Deregulation of signal transduction pathways by the latent viral oncoproteins of Kaposi's sarcoma herpesvirus (KSHV/HHV-8)

Kaposi's sarcoma herpesvirus (KSHV/HHV-8) is a lymphotrophic gamma2-herpesvirus that is etiologically associated with Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL). PEL is a rare subtype of non-Hodgkin's lymphoma that is characterized by lymphomatous effusions in serous cavities often without a distinct solid tumor mass. Most PELs exhibit coinfection with the Epstein-Barr virus (EBV). KSHV establishes a restricted latency in PEL cells, and several latent viral proteins subvert signaling cascades to increase cellular proliferation and survival. We report deregulation of c-Myc and reactive oxygen species (ROS) pathways by the latent KSHV oncoproteins, the latency-associated nuclear antigen (LANA) and viral FLICE-inhibitory protein (vFLIP). Cycloheximide experiments demonstrated that PEL cells have abnormally stable c-Myc protein, and LANA is responsible for this aberration. Using RNA interference we showed that the mechanism of c-Myc stabilization involves LANA's inhibition of glycogen synthase-3beta (GSK-3beta)-mediated phosphorylation of c-Myc on threonine 58, a residue crucial for its proteasomal degradation. Co-imunoprecipitation studies established that c-Myc and LANA are present in the GSK-3beta-containing complexes in PEL cells. Furthermore, our data indicate that KSHV also increases c-Myc transcription through NF-kappaB activation by vFLIP.; Given that viruses can induce ROS production, and that ROS have been implicated in tumorigenesis, we investigated the effect of KSHV on the cellular redox status. Our data showed that PEL cells have higher levels of intracellular ROS than uninfected lymphoma cells. vFLIP could potently induce ROS in a doxycycline-inducible Burkitt lymphoma system. RNA interference studies in PEL cells demonstrated that suppression of endogenous vFLIP leads to a decrease in ROS levels, and tumor necrosis associated factor (TRAF)-mediated pathways might play a role in ROS generation. Inhibition of ROS by antioxidants resulted in decreased PEL constitutive NF-kappaB activity. As PEL cells depend on NF-kappaB for survival, antioxidants may be useful for the treatment of KSHV-associated malignancies.; Finally, we used microarray analysis to determine the viral impact of KSHV and EBV on gene expression in PELs. Our data showed that KSHV plays an important role in the pathogenesis of PEL, as its presence selects for a very distinct cellular gene expression category.