RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression...

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Virus-like particles (VLPs) containing exogenous RNA (armored RNA) serve as valuable RNA controls and standards for virus detection.
• Current methods efficiently package RNA sequences typically under 500 bases into VLPs.
• A need exists for methods to produce longer armored RNA for enhanced control and standardization.

Purpose of the Study:

• To develop a method for producing armored long RNA (L-RNA) with efficient packaging of longer RNA sequences.
• To characterize the produced armored L-RNA and evaluate its utility as a calibrator in multiplex virus assays.

Main Methods:

• Utilized a two-plasmid coexpression system in Escherichia coli to produce MS2 bacteriophage coat protein and maturase from one plasmid.
• Transcribed target RNA sequences, including modified MS2 stem-loop (pac site), from a second plasmid.
• Constructed a 2,248-base 3V armored L-RNA containing gene fragments from Hepatitis C virus, SARS-CoV, avian influenza virus, and H5N1 avian influenza virus.

Main Results:

• Successfully produced a 2,248-base 3V armored L-RNA using the two-plasmid coexpression system.
• Demonstrated that the produced armored L-RNA possesses all characteristics of traditional armored RNA.
• Evaluated the 3V armored L-RNA as a calibrator for multiplex virus assays, showing successful calibration against the WHO International Standard for HCV RNA.

Conclusions:

• The developed method for armored L-RNA preparation is practical and reduces labor and cost for quality control in multiplex RNA virus assays.
• The chimeric armored RNA can be assigned an international unit for quantitative detection.
• This approach enables the use of longer RNA sequences as controls and standards, improving the reliability of molecular diagnostics.