Characterization of nucleolar localization signals in the avian infectious bronchitis virus nucleocapsid protein and their critical role in viral replication.

UNLABELLED: The nucleocapsid (N) protein of infectious bronchitis virus (IBV) is the most abundant viral protein in infected cells and is essential for virion assembly and for modulating host cellular processes. Although previous studies have reported nucleolar accumulation of IBV N, a comprehensive mapping of its nucleolar localization signals (NoLSs) is lacking. Here, we systematically mapped functional NoLSs within the IBV N protein and evaluated their contributions to viral replication. Under both infection and transient-transfection conditions, the N protein exhibited a dual cytoplasmic-nucleolar distribution. Analysis of EGFP-tagged N protein truncation mutants identified four candidate NoLSs (NoLS1-4) and a nuclear localization signal (NLS). Alanine-scanning mutagenesis established that NoLS3 (amino acids [aa] 330-349) and NoLS4 (aa 356-368) were the principal determinants of nucleolar targeting within the full-length protein, with this activity depending on basic residue clusters 2 and 3 in NoLS3 and cluster 1 in NoLS4. Recombinant virus rescue assays demonstrated that the double mutant strain (rYN-N-K356A/R359A) abolished nucleolar accumulation of N protein and showed significantly decreased replication levels at 24 and 36 h post-infection. Collectively, this study identifies specific basic residues in NoLS3 and NoLS4 as the molecular basis for IBV N nucleolar localization and demonstrates that nucleolar targeting is required for optimal IBV replication.

IMPORTANCE: Avian infectious bronchitis virus (IBV) is a highly contagious pathogen that causes severe economic losses to the global poultry industry. While the N protein is known to localize to the nucleolus, the precise sequences of its NoLSs and their impact on viral replication, remain unclear. This study systematically identified the functional NoLSs of the IBV N protein and the essential basic residues within them. Crucially, we demonstrated that the nucleolar localization of the N protein plays an important role in viral replication. Furthermore, elucidating the specific motifs required for N protein functionality may inform the rational design of live-attenuated vaccines or antiviral approaches against IBV.