Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards

Abstract

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. "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 coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at -20 degreesC or for 60 days at 4 degreesC. Additionally, Armored RNA preparations are homogeneous and noninfectious.

FIG. 1

Sequence of the HIV RNA packaged within AR–HIV-B. The sequences with which the primers SK462 and SK431 from the HIV-1 Monitor kit hybridize are indicated.

FIG. 2

Characterization of the recombinant RNA packaged in AR–HIV-B. reRNA was isolated from AR–HIV-B, fractionated in a denaturing 1% agarose gel, stained with ethidium bromide, and detected by UV fluorescence. The reRNA was transferred to a membrane and probed with a ³²P-labeled oligonucleotide to the 3′ end of the HIV-B sequence. Abbreviations: M, RNA markers; AR, AR–HIV-B reRNA.

FIG. 3

Densities of AR–HIV-B and bacteriophage MS2 particles. MS2 and AR–HIV-B were loaded in separate gradients and centrifuged, and then 0.5-ml fractions were collected and weighed to determine the density of the CsCl. The optical density of each fraction at 260 nm was measured to calculate the Armored RNA and MS2 concentrations.

FIG. 4

Resistance of purified Armored RNA particles to nucleases. The particles were mixed with plasmid DNA and purified, naked reRNA. The mixture of plasmid DNA, reRNA, and intact Armored RNA was incubated with DNase I and/or the RNases at 37°C for 1 h, fractionated by gel electrophoresis in a 2.0% nondenaturing agarose gel, and detected by ethidium bromide staining and UV fluorescence. The numbers on the left are in base pairs.

FIG. 5

Stability study of AR–HIV-B spiked into EDTA-anticoagulated human plasma at 4°C. AR–HIV-B was added to clarified plasma to a final concentration of ∼7,500 copies/ml. Samples were incubated at 4°C for 0, 1, 2, 4, 7, 14, 28, 42, and 60 days. Samples from each time point were assayed in duplicate, and the copy number determinations were averaged. The mean for all of the samples was 7,780 copies per ml (3.8 log₁₀; range, 6,530 to 9,020 copies per ml [range, 3.81 to 3.96 log₁₀]), and the coefficient of variation was 10.7%. The dashed line represents the mean.

FIG. 6

Comparison of Armored RNA positive control and the HIV-1 Monitor assay high-positive control (Roche-Hi) used in a clinical setting over 180 days. AR–HIV-B was added to clarified EDTA-anticoagulated plasma to a final concentration of ∼65,000 copies/ml, aliquoted into 0.2-ml samples, and stored at −20°C until it was used in the HIV-1 Monitor assay to determine the RNA copy number. The Armored RNA positive control and the HIV-1 Monitor assay high-positive control were used in clinical runs two to four times per week for 180 days. For the Armored RNA standard, the mean was 64,598 copies/ml, the range was 31,760 to 191,716 copies/ml (4.50 to 5.28 log₁₀), and the coefficient of variation was 40%. For the HIV-1 Monitor assay high-positive control, the mean was 290,537 copies/ml; the range was 94,345 to 544,737 copies/ml (4.97 to 5.74 log₁₀), and the coefficient of variation was 32%. The dashed lines represent the means for the two different positive controls.

FIG. 7

Stability of Armored RNA and HIV coincubated in normal human plasma. AR-QS and HIV-1_MC99 were coincubated in normal human plasma over 30 days at 37°C. The concentrations of the QS and HIV RNA sequences were determined by the HIV-1 Monitor assay.