Development of fluorescent and affinity tagged variants of rotavirus nonstructural protein 4 using the reverse genetics system
Year: 2026
Type: preprint
Abstract: Rotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional viroporin and enterotoxin that also serves an essential structural role in virion assembly, making it both a central virulence factor and a potential therapeutic target. However, studying NSP4 within infectious virus has been hindered by essential RNA packaging signals at the 5' end of gene segment 10 (gs10) and an DLP-binding domain at its C-terminus, both of which constrain conventional mutagenesis. Using a plasmid-based reverse genetics system, we generated six recombinant rotaviruses (rRVs) encoding tagged or reporter-fused NSP4 via three strategies: expression from an alternative segment (gs7) in a bicistronic arrangement, N-terminal tagging of gs10 with translation shifted downstream of the native packaging sequence, and retention of the gs10 5' UTR and fusion of the first 20 amino acids fused to a reporter followed by bicistronic full-length NSP4 expression. All six rRVs were replication competent, with gs10-based constructs closely matching wild-type replication kinetics while gs7-based constructs showed modest attenuation and, in one case, genetic instability upon passage. Engineered NSP4 proteins were robustly expressed, correctly glycosylated, and capable of oligomerization. Live-cell imaging showed reporter fluorescence reliably tracked NSP4 synthesis and preceded NSP4-dependent intercellular calcium signals. Affinity purification of tagged NSP4 recovered viroplasm-associated and structural viral proteins along with candidate host interactors, including ANP32A, ANP32E, PPM1G, and H2AC1. These rRVs constitute a validated toolkit for dissecting NSP4 function during bona fide infection and establish a generalizable strategy for engineering constrained rotavirus gene segments.
Institution: Baylor College of Medicine
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
Awards: G1756901816, G2911158097, G3832887421, G418187648, Role of Rotavirus NSP4 Viroporin and Enterotoxin Activity in Aberrant Calcium Signaling and Replication, Proteomics and Metabolomics Core Facility, THERMO SCIENTIFIC Q EXACTIVE HF-X HYBRID QUADRUPOLE-ORBITRAP MASS SPECTROMETER, G7837447426, G8439629300, G8759711797, Enteric virus exploitation of calcium signaling, The Clinical Translational Research Certificate of Added Qualification Program, Baylor College of Medicine Cancer Center - Cancer Center Support Grant, Dissecting Rotavirus Viroporin and Enterotoxin Calcium Signaling Pathways
OpenAlex ID: W7211963556