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A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants

Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA
UF Genetics Institute, University of Florida, Gainesville, FL 32610, USA
Author to whom correspondence should be addressed.
Present address: Department of Insect Biotechnology, Justus-Liebig University Giessen, Henrich-Buff-Ring 26–28, Giessen, Hesse 35392, Germany.
Academic Editor: Andrew Mehle
Viruses 2016, 8(2), 54;
Received: 17 December 2015 / Revised: 4 February 2016 / Accepted: 14 February 2016 / Published: 19 February 2016
(This article belongs to the Section Animal Viruses)
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. View Full-Text
Keywords: KSHV; human herpesvirus 8; miRNA; bacmid KSHV; human herpesvirus 8; miRNA; bacmid
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Jain, V.; Plaisance-Bonstaff, K.; Sangani, R.; Lanier, C.; Dolce, A.; Hu, J.; Brulois, K.; Haecker, I.; Turner, P.; Renne, R.; Krueger, B. A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants. Viruses 2016, 8, 54.

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