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A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants
Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red reco...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776209/ https://www.ncbi.nlm.nih.gov/pubmed/26907327 http://dx.doi.org/10.3390/v8020054 |
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author | Jain, Vaibhav Plaisance-Bonstaff, Karlie Sangani, Rajnikumar Lanier, Curtis Dolce, Alexander Hu, Jianhong Brulois, Kevin Haecker, Irina Turner, Peter Renne, Rolf Krueger, Brian |
author_facet | Jain, Vaibhav Plaisance-Bonstaff, Karlie Sangani, Rajnikumar Lanier, Curtis Dolce, Alexander Hu, Jianhong Brulois, Kevin Haecker, Irina Turner, Peter Renne, Rolf Krueger, Brian |
author_sort | Jain, Vaibhav |
collection | PubMed |
description | Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community. |
format | Online Article Text |
id | pubmed-4776209 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-47762092016-03-09 A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants Jain, Vaibhav Plaisance-Bonstaff, Karlie Sangani, Rajnikumar Lanier, Curtis Dolce, Alexander Hu, Jianhong Brulois, Kevin Haecker, Irina Turner, Peter Renne, Rolf Krueger, Brian Viruses Article Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community. MDPI 2016-02-19 /pmc/articles/PMC4776209/ /pubmed/26907327 http://dx.doi.org/10.3390/v8020054 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Jain, Vaibhav Plaisance-Bonstaff, Karlie Sangani, Rajnikumar Lanier, Curtis Dolce, Alexander Hu, Jianhong Brulois, Kevin Haecker, Irina Turner, Peter Renne, Rolf Krueger, Brian A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants |
title | A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants |
title_full | A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants |
title_fullStr | A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants |
title_full_unstemmed | A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants |
title_short | A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants |
title_sort | toolbox for herpesvirus mirna research: construction of a complete set of kshv mirna deletion mutants |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776209/ https://www.ncbi.nlm.nih.gov/pubmed/26907327 http://dx.doi.org/10.3390/v8020054 |
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