Cloning and Identification of a MicroRNA Cluster within the Latency-Associated Region of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

MicroRNAs (miRNAs) are small, noncoding regulatory RNA molecules that bind to 3 untranslated regions (UTRs) of mRNAs to either prevent their translation or induce their degradation. Previously identified in a variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by viruses. The human gammaherpesvirus Epstein-Barr virus has been shown to encode miRNAs, which potentially regulate both viral and cellular genes. To determine whether Kaposi's sarcoma-associated herpesvirus (KSHV) encodes miRNAs, we cloned small RNAs from KSHV-positive primary effusion lymphoma-derived cells and endothelial cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align with KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latencyassociated region of KSHV. These data suggest that viral and host cellular gene expression may be regulated by miRNAs during both latent and lytic KSHV replication.MicroRNAs (miRNAs) range from 19 to 24 nucleotides (nt) in length and are small, noncoding RNA molecules which bind complementary mRNAs to regulate gene expression at the posttranscriptional level. In plants, miRNAs bind precisely to complementary sequences within the 3Ј untranslated region (UTR) of a target gene and induce mRNA degradation through the RNA interference pathway. Most animal miRNAs have limited complementarity to their target sequences within the 3Ј UTR and either degrade mRNA via the RNA interference pathway or down-regulate translation by a mechanism not yet understood. In human cells, over 235 miRNAs have been identified to date (for review, see references 1 and 4). Targets and functions of very few miRNAs have been experimentally determined thus far, yet some molecules, such as human hsamiR-14 and hsa-miR-181, are known to have roles in fundamental biological processes like apoptosis, cell proliferation, and hematopoiesis (6, 9).Most recently, miRNAs have been identified and isolated in the gammaherpesvirus Epstein-Barr virus (EBV) (21) and predicted for the human immunodeficiency virus using in silico methods (5). Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also called human herpesvirus type 8 (HHV-8), is another gammaherpesvirus. The virus is associated with KS and two lymphoproliferative diseases: primary effusion lymphomas (PELs) and a subset of multicentric Castleman's disease (7,8,26). In this report, we demonstrate that KSHV, like EBV, encodes miRNAs.Cloning of small RNAs from KSHV-infected cells. To determine whether KSHV encodes miRNAs, we generated small RNA libraries by positional cDNA cloning from a primary effusion lymphoma-derived cell line (BCBL-1) undergoing either latent or tetradecanoyl phorbol acetate (TPA)-induced lytic KSHV infection (23). Additionally, we cloned small RNAs from a telomerase-immortalized endothelial cell line latently infected with KSHV (TIVE-LTC) (F. Q. An and R. Renne, data to be published elsewhere). Cloning was performed as described in reference 16, with minor modific...

Cellular cofactors affecting hepatitis C virus infection and replication

“…Thus, although replicating HCV was shown to be susceptible to transfected HCV-specific siRNAs and not inhibitory to RNAi, replicating HCV did not appear to be susceptible to endogenous RNAi. Similar studies with other viruses revealed a surprising dependence on the RNAi machinery: HCMV, HHV8, MHV68, SV40, and HIV encode viral miRNAs (13,(52)(53)(54)(55)(56)(57).…”

Expression and Function of MicroRNAs Encoded by Kaposi's Sarcoma-associated Herpesvirus

“…2). Work from other investigators subsequently identified two more KSHV miRNAs, miR-K11 and miR-K12, that were missed in our original analysis (Pfeffer et al 2005;Samols et al 2005;Grundhoff et al 2006). All 12 KSHV miRNAs form part of one arm of an approximately 80-nucleotide RNA hairpin structure, and in several cases, the predicted "passenger" strand, that is, the strand of the miRNA duplex intermediate that is normally excluded from RISC and degraded, was also recovered (Fig.…”

The functions of herpesvirus-encoded microRNAs

“…Cloning studies by multiple groups identiWed viral miRNAs encoded by a second human gamma herpesvirus, Karposi's sarcoma-associated herpesvirus (KSHV) [26,36,37]. Simi-lar to EBV, KSHV infection is associated with a number of tumors, most notably Kaposi's sarcoma, a skin malignancy common amongst AIDS patients and older men of Mediterranean descent [38].…”