GC content mismatch of transgene destabilizes RNA virus genomes

GC content-the proportion of guanine and cytosine nucleotides-varies widely among organisms and viruses. Although GC content is recognized to have biological significance, its functional role in viruses remains poorly understood. Here, we examined the impact of GC-content bias using three positive-sense single-stranded RNA viruses with distinct GC profiles: severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), Japanese encephalitis virus (JEV), and hepatitis C virus (HCV). Variants of the NanoLuc (Nluc) reporter gene, engineered with synonymous mutations to alter GC content, were inserted into each viral genome. Serial passaging experiments revealed strong effects on genome stability and viral fitness. In SARS-CoV-2, which has low GC content, the introduction of a GC-rich Nluc gene disrupted genome stability, leading to frequent deletions of Nluc. In both SARS-CoV-2 and HCV, Nluc with mismatched GC levels accumulated substitutions that optimized GC content toward that of the viral genome, predominantly at the third codon position. tRNA-seq analysis revealed a shift in the host tRNA pool toward a more GC-rich composition during HCV infection, consistent with this substitution bias. In contrast, JEV (intermediate GC content) maintained the Nluc variants across serial passages, suggesting reduced selective pressure. Taken together, these findings demonstrate that mismatches between viral genome GC content and inserted sequences profoundly affect genetic stability, suggesting evolutionary constraints that may shape RNA virus composition more broadly. This study provides mechanistic insight into how GC content influences viral genome maintenance and offers a framework for designing genetically stable recombinant reporter viruses.IMPORTANCEGC content is a fundamental genomic feature that influences gene expression, genome architecture, and adaptation. Although its role in cellular genomes has been extensively studied, the functional significance of GC content in viral genomes remains poorly understood. In this study, we show that incompatibility in GC content between a viral genome and an inserted transgene destabilizes RNA secondary structures, compromising viral genome integrity, and is accompanied by changes in the host tRNA pool consistent with this compositional bias. These findings identify GC content as a previously underappreciated determinant of virus-host compatibility and viral fitness. Importantly, our results provide mechanistic insight into how the GC content of a viral genome can be matched to its host cellular environment. By demonstrating that GC content matching is critical for the stability of recombinant viruses, this study provides a basis for the rational design of reporter viruses, vaccine platforms, and antiviral screening tools.