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

Vaibhav Jain,Curtis Lanier,Karlie Plaisance-Bonstaff,Alexander Dolce,Rolf Renne,Brian Krueger,Rajnikumar Sangani,Kevin Brulois,Irina Haecker,Jianhong Hu,Peter Turner

doi:10.3390/v8020054

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.

Highlights

Kaposi’s sarcoma-associated herpes virus (KSHV) is the etiological agent causing Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and a subset of multicentric Castleman’s disease (MCD) [1,2,3]
Intra-molecular recombination is facilitated by a duplicated sequence flanking the mutagenesis cassette which results in the removal of the positive selection marker and the deletion of the target sequence
The objective of this work was to generate a set of quality controlled recombinant viruses that contain defined miRNA mutations on a single genetic backbone of Kaposi’s sarcoma-associated herpesvirus (KSHV) (BAC16) [20]

Summary

Introduction

Kaposi’s sarcoma-associated herpes virus (KSHV) is the etiological agent causing Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and a subset of multicentric Castleman’s disease (MCD) [1,2,3]. Most cells in KSHV-associated malignancies are latently infected and express only a very limited number of viral genes, which contribute to viral pathogenesis and/or tumorigenesis by Viruses 2016, 8, 54; doi:10.3390/v8020054 www.mdpi.com/journal/viruses. After the initial discovery of virally-encoded miRNAs in EBV in 2004 [7], several laboratories [8,9,10,11] identified 12 miRNA genes that theoretically can give rise to 25 mature miRNAs in lymphoma cells latently infected with KSHV. A large number of studies have reported viral miRNA/mRNA target interactions for either viral or host cellular genes, and much of these data suggest that KSHV miRNAs target a number of central signal transduction pathways as well as host and/or viral genes controlling latency/lytic viral replication

Objectives

Methods

Conclusion