan-specific N-terminal domain of LysRS. The two catalytic steps of tRNALys3 aminoacylation are structurally ordered; docking of the 3'-CCA end in the active site cannot proceed until the lysyl-adenylate intermediate is formed and the pyrophosphate byproduct is released. Association of the h-LysRS-tRNALys3 complex with a multi-tRNA synthetase complex-derived peptide shifts the equilibrium towards the 3'-CCA end docked conformation and allosterically enhances h-LysRS catalytic efficiency. The insights presented here have broad implications for understanding the role of tRNA modifications in protein synthesis, the human aminoacylation machinery, and the growing catalog of metabolic and neurological diseases linked to it.

biochemistry↗

bioRxiv2020

Human trans-editing enzyme displays tRNA acceptor stem specificity and relaxed amino acid selectivity

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase (ProRS). Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, while tRNAPro is selected via recognition of tRNA acceptor stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family and provides the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.

biochemistry↗

bioRxiv2020

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (Gag{Delta}p6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. Gag{Delta}p6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically nucleates Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

biochemistry↗

bioRxiv2018

Analysis of Local Variability and Allostery in Macromolecular Assemblies using Cryo-EM and Focused Classification

Single-particle electron cryo-microscopy and computational image classification can be used to analyze structural variability in macromolecules and their assemblies. In some cases, a particle may contain different regions that each display a range of distinct conformations. We have developed strategies, implemented within the Frealign and cisTEM image processing packages, to focus classify on specific regions of a particle and detect potential covariance. The strategies are based on masking the region of interest using either a 2-D mask applied to reference projections and particle images, or a 3-D mask applied to the 3-D volume. We show that focused classification approaches can be used to study structural allostery, a concept that is likely to gain more importance as datasets grow in size, allowing the distinction of more structural states and smaller differences between states. Finally, we apply the approaches to an experimental dataset containing the HIV-1 Transactivation Response (TAR) element RNA fused into the large bacterial ribosomal subunit, to deconvolve structural mobility within localized regions of interest.\n\nHighlightsO_LIDescription of different image classification strategies in single-particle cryo-EM\nC_LIO_LIQuantitative evaluation of two classification methods using simulated data\nC_LIO_LIApplication of the two classification methods to an experimental dataset\nC_LI

biophysics↗