Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target GADD45B To Protect Infected Cells from Cell Cycle Arrest and Apoptosis

Abstract

Kaposi's sarcoma is one of the most common malignancies in HIVinfected individuals. The responsible agent, Kaposi's sarcoma-associated herpesvirus (KSHV; HHV8), expresses multiple microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. After infection in primary endothelial cells with KSHV, growth arrest DNA damage-inducible gene 45 beta (GADD45B) is one of the most repressed genes using genomic expression profiling. GADD45B was also repressed in mRNA expression profiling experiments when KSHV miRNAs were introduced to uninfected cells. We hypothesized that KSHV miRNAs target human GADD45B to protect cells from consequences of DNA damage, which can be triggered by viral infection. Expression of GADD45B protein is induced by the p53 activator, Nutlin-3, and KSHV miRNA-K9 inhibits this induction. In addition, Nutlin-3 increased apoptosis and cell cycle arrest based on flow cytometry assays. KSHV miR-K9 protected primary endothelial cells from apoptosis and cell cycle arrest following Nutlin-3 treatment. Similar protective phenotypes were seen for targeting GADD45B with short interfering RNAs (siRNAs), as with miR-K9. KSHV miR-K9 also decreased the protein levels of cleaved caspase-3, cleaved caspase-7, and cleaved poly(ADP-ribose) polymerase (PARP). In B lymphocytes latently infected with KSHV, specific inhibitors of KSHV miR-K9 led to increased GADD45B expression and apoptosis, indicating that 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 GADD45B 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.

Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

“…The requirement for IL-6 during KSHV infection is well documented [30][31][32][33][34][35][36][37][38], but the role of GADD45B may be more nuanced. While we demonstrated that it escapes host shutoff during lytic KSHV infection and appears important for viral reactivation in the iSLK.219 model, GADD45B expression is repressed by viral miRNAs during latency to avoid its cell cycle arrest and pro-apoptotic functions [73]. However, GADD45B has a number of additional activities that could be necessary for the viral lytic cycle.…”

Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

“…The requirement for IL-6 during KSHV infection is well documented [30][31][32][33][34][35][36][37][38], but the role of GADD45B may be more nuanced. While we demonstrated that it escapes host shutoff during lytic KSHV infection and appears important for viral reactivation in the iSLK.219 model, GADD45B expression is repressed by viral miRNAs during latency to avoid its cell cycle arrest and pro-apoptotic functions [73]. However, GADD45B has a number of additional activities that could be necessary for the viral lytic cycle.…”

C19ORF66 Broadly Escapes Virus-Induced Endonuclease Cleavage and Restricts Kaposi’s Sarcoma-Associated Herpesvirus

“…Functionally, while the beneficial role of IL-6 for KSHV during infection is well documented (25)(26)(27)(28)(29)(30)(31)(32)(33), the role of GADD45B is still unclear. In fact, GADD45B is repressed during KSHV latency (34), and GADD45B known proapoptotic roles may indicate that this transcript escapes to participate in an antiviral response to host shutoff.…”