title: A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. vaibhavjain@ufl.edu.
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Jain
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. karliebonstaff@gmail.com.
authors.forename: Karlie
authors.initials: K
authors.lastname: Plaisance-Bonstaff
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. rsangani@gru.edu.
authors.forename: Rajnikumar
authors.initials: R
authors.lastname: Sangani
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. laeucu@ufl.edu.
authors.forename: Curtis
authors.initials: C
authors.lastname: Lanier
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. adolce@ufl.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dolce
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. jianhong.hu@bcm.edu.
authors.forename: Jianhong
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. kbrulois@stanford.edu.
authors.forename: Kevin
authors.initials: K
authors.lastname: Brulois
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. irina25@me.com.
authors.forename: Irina
authors.initials: I
authors.lastname: Haecker
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. pturner@mgm.ufl.edu.
authors.forename: Peter
authors.initials: P
authors.lastname: Turner
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. rrenne@ufl.edu.|UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA. rrenne@ufl.edu.|UF Genetics Institute, University of Florida, Gainesville, FL 32610, USA. rrenne@ufl.edu.
authors.forename: Rolf
authors.initials: R
authors.lastname: Renne
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. brian.j.krueger@gmail.com.
authors.forename: Brian
authors.initials: B
authors.lastname: Krueger
mesh_terms: D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012514:Sarcoma, Kaposi / Q000821:virology*; D017384:Sequence Deletion
chemical_list: D035683:MicroRNAs; D012367:RNA, Viral
keywords: KSHV; bacmid; human herpesvirus 8; miRNA