A Replication-Competent Flavivirus Genome with a Stable GFP Insertion at the NS1-NS2A Junction

The flavivirus NS1 protein is a component of the viral replication complex and plays diverse, yet poorly understood, roles in the viral life cycle. To enable real-time visualization of the developing replication organelle and biochemical analysis of tagged NS1 and its interacting partners, we engineered a replication-competent yellow fever virus (YFV) replicon encoding a C-terminal fusion of NS1 with green fluorescent protein (NS1-GFP). The initial variant was non-viable in the absence of trans-complementation with wild-type NS1; however, viability was partially restored through the introduction of co-adaptive mutations in GFP (Q204R/A206V) and NS4A (M108L). Subsequent cell culture adaptation generated a 17-nucleotide frameshift within the NS1-GFP linker, resulting in a more flexible and less hydrophobic linker sequence. The optimized genome, in the form of a replicon, replicates in packaging cells that produce YFV structural proteins, as well as in naive BHK-21 cells. In the packaging cells, the adapted NS1-GFP replicon produces titers of infectious particles of approximately 10⁶ FFU/mL and is genetically stable over five passages. The expressed NS1-GFP fusion protein localizes to the endoplasmic reticulum and co-fractionates with detergent-resistant heavy membranes, a hallmark of flavivirus replication organelles. This NS1-GFP replicon provides a novel platform for studying NS1 functions and can be further adapted for proximity-labeling strategies aimed at identifying the still-unknown protease responsible for NS1-NS2A cleavage.