Rewriting nature's assembly manual for a ssRNA virus

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Satellite tobacco necrosis virus (STNV) has a minimal genome encoding only its coat protein (CP).
• STNV replication depends on a helper virus, and its genome contains dispersed RNA packaging signals (PSs) with AXXA motifs.

Purpose of the Study:

• To investigate critical assembly features within the STNV genomic fragment (nucleotides 1-127).
• To determine the role of CP-binding motifs, PS stem-loop placement, number, and folding in STNV assembly.

Main Methods:

• Site-directed mutagenesis of STNV RNA to alter packaging signals.
• Assembly assays comparing wild-type and mutant STNV RNAs.
• Competition assays between modified and wild-type STNV genomes.

Main Results:

• Assembly nucleation is primarily driven by the recognition of folded PSs, not just CP binding.
• Mutations removing AXXA motifs significantly impair RNA assembly.
• A synthetic fragment with improved PSs partially restored assembly and produced incomplete virus-like particles.
• Swapping the improved fragment into wild-type STNV enhanced capsid production and outcompeted the wild-type genome.

Conclusions:

• Confirms the PS-mediated assembly mechanism for STNV.
• Demonstrates that optimized PSs can improve virus-like particle production.
• Identifies a strategy for producing virus-like particles for encapsulating nonnative RNAs or cargoes.