JVI Accepts, published online ahead of print on 4 November 2009

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J. Virol. doi:10.1128/JVI.02013-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

In-depth analysis of KSHV microRNA expression provides insights into the mammalian microRNA processing machinery

Jennifer L. Umbach and Bryan R. Cullen^*

Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Box 3025, Durham, NC 27710

^* To whom correspondence should be addressed. Email: bryan.cullen{at}duke.edu.

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 10⁶ 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 ten 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 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 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.