[PMC free article] [PubMed] [CrossRef] [Google Scholar] 30

[PMC free article] [PubMed] [CrossRef] [Google Scholar] 30. miR-K9 is important for reducing apoptosis in infected cells. Furthermore, ectopic expression of GADD45B in KSHV-infected cells promoted apoptosis. Together, these results identify a new miRNA target and demonstrate that KSHV miRNAs are important for protecting infected cells from DNA damage responses. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus is a leading cause of cancers in individuals with AIDS. Promoting survival of infected cells is essential for maintaining viral infections. A virus needs to combat various cellular defense mechanisms designed to eradicate the viral infection. One such response can include DNA damage response factors, which can promote an arrest in cell growth and trigger cell death. We used a new approach to search for human genes repressed by small nucleic acids (microRNAs) expressed by a gammaherpesvirus (KSHV), which identified a gene called as a target of microRNAs. Repression of GADD45B, which is expressed in response to DNA damage, benefited survival of infected cells in response to a DNA damage response. This information could be used to design new treatments for herpesvirus infections. (family proteins are commonly repressed in multiple types of cancers (10). This family of proteins can cooperate to repress cell growth in response to various stress inducers Eslicarbazepine (11, 12). GADD45B can also regulate inflammatory responses from interleukin-6 (IL-6), IL-18, and IL-12, tumor necrosis factor (TNF), and transforming growth factor 1 (TGF-1) (13,C16). Furthermore, can be induced by the innate immune activator lipopolysaccharide (LPS) (17). GADD45B has also been shown to be important for production of gamma interferon in response to cytokines (14). Mice deficient for GADD45B have granulocytes and macrophages that are defective in their chemotactic responses to lipopolysaccharide and IL-8 (18). Hypoxia, which is an inducer of GADD45B expression (19), can also stimulate lytic replication in KSHV infections (20). Eslicarbazepine KSHV infection can also upregulate hypoxia inducible factor (HIF), and both hypoxia and HIF-2 have been shown to induce GADD45B expression (21). Here, we report that is targeted by multiple KSHV miRNAs for repression. Eslicarbazepine We show that repression of by KSHV miRNAs can inhibit apoptosis induced by a p53 activator, Nutlin-3. In Eslicarbazepine the context of KSHV infection, both an antisense inhibitor of a specific KSHV miRNA and ectopic expression of GADD45B promote apoptosis. These results suggest that some KSHV miRNA functions include modulating DNA damage response factors to promote survival of infected cells in the face of stress signals. RESULTS GADD45B expression is repressed by KSHV infection and specific KSHV miRNAs. We utilized our previously published data sets that investigated changes in human gene expression in response to KSHV infection or in separate assays with cells transfected with KSHV miRNA mimics. We focused on mRNA expression changes after KSHV infection (22) or after transfection of a pool of KSHV miRNAs (23) and found that the (suggested that this gene was directly targeted for repression by KSHV miRNAs. Transfection of individual KSHV miRNAs in primary endothelial cells revealed that multiple KSHV miRNAs repressed endogenous GADD45B protein (Fig. 1B and ?andCC). Open in a separate window FIG 1 GADD45B expression is repressed by KSHV infection and specific KSHV miRNAs. (A) Microarray data were analyzed for changes after KSHV infection (48 h) or transfection of KSHV miRNAs (30 h). Average expression changes are shown from the two conditions and sorted by expression change. The arrow shows the location of the probe corresponding to GADD45B. (B) Primary endothelial cells were transfected with individual miRNA mimics and harvested 48 h after transfection. Protein expression changes of GADD45B (normalized to the loading control beta-actin) were obtained by immunoblotting using fluorescently labeled secondary antibodies and normalized to a nontargeting negative-control miRNA (miR-Neg). ?, value of <0.05 compared to miR-Neg using the Student test. (C) A representative Western blot is shown for GADD45B (18 kDa) and beta-actin (45 kDa). The loading control was Eslicarbazepine beta-actin. KSHV miRNAs target the 3UTR of for miRNA PPARG1 seed-matching sequences revealed multiple potential target sites for KSHV miRNAs (Fig. 2A). We.