A Kaposi's Sarcoma-Associated Herpesvirus MicroRNA and Its Variants Target the Transforming Growth Factor β Pathway To Promote Cell Survival

Transforming growth factor β (TGF-β) signaling regulates cell growth and survival. Dysregulation of the TGF-β pathway is common in viral infection and cancer. Latent infection by Kaposi''s sarcoma-associated herpesvirus (KSHV) is required for the development of several AIDS-related malignancies, including Kaposi''s sarcoma and primary effusion lymphoma (PEL). KSHV encodes more than two dozen microRNAs (miRs) derived from 12 pre-miRs with largely unknown functions. In this study, we show that miR variants processed from pre-miR-K10 are expressed in KSHV-infected PEL cells and endothelial cells, while cellular miR-142-3p and its variant miR-142-3p_-1_5, which share the same seed sequence with miR-K10a_ +1_5, are expressed only in PEL cells and not in uninfected and KSHV-infected TIME cells. KSHV miR-K10 variants inhibit TGF-β signaling by targeting TGF-β type II receptor (TβRII). Computational and reporter mutagenesis analyses identified three functional target sites in the TβRII 3′ untranslated region (3′UTR). Expression of miR-K10 variants is sufficient to inhibit TGF-β-induced cell apoptosis. A suppressor of the miRs sensitizes latent KSHV-infected PEL cells to TGF-β and induces apoptosis. These results indicate that miR-K10 variants manipulate the TGF-β pathway to confer cells with resistance to the growth-inhibitory effect of TGF-β. Thus, KSHV miRs might target the tumor-suppressive TGF-β pathway to promote viral latency and contribute to malignant cellular transformation.     

KSHV MicroRNAs Repress Tropomyosin 1 and Increase Anchorage-Independent Growth and Endothelial Tube Formation

Kaposi’s sarcoma (KS) is characterized by highly vascularized spindle-cell tumors induced after infection of endothelial cells by Kaposi’s sarcoma-associated herpesvirus (KSHV). In KS tumors, KSHV expresses only a few latent proteins together with 12 pre-microRNAs. Previous microarray and proteomic studies predicted that multiple splice variants of the tumor suppressor protein tropomyosin 1 (TPM1) were targets of KSHV microRNAs. Here we show that at least two microRNAs of KSHV, miR-K2 and miR-K5, repress protein levels of specific isoforms of TPM1. We identified a functional miR-K5 binding site in the 3’ untranslated region (UTR) of one TPM1 isoform. Furthermore, the inhibition or loss of miR-K2 or miR-K5 restores expression of TPM1 in KSHV-infected cells. TPM1 protein levels were also repressed in KSHV-infected clinical samples compared to uninfected samples. Functionally, miR-K2 increases viability of unanchored human umbilical vein endothelial cells (HUVEC) by inhibiting anoikis (apoptosis after cell detachment), enhances tube formation of HUVECs, and enhances VEGFA expression. Taken together, KSHV miR-K2 and miR-K5 may facilitate KSHV pathogenesis.     

Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target IRAK1 and MYD88, Two Components of the Toll-Like Receptor/Interleukin-1R Signaling Cascade,To Reduce Inflammatory-Cytokine Expression

Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3′-untranslated region (3′UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3′UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.     

Kaposi's sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.     

A microRNA Encoded by Kaposi Sarcoma-Associated Herpesvirus Promotes B-Cell Expansion In Vivo

The human gammaherpesvirus Kaposi sarcoma-associated herpesvirus is strongly linked to neoplasms of endothelial and B-cell origin. The majority of tumor cells in these malignancies are latently infected, and latency genes are consequently thought to play a critical role in virus-induced tumorigenesis. One such factor is kshv-miR-K12-11, a viral microRNA that is constitutively expressed in cell lines derived from KSHV-associated tumors, and that shares perfect homology of its seed sequence with the cellular miR-155. Since miR-155 is overexpressed in a number of human tumors, it is conceivable that mimicry of miR-155 by miR-K12-11 may contribute to cellular transformation in KSHV-associated disease. Here, we have performed a side-by-side study of phenotypic alterations associated with constitutive expression of either human miR-155 or viral miR-K12-11 in bone marrow-derived hematopoietic stem cells. We demonstrate that retroviral-mediated gene transfer and hematopoietic progenitor cell transplantation into C57BL/6 mice leads to increased B-cell fractions in lymphoid organs, as well as to enhanced germinal center formation in both microRNA-expressing mouse cohorts. We furthermore identify Jarid2, a component of Polycomb repressive complex 2, as a novel validated target of miR-K12-11, and confirm its downregulation in miR-K12-11 as well as miR-155 expressing bone marrow cells. Our findings confirm and extend previous observations made in other mouse models, and underscore the notion that miR-K12-11 may have arisen to mimic miR-155 functions in KSHV-infected B-cells. The expression of miR-K12-11 may represent one mechanism by which KSHV presumably aims to reprogram naïve B-cells towards supporting long-term latency, which at the same time is likely to pre-dispose infected lymphocytes to malignant transformation.     

Divergent target recognition by coexpressed 5′-isomiRs of miR-142-3p and selective viral mimicry

Sequence heterogeneity at the ends of mature microRNAs (miRNAs) is well documented, but its effects on miRNA function are largely unexplored. Here we studied the impact of miRNA 5′-heterogeneity, which affects the seed region critical for target recognition. Using the example of miR-142-3p, an emerging regulator of the hematopoietic lineage in vertebrates, we show that naturally coexpressed 5′-variants (5′-isomiRs) can recognize largely distinct sets of binding sites. Despite this, both miR-142-3p isomiRs regulate exclusive and shared targets involved in actin dynamics. Thus, 5′-heterogeneity can substantially broaden and enhance regulation of one pathway. Other 5′-isomiRs, in contrast, recognize largely overlapping sets of binding sites. This is exemplified by two herpesviral 5′-isomiRs that selectively mimic one of the miR-142-3p 5′-isomiRs. We hypothesize that other cellular and viral 5′-isomiRs can similarly be grouped into those with divergent or convergent target repertoires, based on 5′-sequence features. Taken together, our results provide a detailed characterization of target recognition by miR-142-3p and its 5′-isomiR-specific viral mimic. We furthermore demonstrate that miRNA 5′-end variation leads to differential targeting and can thus broaden the target range of miRNAs.     

Kaposi's sarcoma-associated herpesvirus-encoded microRNA miR-K12-11 attenuates transforming growth factor beta signaling through suppression of SMAD5

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs (pre-miRNAs). Current studies have shown that these miRNAs are involved in regulation of viral and host gene expression, implicating a role in the maintenance of viral latency and suppression of antiviral innate immunity. However, the functions of these miRNAs remain largely unknown. On the basis of the sequence homology between oncogenic miR-155 and KSHV-encoded miR-K12-11, we hypothesized that miR-K12-11 could attenuate transforming growth factor β (TGF-β) signaling, facilitating viral infection and tumorigenesis. In the present study, we demonstrated that ectopic expression of miR-K12-11 in Ramos, a TGF-β-sensitive cell line, downregulated TGF-β signaling and facilitated cell proliferation upon TGF-β treatment by directly targeting SMAD5, an important mediator in TGF-β signaling. In addition, the downregulation of SMAD5 by miR-K12-11 was further confirmed in a de novo KSHV infection system or latently infected KSHV-positive B-lymphoma cell lines. More importantly, repression of miR-K12-11 by a specific sponge inhibitor restored the expression of SMAD5 in both de novo-infected and latently infected cells. Finally, we found that restoration of SMAD5, in addition to the TGF-β type II receptor, which was epigenetically silenced by the latent viral protein latency-associated nuclear antigen, sensitized BC3 cells to the cytostatic effect of TGF-β signaling. Taken together, our findings highlight a novel mechanism in which miR-K12-11 downregulates TGF-β signaling and suggest that viral miRNAs and proteins may exert a dichotomy regulation in virus-induced oncogenesis by targeting the same signaling pathway.