Background:  Herpesviruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein Barr virus (EBV), have been thought to be linked to AIDS-related malignancies. MicroRNA (miRNA) are a novel class of regulatory RNA, which can down-regulate target gene expression through RNA silencing. Vertebrate miRNA have been implicated in developmental timing, cell differentiation, and tumorigenesis. Here we show that both KSHV and EBV express several viral miRNA in latently infected cells. These viral miRNA may have an important role in the modulation of the virus life cycle.

Methods:  Small RNA (18 to 24 nt) were size selected and cDNA cloned from BC1 cells latently co-infected with KSHV and EBV. mFOLD analysis of miRNA precursor sequences was performed to validate the authenticity of candidate viral miRNA. Existence of viral miRNA was also confirmed by Northern analysis.

Results:  Of 959 small cDNA clones sequenced, 167 (18%) were derived from KSHV, consisting of 11 distinct miRNA sequences; and 222 (23%) were of EBV origin, including 15 different miRNA. All 11 KSHV miRNA are readily detectable by Northern blotting. They are arrayed in 1 cluster between the v-cyclin and K12/Kaposin genes in the viral latency-associated region. Transcript mapping studies indicate that these KSHV miRNA are originally derived from 3 latent transcripts, which also function as Kaposin pre-mRNA. We identified by miRNA cloning 12 novel EBV miRNA. Combined with several EBV miRNA previously reported, EBV expresses at least 17 distinct miRNA in latently infected cells. They are located in 2 clusters of the viral genome:  14 miRNA are clustered in the introns of the viral BART gene, while 3 are located adjacent to the BHRF1 gene. The BART miRNA are highly expressed in latently infected epithelial cells, although they are also detectable in infected B cells. In contrast, the BHRF1 miRNA are only expressed in B cells at latency stage III.

Conclusions:  For the first time, we report the identification of an array of KSHV and EBV miRNA from latently infected cells. EBV-encoded BART miRNA are highly expressed in nasopharyngeal carcinoma cells while BHRF miRNA can only be detected in B cells at latency phase III. These viral miRNA may play a critical role in the maintenance of the viral life cycle and virus-induced tumorgenesis.