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

## TL;DR

Scientists created a modified yellow fever virus that includes a glowing protein to study how the virus replicates and identify a key enzyme involved in its life cycle.

## Contribution

The first replication-competent flavivirus genome with a stable GFP insertion near the NS1-NS2A cleavage site.

## Key findings

- Co-adaptive mutations in GFP and NS4A partially restored viability of the modified virus.
- A frameshift in the NS1-GFP linker improved replicon stability and flexibility.
- The NS1-GFP fusion localizes to the endoplasmic reticulum and replication organelles.

## Abstract

Viruses rely on molecular machines to replicate in living cells. The yellow fever virus and some others from fifty related viruses cause human disease, making it important to understand their replication machinery for therapeutic purposes. This machinery includes two proteins: nonstructural proteins one and two, which act as gears in a molecular clockwork. While their functions are partly understood, it remains unclear how these proteins are produced. They are generated by cleavage of a single precursor, yet the cellular “tool” responsible for this cut (a protease)—a potential antiviral target—has not been identified. In this study, we aimed to modify the precursor at an unusual site—very close to the cleavage site—by fusing nonstructural protein one to a green fluorescent protein. This forced the viral machinery to adjust by accumulating adaptive mutations. We describe the mutations in the replication machinery that allowed the modified virus to regain viability. This viral system is the first in its genus to have a large, functional foreign protein inserted within the replication machinery near the cleavage site, providing a platform to identify the elusive protease.

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.