期刊
VIRUSES-BASEL
卷 8, 期 2, 页码 -出版社
MDPI AG
DOI: 10.3390/v8020054
关键词
KSHV; human herpesvirus 8; miRNA; bacmid
类别
资金
- NIH/NCI ARRA [RC2 CA148407]
- NIH/NCI [5R01 CA119917]
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 (miR-K12-1 through -12), and for mutants deleted for 10 of 12 (miR-cluster) or all 12 miRNAs (miR-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.
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