Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells

RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5’UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets

Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1CVB3. CVB3 persistence in HL-1CVB3 cells represented a typical carrier-state infection with high levels (106 to 108 PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1CVB3 cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1CVB3 cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence

RNA reference materials with defined viral RNA loads of SARS-CoV-2—A useful tool towards a better PCR assay harmonization

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.Peer Reviewe

Mapping of accessible sites for oligonucleotide hybridization in the 5’UTRcvb3 RNA.

<p>(A) Schematic representation of the two methods applied to map regions which are accessible to oligonucleotide hybridization. Thick grey lines—complementary oligomers hybridized to RNA; star—radioactive label; dashed arrow—a direction of primer extension; triangle—a site of RNase H induced RNA cleavage. Grey or black lines with an arrow at the end—dsDNA or RNA products of the procedures, respectively. (B) Analysis of Reverse Transcription with Random Oligonucleotide Libraries (RT-ROL) products by sequencing gel electrophoresis. The RT-ROL products were generated with 8- and 12-mer libraries followed by PCR amplification with the radiolabeled RNA-specific primers and the tag primer. Lanes: <i>(-)</i>—reaction control without DNA library; A, G, U, C—RNA sequencing lines; <i>8</i>—random 8-mer library; <i>12</i>—random 12-mer library; a—reactions in the presence of the antitag-oligomer. Selected nucleotide residues are labeled on the left. Figure shows a typical autoradiogram. The other autoradiograms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136395#pone.0136395.s001" target="_blank">S1 Fig</a>. (C) Cleavage sites induced by RNase H in the presence of semi-random libraries of deoxynucleotide 6-mers: <i>a</i>, <i>c</i>, <i>t</i>, or <i>g</i>. The reactions were carried out at 37°C for 10 and 30 min with the 5'-end-³²P-labeled 5’UTRcvb3 RNA: Lanes: <i>(-)</i>—reaction control without DNA library; L—formamide ladder; T₁—limited hydrolysis by RNase T1. Selected guanine residues are labeled on the left. The short and long run of the gel is shown.</p

Helper antisense oligonucleotides and siRNAs against the 5’UTRcvb3 RNA.

<p>Helper antisense oligonucleotides and siRNAs against the 5’UTRcvb3 RNA.</p

Sites in the 5’UTRcvb3 RNA that are accessible for hybridization to complementary oligonucleotides.

<p>Thick grey lines indicate sites that were mapped as accessible in the secondary structure models proposed by Bailey and Tapprich (2007). Potential target sites for siRNAs are indicated by thin black lines along the structure. (A) The structure model of the 5' UTR of CV-B3 generated by comparative sequence analysis and energy minimization. (B) Experimentally validated structure model.</p

Opening of the new accessible sites for oligonucleotide hybridization in the tightly structured 5’UTRcvb3 <i>in vitro</i>.

<p>(A) Schematic secondary structure model of the 5'UTR of CV-B3 according to Bailey and Tapprich (2007). Target sites of 2’-O-methyl-16-mers are indicated with grey thick lines and target sites of DNA 19-mers are indicated with black lines. (B) Representative agarose gels showing digestion products of the 5’UTRcvb3 RNA by RNase H in the presence of particular DNA 19-mer. Numbers denote particular oligonucleotides present in the mixture: bold number—name of DNA 19-mer; number in italics—name of a helper oligomer (2’-O-methyl-16-mer). C or <i>C</i>—control reaction with an unspecific oligonucleotide. Upper panel—reactions performed at 37°C; lower panel—reactions performed at 23°C.</p

Induction of RNase H cleavage by potential helper oligomers.

<p>(A) Nucleotide sequence of the 5’UTRcvb3 RNA. Sites mapped as accessible to oligonucleotide hybridization are indicated with gray rectangles. Target sites of DNA 16-mers, potential helper oligomers, are marked with black lines below the sequence and numbered 1–19. (B) Representative agarose gels showing degradation of 5’UTRcvb3 RNA by RNase H in the presence of one of the DNA 16-mers. Numbers correspond to target sites indicated in the sequence in (A). Ctr—control reaction without DNA oligonucleotide.</p

siRNA-induced silencing of the reporter construct hrGFP-5’UTRcvb3 in the absence and presence of helper oligomers.

<p>(A) Secondary structure model of the 5' UTR of CV-B3 according to Bailey and Tapprich (2007). Results of the <i>in vivo</i> structure probing are displayed on the model with the circles: black circle—strong DMS-induced A or C modification; empty circle—weak A or C modification. Thick gray lines indicate the target sites of helper 2’-O-methyl-16-mers (light gray) and siRNAs (dark gray). (B) Representative Western blot showing down regulation of the hrGFP-5’UTRcvb3 construct in HeLa cells. The cells were co-transfected with the combinations of 50 nM helper oligomers (name in italics) and 0.1 or 0.25 nM siRNA, respectively (name in black.: si162—siRNA_162, si213—siRNA_213, si420—siRNA_420), as indicated in the figure; GAPDH—loading control; hrGFP—reporter protein. Numbers under blots respect bars showed in graph in panel C. (C) Graph showing relative hrGFP band density normalized to GAPDH. Data were obtained in Western blots for 50 nM helper oligomers and 0.25 nM siRNA. Values are the averages from three independent experiments. Numbers under X axis respect lines showed in Western blot in panel B. The p-values were calculated using Student’s t-test for the following samples: Lanes 3:4, p = 0.062; lanes 6:7, p = 0.209; lanes 9:10, p = 0.756. (D) Graphs showing relative fluorescence intensity exhibited by HeLa cells co-transfected with oligomer combinations and a reporter fusion construct hrGFP-5’UTRcvb3, measured by plate reader. Components of the oligonucleotide mixture indicated in the figure as described above. The p-values were calculated using Student’s t-test for the following samples: 0.1 nM si162+<i>me_C</i>: 0.1 nM si162+<i>me_5</i>, p = 0.152; 0.1 nM si213+<i>me_C</i>: 0.1 nM si213+<i>me_8</i>, p = 0.147; 0.1 nM si420+<i>me_C</i>: 0.1 nM si420+<i>me_9</i>, p = 0.128; 0.25 nM si162+<i>me_C</i>: 0.1 nM si162+<i>me_5</i>, p = 0.679; 0.25 nM si213+<i>me_C</i>: 0.1 nM si213+<i>me_8</i>, p = 0.379; 0.25 nM si420+<i>me_C</i>: 0.1 nM si420+<i>me_9</i>, p = 0.451.</p