Biology subjects

Rosecrans, J.

Publications and source records attributed to Rosecrans, J..

7 recordsLinked to original sources

bioRxiv2026

Epistasis between SARS-CoV-2 M and N Proteins Balances Particle Assembly and Immune Evasion

Since its emergence in the human population, SARS-CoV-2 has continuously evolved to evade immune responses and robustly establish global circulation. In this process, the structural viral membrane (M) protein has accumulated amino acid changes whose impact on viral particle assembly and innate immune evasion remains incompletely understood. Here, we designed a SARS-CoV-2 replicon system lacking M that assesses the influence of transiently transfected M protein variants on viral particle production independently of viral RNA replication. We found that M protein variants have reduced particle assembly while innate immune antagonism functions are strengthened. Notably, the assembly defect is rescued by co-evolving N protein variants, highlighting how SARS-CoV-2 evolution coordinates between two of its structural proteins to optimize viral infection. Our work underscores the complex evolutionary trajectories of SARS-CoV-2 variants across different viral proteins and informs future therapeutic strategies targeting viral assembly and limiting infection. Author summarySince the onset of the COVID-19, SARS-CoV-2 has been changing continuously in ways that help it spread efficiently and evade human defenses. The viral membrane (M) protein is a key structural component required to form new viral particles. Nevertheless, it has accumulated changes over time with overlooked functions. In this study, we developed an experimental system to examine the impact of these changes on virus assembly and host immune system. We found that recent M variants are better than older variants at suppressing immune responses but less efficient at forming viral particles. This defect seems to be compensated for by coordinated changes in another structural protein, nucleocapsid (N). These findings reveal that SARS-CoV-2 evolution involves trade-offs between different viral characteristics, with changes in one protein balancing changes in another. Understanding these trade-offs provides new insight into how the virus adapts to humans and may help guide therapeutic strategies to limit infection.

microbiology↗

bioRxiv2026

Pandemic Coronavirus Genome Packaging Relies on Multiple Dispersed Packaging Signals

Packaging of viral genomes into progeny virions is a critical step in the viral life cycle. Coronaviruses such as SARS-CoV-2 possess unusually large RNA genomes ([~]30 kb), yet the mechanism by which these genomes are selectively condensed and incorporated into virions remains poorly understood. Here, we demonstrate that the SARS-CoV-2 genome contains multiple dispersed RNA structures, termed packaging signals (PSs), which cooperate with a dominant central PS to direct genomic RNA incorporation into infectious particles. We identify the dominant PS within the coding region of the nsp15 gene, downstream of a packaging signal previously described in Embecoviruses. Using virus-like particles (VLPs), we investigate its role in virion assembly and selective genomic RNA packaging, and show that it promotes the formation of ribonucleoprotein (RNP) complexes with the viral nucleocapsid protein (N), as revealed by mass photometry. Notably, we uncover a unique N-induced conformational rearrangement of the PS RNA, from an extended structure to a double stem-loop architecture. This dominant PS acts together with a nearby stem-loop element to assemble a higher-order RNP complex containing 12 N-protein dimers. Using a SARS-CoV-2 replicon system, we further demonstrate functional cooperativity between the dominant PS, its proximal partner stem-loop, and additional packaging elements located approximately 10 kb upstream. Collectively, our findings support a highly dynamic and cooperative mechanism of SARS-CoV-2 genome packaging that relies on multiple dispersed packaging signals organized around a dominant central PS. These insights provide a mechanistic framework for understanding coronavirus genome packaging and reveal new opportunities for antiviral intervention through disruption of this process. They also have important implications for vaccine development and may enable the design of membrane-based vector systems capable of efficiently delivering large nucleic acid cargoes, expanding the potential of bionanotechnology and genetic medicine.

microbiology↗

bioRxiv2026

Coupling high-throughput protease enzymology with viral replication reveals biochemical constraints of viral fitness

Proteases govern essential biological processes and are key drug targets, yet how protease sequence variation quantitatively reshapes biochemical parameters and constrains biological fitness remains poorly understood. Here, we integrate high-throughput in vitro enzymology with cellular assays to link protease sequence, biochemistry, and fitness. We extend a microfluidic platform for high-throughput protease enzymology (HT-MEKpro), which is broadly applicable across protease families and catalytic classes, enabling measurement of catalytic turnover (kcat), Michaelis constant (KM), inhibitor potency (IC50), and relative substrate specificity for 102-103 variants. Applied to the SARS-CoV-2 main protease (Mpro), HT-MEKpro generated parallel catalytic and inhibitory landscapes for >400 variants. Integration with viral replication and in-cell cleavage assays reveals that variants with altered substrate specificity fail to support replication, suggesting imbalanced polyprotein processing as a constraint on viral fitness. More broadly, these data can enable mechanistically grounded modeling of protease sequence-property relationships and inform strategies for pharmacological modulation beyond active-site inhibition.

biochemistry↗

bioRxiv2025

Discovery of AVI-6451, a Potent and Selective Inhibitor of the SARS-CoV-2 ADP-Ribosylhydrolase Mac1 with Oral Efficacy in vivo

The COVID-19 pandemic made plain the need for effective antivirals acting on novel antiviral targets, among which viral macrodomains have attracted considerable attention. We recently described AVI-4206 (1), a potent and selective inhibitor of the SARS-CoV-2 ADP-ribosylhydrolase Mac1 based on a 9H-pyrimido[4,5-b]indole core, the first Mac1 inhibitor to demonstrate antiviral efficacy in mouse models of SARS-CoV-2 infection, but requiring IP administration and frequent dosing. Herein we describe an extensive, structurally enabled medicinal chemistry effort to identify orally bioavailable Mac1 inhibitors by addressing permeability and efflux liabilities of 1 and many of its analogs. Multiple strategies were pursued to overcome these issues, including replacing a urea function to reduce hydrogen bond donor count. While heterocyclic urea mimetics could deliver analogs like AVI-6318 (3) with potencies and ADME profiles similar to 1, abrogation of the P-gp liability was finally achieved with entirely non-polar substituents in place of urea. Thus, AVI-6451 (4) is a potent Mac1 inhibitor lead with low intrinsic clearance, high oral bioavailability, and antiviral efficacy with once-daily oral administration in a mouse model of SARS-CoV-2 infection.

pharmacology and toxicology↗

bioRxiv2025

A non-spike nucleocapsid R204P mutation in SARS-CoV-2 Omicron XEC enhances inflammation and pathogenicity

The global circulation of SARS-CoV-2 in human populations has driven the emergence of Omicron subvariants, which have become highly diversified through recombination. In late 2024, SARS-CoV-2 Omicron XEC variant emerged from the recombination of two JN.1 progeny, KS.1.1 and KP.3.3, and became predominant worldwide. Here, we investigated virological features of the XEC variant. Epidemic dynamics modeling suggested that spike substitutions in XEC mainly contribute to its increased viral fitness. Additionally, four licensed antivirals were effective against XEC. Although the fusogenicity of XEC spike is comparable to that of the JN.1 spike, the intrinsic pathogenicity of XEC in hamsters was significantly higher than that of JN.1. Notably, we found that the nucleocapsid R204P mutation of XEC enhanced inflammation through NF-{kappa}B activation. Recent studies suggest that the evolutionary potential of spike protein is reaching its limit. Indeed, our findings highlight the critical role of non-spike mutations in the future evolution of SARS-CoV-2.

microbiology↗

bioRxiv2024

Torsional Twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation.

microbiology↗

bioRxiv2024

The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Standard cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner. However, a stronger antiviral effect for AVI-4206 is observed in human airway organoids and peripheral blood monocyte-derived macrophages. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.

immunology↗