Biology subjects

Datta, S. A.

Publications and source records attributed to Datta, S. A..

3 recordsLinked to original sources

bioRxiv2026

Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation

The nucleocapsid protein of SARS-CoV-2 scaffolds genomic RNA into ribonucleoprotein complexes (RNP) for assembly in the virion, but also fulfills critical intracellular functions in replication and the suppression of host defense. It is comprised of a folded nucleic acid binding domain (NTD) and a dimerization domain, connected by a disordered linker containing a serine/arginine-rich (SR) region and a leucine-rich sequence (LRS). The switch between intracellular and assembly functions of N-protein is controlled by phosphorylation of the SR region, but the molecular details are unclear. Here we describe a model in which two mutually exclusive intramolecular loops bind the NTD and dynamically control self-association and nucleic acid binding properties dependent on the SR linker phosphorylation state. The model is supported by biophysical properties and interactions of full-length protein, point mutants, and peptide fragments. We find SR linker phosphorylation compacts the protein and inhibits nucleic acid binding and RNP formation, while enhancing self-association through promotion of transient coiled-coils in the LRS of the linker. These changes shift the nucleocapsid protein to a configuration poised for multi-valent interactions that support intracellular functions.

biophysics↗

bioRxiv2025

Evolution of a fuzzy ribonucleoprotein complex in viral assembly

Previously we showed that the genetic diversity of SARS-CoV-2 nucleocapsid (N) protein explores a wide range of biophysical properties facilitated by non-local impact of point mutations to its intrinsically disordered regions (Nguyen et al., 2024). This includes modulation of self-association, such as the creation of a de novo binding interface through the P13L mutation characteristic of Omicron variants. In the present work we focus on the key function of N condensing viral RNA into ribonucleoprotein particles (RNPs) for viral assembly. Lacking high-resolution structural information, biochemical and biophysical approaches have revealed architectural principles of RNPs, which involve cooperative interactions of several protein-protein and protein-RNA interfaces, initiated through oligomerization of conserved transient helices in the central disordered linker of N. Here we study the impact of defining N-protein mutations in variants of concern on RNP formation, using biophysical tools, a virus-like particle assay, and reverse genetics experiments. We find convergent evolution in repeated, independent introduction of amino acid substitutions strengthening existing binding interfaces, compensating for other substitutions that promote viral replication but decrease RNP stability. Furthermore, we show that the P13L mutation of Omicron variants enhances RNP assembly and increases viral fitness. Overall, our data reveal RNP complexes to be highly variable not only in sequence and conformations, but also in thermodynamic and kinetic stability, with its pleomorphism affecting basic architectural principles. We hypothesize that the formation of polydisperse, fuzzy N-RNA clusters with multiple distributed weak binding interfaces optimizes reversible RNA condensation, while supporting host adaptation and allowing for a large sequence space to be explored.

biophysics↗

bioRxiv2024

SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

The CD225/Dispanins superfamily consists of membrane proteins that regulate vesicular transport and membrane fusion events driving neurotransmission, glucose transport, and antiviral immunity. However, how the CD225 domain controls membrane trafficking was unknown. We reveal that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens. Proline rich transmembrane protein 2 (PRRT2) encodes a SNARE-like motif that enables interaction with neuronal SNARE proteins, and mutations therein disrupt SNARE binding and are linked to neurological disease. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), protects cells against Influenza A virus infection. IFITM3 interacts with SNARE proteins that mediate late endosome-late endosome (homotypic) fusion and late endosome-lysosome (heterotypic) fusion. IFITM3 binds to syntaxin 7 (STX7) in cells and in vitro, and mutations that abrogate STX7 binding cause loss of antiviral activity against Influenza A virus. Mechanistically, IFITM3 disrupts assembly of the SNARE complex controlling homotypic fusion and accelerates the trafficking of endosomal cargo to lysosomes. Our results suggest that SNARE modulation plays a previously unrecognized role in the diverse functions performed by CD225 proteins.

cell biology↗