Cloning and identification of a microRNA cluster within the latency-associated region of Kaposi's sarcoma-associated herpesvirus.

Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA.
Journal of Virology (Impact Factor: 4.65). 08/2005; 79(14):9301-5. DOI: 10.1128/JVI.79.14.9301-9305.2005
Source: PubMed

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 latency-associated 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.

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    ABSTRACT: Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (γHVs), including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine γHV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes in vitro, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to γHV infection, we have used γHV68, a small-animal model of γHV pathogenesis. γHV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the γHV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to γHV acute infection and pathogenesis, we generated and characterized a recombinant γHV68 strain devoid of all eight TMERs. TMER-deficient γHV68 has wild-type levels of lytic replication in vitro and normal establishment of latency in B cells early following acute infection in vivo. In contrast, during acute infection of immunodeficient mice, TMER-deficient γHV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that γHV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate γHV-induced infection and pathogenesis. The gammaherpesviruses (γHVs) are a subfamily of viruses associated with chronic inflammatory diseases and cancer, particularly in immunocompromised individuals. These viruses uniformly encode multiple types of noncoding RNAs (ncRNAs) that are not translated into proteins. It remains unclear how virus-expressed ncRNAs influence the course and outcome of infection in vivo. Here, we generated a mouse γHV that lacks the expression of multiple ncRNAs. Notably, this mutant virus is critically impaired in the ability to cause disease in immunocompromised hosts yet shows a paradoxical increase in infected cells early during infection in these hosts. While the original mouse virus encodes multiple ncRNAs, the expression of a single domain of one ncRNA can partially reverse the defects of the mutant virus. These studies demonstrate that γHV ncRNAs can directly contribute to virus-induced disease in vivo and that these RNAs may be multifunctional, allowing the opportunity to specifically interfere with different functional domains of these RNAs. Copyright © 2015 Diebel et al.
    mBio 02/2015; 6(1):e01670-14. DOI:10.1128/mBio.01670-14 · 6.88 Impact Factor
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    ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNA species that can bind to both untranslated and coding regions of target mRNAs, causing their degradation or post-transcriptional modification. Currently, over 2500 miRNAs have been identified in the human genome. Burgeoning evidence suggests that dysregulation of human miRNAs can play a role in the pathogenesis of a variety of diseases, including cancer. In contrast, only a small subset of human miRNAs has been functionally validated in the pathogenesis of oncogenic viruses, in particular, Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of several human cancers, such as primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS), which are mostly seen in acquired immune deficiency syndrome (AIDS) patients or other immuno-suppressed subpopulation. This review summarizes recent literature outlining mechanisms for KSHV/viral proteins regulation of cellular miRNAs contributing to viral pathogenesis, as well as recent findings about the unique signature of miRNAs induced by KSHV infection or KSHV-related malignancies.
    Viruses 11/2014; 6(11):4571-4580. DOI:10.3390/v6114571 · 3.28 Impact Factor
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    ABSTRACT: The human oncogenic Kaposi's Sarcoma-associated herpesvirus (KSHV) expresses a set of ∼20 viral miRNAs. miR-K10a stands out among these miRNAs because its entire stem-loop precursor overlaps the coding sequence for the Kaposin (Kap) A/C proteins. The ectopic expression of KapA has been reported to lead to transformation of rodent fibroblasts. However, these experiments inadvertently also introduced miR-K10a, which raises the question whether the transforming activity of the locus could in fact be due to miR-K10a expression. To answer this question, we have uncoupled miR-K10a and KapA expression. Our experiments revealed that miR-K10a alone transformed cells with similar efficiency as its co-expression with KapA. Maintenance of the transformed phenotype was conditional upon continued miR-K10a but not KapA protein expression, consistent with its dependence on miRNA-mediated changes in gene expression. Importantly, miR-K10a taps into an evolutionary conserved network of miR-142-3p targets, several of which are expressed in 3T3 cells and are also known inhibitors of cellular transformation. In summary, our studies of miR-K10a serve as an example of an unsuspected function of a miRNA whose precursor is embedded within a coding transcript. In addition, our identification of conserved miR-K10a targets that limit transformation will point the way to a better understanding of the role of this miRNA in KSHV-associated tumors. Kaposi's Sarcoma-associated herpesvirus (KSHV) is a human tumor virus. The viral Kaposin locus has known oncogenic potential, which has previously been ascribed to the encoded KapA protein. Here we show that the virally encoded miR-K10a miRNA, whose precursor overlaps the KapA coding region, may account for the oncogenic properties of this locus. Our data suggest that miR-K10a mimics the cellular miRNA miR-142-3p and thereby represses several known inhibitors of oncogenic transformation. Our work demonstrates that functional properties ascribed to a coding region may in fact be carried out by an embedded non-coding element and sheds light on the functions of viral miR-K10a. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Journal of Virology 12/2014; DOI:10.1128/JVI.03317-14 · 4.65 Impact Factor

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