Chantal AGBEMABIESE, NOGUCHI MEMORIAL INSTITUTE FOR MEDICAL RESEARCH

Chantal Ama Agbemabiese (Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana). Asha Ann Philip (CSL Seqirus, MA, USA) George Enyimah Armah (Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana) John Thomas Patton (Department of Biology, Indiana University, Bloomington, Indiana, USA)

Virus Replication: Entry, Exit and Everything in Between

The Rotavirus A genome comprises 11 double-stranded RNA segments (encoding six structural and six non-structural proteins), each with conserved terminal sequences (5′-GGC…UGUGG/ACC-3′). The NSP3 enhances viral mRNA translation while suppressing host protein synthesis by outcompeting eukaryotic poly-A-binding protein (PABP) for eIF4G binding. Although in vitro studies show that 3′-consensus sequence (3′-CS) alterations impair NSP3 binding and reduce translational efficiency, the lack of a fully plasmid-based reverse genetics (RG) system made it impossible to generate the appropriate mutant rotaviruses required to evaluate the importance of the individual residues of the 3’-CS on replication and translation of viral (+)RNA within the context of infection. Using RG, we generated recombinant SA11 strains (wildtype and 3′-CS mutants). A mutant lacking the UGUG RdRp recognition motif was rescued but exhibited low viral yield. The mutant displayed reduced protein expression, smaller plaques, and lower peak titers than the wildtype. Though viable, the 3′-CS mutant lacking UGUG was genetically unstable, acquiring compensatory mutations suggesting adaptive evolution to restore fitness during subsequent plaque purification and amplification. Our findings demonstrate that 3′-CS mutations impact replication efficiency but do not abolish virus viability. Nucleotides upstream of the CC in the heptameric 3′-CS motif (UGUGG/ACC) need additional investigations to ascertain their need for rotavirus replication. The successful recovery of the mutant viruses highlights the potential flexibility of nucleotides upstream of the CC in the heptameric 3′-CS motif. This study advances understanding of rotavirus genetic plasticity and the functional constraints of conserved regulatory sequences in viral replication.