RNase-Resistant Virus-Like Particles Containing Long Chimeric RNA Sequences Produced by Two-Plasmid Coexpression System

Abstract

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragmentshepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)-was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 10 6 to 10 2 copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.Armored RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the bacteriophage sequence consisting of the maturase, the coat protein, the pac site, and an exogenous RNA sequence. This method produces recombinant virus-like particles that are noninfectious and contain predefined RNA (2-6, 8, 11, 12, 15, 16, 28). These armored RNAs are RNase resistant by virtue of their encapsulation within an MS2 coat protein, and they have been widely used as controls, standards, or calibrators for the detection of hepatitis C virus (HCV) (11,12,28), human immune-deficiency virus (11, 15), severe acute respiratory syndrome coronavirus (SARS-CoV) (3, 11), enterovirus (2, 5), avian influenza virus 5 (4), and West Nile virus (6) using reverse transcription-PCR (RT-PCR), real-time RT-PCR, and branched DNA assays (2-6, 11, 12, 15, 28).The MS2 bacteriophage consists of 180 U of the bacteriophage coat protein that encapsulates the bacteriophage genome (25). The MS2 phage RNA genome comprises a single plus-sense strand encoding 3,569 nucleotides. The genes are organized from the 5Ј end as follows: the maturase or A protein, the bacteriophage coat protein, a 75-amino-acid lysis protein, and a replicase subunit. Packaging of the RNA genome by coat protein is initiated by high-specificity binding to a unique site on the RNA, a single stem-loop structure, containi...

External Quality Assessment for Avian Influenza A (H7N9) Virus Detection Using Armored RNA

“…As expected, the data obtained from stability analyses revealed that at concentrations of 10 9 and 10 6 copies/ml, the VLPs were stable for at least 2 weeks at 37°C, 4 weeks at room temperature, and 2 months at both 4°C and Ϫ20°C. These were consistent with the previously reported results (11). Stability was defined as a decrease of Ͻ0.5 log 10 copies/ml compared to the control that was stored at Ϫ80°C for the entire duration.…”

“…The MS2 virus-like particles (VLPs) were expressed and purified as in previous studies in our laboratory (10)(11)(12). Briefly, the recombinant plasmids pACYC-MS2-HA, pACYC-MS2-NA, pACYC-MS2-MP, and pACYC-MS2-NP were transformed into the Escherichia coli strain BL21(DE3).…”

Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1

“…We have demonstrated that 1,981-base chimeric RNA can be successfully packaged into the MS2 coat protein by utilizing a one-plasmid expression system with two C-variant pac sites (32). The 2,248-base armored RNA was also successfully expressed by a two-plasmid coexpression system with one C-variant pac site (33). This paper reports the development of armored long RNA (armored L-RNA) controls or standards for the bDNA assay for HIV-1 (AR-HIV-pol-3034b; 3,034 bases).…”

The True Story and Advantages of RNA Phage Capsids as Nanotools

“…Recently, the creation of armored dsDNA using HBV and HPV particles was accomplished on the basis of previous work using MS2 VLPs [275]. Armored RNAs have multiple uses, such as for the detection of HCV, severe acute respiratory syndrome coronavirus, and influenza virus RNA [276] or for the creation of unique RNA molecules harboring both tRNA and mRNA functions [277]. Perspectives on the production and practical use of MS2 VLPs for routine diagnostics including food quality control were discussed in a recent review [278].…”