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Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA

Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense respons...

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Detalles Bibliográficos
Autores principales: Wiehler, Shahina, Proud, David
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006032/
https://www.ncbi.nlm.nih.gov/pubmed/33670292
http://dx.doi.org/10.3390/mps4010013
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author Wiehler, Shahina
Proud, David
author_facet Wiehler, Shahina
Proud, David
author_sort Wiehler, Shahina
collection PubMed
description Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. Despite the central role of dsRNA in regulating host cell responses, no method for the quantitative assessment of dsRNA levels during HRV infections has been developed. Conventional RT-PCR for the negative strand template is not effective as self-priming results in apparent signals, even in the absence of primer during reverse transcription. To avoid these issues, we developed a selective assay for the negative strand template that uses a chimeric primer containing a 5′ non-viral sequence for reverse transcription and a primer using the non-viral sequence during subsequent PCR. We established that this assay avoided issues of self-priming and is strand specific, as it is unaffected even in the presence of a 1000-fold excess of positive strand. Assays in primary human airway epithelial cells showed that negative strand was detectable within 6 h of virus exposure and peaked at 18 h after virus exposure. The temporal pattern of negative strand induction mirrored that of genomic RNA but was always 1000-fold lower than positive strand, indicating that the negative strand levels regulate levels of dsRNA formation. This assay will permit relative quantification of dsRNA during studies of HRV regulation of epithelial cell function.
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spelling pubmed-80060322021-03-30 Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA Wiehler, Shahina Proud, David Methods Protoc Communication Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. Despite the central role of dsRNA in regulating host cell responses, no method for the quantitative assessment of dsRNA levels during HRV infections has been developed. Conventional RT-PCR for the negative strand template is not effective as self-priming results in apparent signals, even in the absence of primer during reverse transcription. To avoid these issues, we developed a selective assay for the negative strand template that uses a chimeric primer containing a 5′ non-viral sequence for reverse transcription and a primer using the non-viral sequence during subsequent PCR. We established that this assay avoided issues of self-priming and is strand specific, as it is unaffected even in the presence of a 1000-fold excess of positive strand. Assays in primary human airway epithelial cells showed that negative strand was detectable within 6 h of virus exposure and peaked at 18 h after virus exposure. The temporal pattern of negative strand induction mirrored that of genomic RNA but was always 1000-fold lower than positive strand, indicating that the negative strand levels regulate levels of dsRNA formation. This assay will permit relative quantification of dsRNA during studies of HRV regulation of epithelial cell function. MDPI 2021-02-11 /pmc/articles/PMC8006032/ /pubmed/33670292 http://dx.doi.org/10.3390/mps4010013 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Wiehler, Shahina
Proud, David
Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA
title Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA
title_full Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA
title_fullStr Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA
title_full_unstemmed Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA
title_short Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA
title_sort specific assay of negative strand template to quantify intracellular levels of rhinovirus double-stranded rna
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006032/
https://www.ncbi.nlm.nih.gov/pubmed/33670292
http://dx.doi.org/10.3390/mps4010013
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