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

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          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.

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          Most cited references22

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          Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.

          Representational difference analysis was used to isolate unique sequences present in more than 90 percent of Kaposi's sarcoma (KS) tissues obtained from patients with acquired immunodeficiency syndrome (AIDS). These sequences were not present in tissue DNA from non-AIDS patients, but were present in 15 percent of non-KS tissue DNA samples from AIDS patients. The sequences are homologous to, but distinct from, capsid and tegument protein genes of the Gammaherpesvirinae, herpesvirus saimiri and Epstein-Barr virus. These KS-associated herpesvirus-like (KSHV) sequences appear to define a new human herpesvirus.
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            A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses.

            We have developed an approach to identify microRNAs (miRNAs) that is based on bioinformatics and array-based technologies, without the use of cDNA cloning. The approach, designed for use on genomes of small size (<2 Mb), was tested on cells infected by either of two lymphotropic herpesviruses, KSHV and EBV. The viral genomes were scanned computationally for pre-miRNAs using an algorithm (VMir) we have developed. Candidate hairpins suggested by this analysis were then synthesized as oligonucleotides on microarrays, and the arrays were hybridized with small RNAs from infected cells. Candidate miRNAs that scored positive on the arrays were then subjected to confirmatory Northern blot analysis. Using this approach, 10 of the known KSHV pre-miRNAs were identified, as well as a novel pre-miRNA that had earlier escaped detection. This method also led to the identification of seven new EBV-encoded pre-miRNAs; by using additional computational approaches, we identified a total of 18 new EBV pre-miRNAs that produce 22 mature miRNA molecules, thereby more than quadrupling the total number of hitherto known EBV miRNAs. The advantages and limitations of the approach are discussed.
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              In-depth analysis of Kaposi's sarcoma-associated herpesvirus microRNA expression provides insights into the mammalian microRNA-processing machinery.

              We have used deep sequencing to analyze the pattern of viral microRNA (miRNA) expression observed in the B-cell line BC-3, which is latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV). We recovered 14.6 x 10(6) total miRNA cDNA reads, of which a remarkable 92% were of KSHV origin. We detected 11 KSHV miRNAs as well as all 11 predicted miRNA or passenger strands from the miRNA duplex intermediate. One previously reported KSHV miRNA, miR-K9, was found to be mutationally inactivated. This analysis revealed that the 5' ends of 10 of the 11 KSHV miRNAs were essentially invariant, with significantly more variation being observed at the 3' end, a result which is consistent with the proposal that the 5'-proximal region of miRNAs is critical for target mRNA recognition. However, one KSHV miRNA, miR-K10-3p, was detected in two isoforms differing by 1 nucleotide (nt) at the 5' end that were present at comparable levels, and these two related KSHV miRNAs are therefore likely to target at least partially distinct mRNA populations. Finally, we also report the first detection of miRNA offset RNAs (moRs) in vertebrate somatic cells. moRs, which derive from primary miRNA (pri-miRNA) sequences that immediately flank the mature miRNA and miRNA strands, were identified flanking one or both sides of nine of the KSHV miRNAs. These data provide new insights into the pattern of miRNA processing in mammalian cells and indicate that this process is highly conserved during animal evolution.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Viruses
                Viruses
                viruses
                Viruses
                MDPI
                1999-4915
                19 February 2016
                February 2016
                : 8
                : 2
                : 54
                Affiliations
                [1 ]Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA; vaibhavjain@ 123456ufl.edu (V.J.); karliebonstaff@ 123456gmail.com (K.P.-B.); rsangani@ 123456gru.edu (R.S.); laeucu@ 123456ufl.edu (C.L.); adolce@ 123456ufl.edu (A.D.); jianhong.hu@ 123456bcm.edu (J.H.); irina25@ 123456me.com (I.H.); pturner@ 123456mgm.ufl.edu (P.T.); brian.j.krueger@ 123456gmail.com (B.K.)
                [2 ]Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; kbrulois@ 123456stanford.edu
                [3 ]UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA
                [4 ]UF Genetics Institute, University of Florida, Gainesville, FL 32610, USA
                Author notes
                [* ]Correspondence: rrenne@ 123456ufl.edu ; Tel.: +1-352-273-8204
                [†]

                Present address: Department of Insect Biotechnology, Justus-Liebig University Giessen, Henrich-Buff-Ring 26–28, Giessen, Hesse 35392, Germany.

                Article
                viruses-08-00054
                10.3390/v8020054
                4776209
                26907327
                c8c19b22-da52-487c-9ea0-49ad00b954e8
                © 2016 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 17 December 2015
                : 14 February 2016
                Categories
                Article

                Microbiology & Virology
                kshv,human herpesvirus 8,mirna,bacmid
                Microbiology & Virology
                kshv, human herpesvirus 8, mirna, bacmid

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