Biology subjects

Gramm, A. J.

Publications and source records attributed to Gramm, A. J..

3 recordsLinked to original sources

bioRxiv2025

Identification of a Broadly Acting Inhibitor of the Alphavirus Non-Structural Protein 2 Helicase

Alphaviruses are mosquito-borne viruses that have caused significant outbreaks in the 21st century. Despite multiple recent outbreaks, there are no approved antiviral drugs to treat any alphavirus infection. Therefore, developing broadly acting antiviral drugs effective against multiple alphaviruses is necessary and could provide protection from both current and emerging alphavirus threats. A critical component of the alphavirus replication complex is non-structural protein 2 (nsP2), which is a multifunctional enzyme containing a helicase domain connected to a protease domain by a flexible linker. nsP2 functions as an ATP-dependent helicase, is conserved across the alphavirus genus, and is essential for virus replication, making it a promising target for development of alphavirus broad-acting antiviral drugs. Previous studies identified an enantioselective compound RA-0025298 that inhibited nsP2 ATPase activity and chikungunya virus CHIKV replication. Antiviral testing of RA-25298. against a diverse group of alphaviruses found broad activity except for Sindbis-like viruses. Using this information along with mutational profiling of virus passaged with RA-0025298 we identified the site of RA-0025298 action and confirmed the binding site via biophysical analyses. Finally, we found that the active enantiomer of RA-0025298 (SGC-NSP2hel-1) reduced viral loads in vivo and protected mice from tissue damage and disease caused by CHIKV infection. These findings further describe the mechanism of action of a first-in-class nsP2 helicase inhibitor with the potential for development as a broad spectrum drug for treating or preventing disease caused by current and emerging alphaviruses. One Sentence SummaryThis study describes the mechanism of action and in vivo efficacy of a first in class broadly acting inhibitor of alphavirus nsP2 helicase activity.

microbiology↗

bioRxiv2025

D614G reshapes allosteric networks and opening mechanisms of SARS-CoV-2 spikes

The SARS-CoV-2 spike glycoprotein binds human epithelial cells and enables infection through a key conformational transition that exposes its receptor binding domain (RBD). Experimental evidence indicates that spike mutations, particularly the early D614G variant, alter the rate of this conformational shift, potentially increasing viral infectivity. To investigate how mutations reshape the conformational landscape, we conducted extensive weighted ensemble simulations of the Ancestral, Delta, and Omicron BA.1 spike strains along the RBD opening pathway. We observe that Ancestral, Delta, and Omicron BA.1 spike RBDs open differently, with Omicron BA.1 following a more direct opening profile until it reaches a "super-open" state wherein it begins to "peel", suggesting increased S1 flexibility. Via dynamical network analysis, we identified two allosteric communication networks uniting all S1 domains: the established N2R linker and a newly discovered anti-parallel R2N linker. In Delta and Omicron BA.1 variant spikes, RBD opening is facilitated by both linkers, while the Ancestral strain relies predominantly on the N2R linker. In the ancestral spike, the D614-K854 salt bridge impedes allosteric communication through the R2N linker, whereas the loss of this salt bridge in all subsequent VOCs alleviates local frustration and, we believe, accelerates RBD opening. Hydrogen-deuterium mass spectrometry experiments validate these altered dynamics in the D614 region across Ancestral, D614G, and Omicron BA.1 spikes. This study unveils a hidden allosteric network, connecting the NTD to the RBD via the 614-proximal region, and the D614G mutation reshapes the fitness landscape of these critical viral glycoproteins. Significance StatementOur work reveals how the D614G mutation in the SARS-CoV-2 spike protein reshapes its internal communication pathways and speeds up receptor binding domain (RBD) opening, providing mechanistic insight into the evolution and enhanced infectivity of SARS-CoV-2 variants of concern. We also describe differences in opening pathways and relative rates of opening for Delta and Omicron BA.1 spike RBDs relative to the original (Ancestral) coronavirus strain from Wuhan, China.

biophysics↗

bioRxiv2025

SARS CoV-2 spike adopts distinct conformational ensembles in situ

Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.

biophysics↗