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.

Keywords: KSHV; bacmid; human herpesvirus 8; miRNA.

Figure 1

KSHV Latency-Associated Region (KLAR) showing open reading frames (black arrows) and miRNAs (red arrows). The region between LANA and K12 on the minus strand is located between nts position 129,256 (LANA ATG) and 117,738 (K12 UGA) on the KSHV Bac16 genome (Accession number GQ994935).

Figure 2

Two-step recombineering. Primers are designed to PCR amplify the kanamycin-resistance cassette. The primer tails match KSHV sequences (abd for the forward primer and bde for the reverse primer). Note that sequence c will be deleted in the final construct. The PCR product is electroporated into E. coli cells carrying BAC16, and intermolecular recombination generates a kan-resistant bacmid. Induction of I-SceI results in linearization of the bacmid. Intramolecular recombination between sequences bd near the ends of the molecule produces the final markerless KSHV mutant bacmid, with sequences abde. Modified from [23,24].

Figure 3

Testing of recombineering products to confirm desired deletion and integrity of terminal repeats. (A) PCR products following intermolecular recombination, showing that the kan cassette is present at the intended location. The cartoon below shows primer locations; (B) pulsed field gel electrophoresis (PFGE) of NheI-digested bacmid DNA to verify integrity of TRs in the products of the first recombination step. Clones with the desired NheI restriction pattern are indicated by asterisks; (C) PCR analysis of products from the second (intramolecular) recombination verifying that the kan cassette has been removed. The primers used are represented in the cartoon above; (D) confirmation of the deletion by PCR with primers flanking the deleted region, as shown in the diagram below. The wild-type BAC16 gives an 80 bp product and the BAC19ΔmiR-K12-1 mutant a 60 bp product; and (E) PFGE to verify TR integrity in the final bacmid recombination products.

Figure 4

Generation of iSLK producer cell lines by co-culture with bacmid-transfected 293 cells.

Figure 5

Confirmation of loss of miRNA expression in deletion mutants for miR-K12-1, miR-K12-3, and miR-K12-11. TaqMan miRNA assay was used to determine the levels of mature miRNAs K12-1 through K12-12 (strand-specificity for each Taqman miRNA assay is listed in Table S1). The blue bars show expression for wtKSHV normalized to 1. The cyan bars show expression levels for KSHV deleted for miR-K12-1 (top), miR-K12-3 (center), or miR-K12-11 (bottom).