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

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• The flavivirus NS1 protein is crucial for viral replication but its functions remain unclear.
• Understanding NS1's role requires tools for real-time visualization and biochemical analysis.

Purpose of the Study:

• To engineer a replication-competent yellow fever virus (YFV) replicon for visualizing NS1 protein dynamics.
• To create a tool for biochemical analysis of NS1 and its interacting partners.

Main Methods:

• Engineered a YFV replicon encoding a NS1-green fluorescent protein (GFP) fusion.
• Introduced co-adaptive mutations to restore viability.
• Adapted the replicon through cell culture to enhance replication efficiency and genetic stability.

Main Results:

• The optimized NS1-GFP replicon replicates efficiently in packaging and naive cells.
• In packaging cells, the replicon produced high titers of infectious particles (approx. 10^6 FFU/mL) and remained stable over five passages.
• NS1-GFP localized to the endoplasmic reticulum, co-fractionating with replication organelles.

Conclusions:

• The developed NS1-GFP YFV replicon is a viable tool for studying flavivirus replication.
• This platform facilitates real-time visualization and biochemical studies of NS1.
• It can be further adapted for proximity-labeling to identify the protease involved in NS1-NS2A cleavage.