Human Herpesvirus-8 (HHV-8/KSHV) Induces Reactive Oxygen Species in Endothelial Cells That Facilitate Virus Infection.

Reactive oxygen species (ROS) can activate replication of certain viruses, induce the production of various inflammatory mediators and play a critical role in carcinogenesis and tumor development. Kaposi’s sarcoma (KS) is the most prevalent HIV-associated cancer and is caused by infection with human herpesvirus-8 (HHV-8/KSHV). KS tissue has been reported to possess increased levels of ROS. We studied if ROS generation is related to HHV-8 infection and its role in virus entry into endothelial cells. Incubation of dermal microvascular endothelial cells (DMECs) with highly purified HHV-8 induced rapid increases in the production of intracellular hydrogen peroxide (H₂O₂), one of the major forms of ROS. Intracellular H₂O₂ was also induced by the treatment of DMECs with the HHV-8 envelope glycoprotein B (gB). The gB protein possesses an RGD motif, binds to the integrin molecules, alpha3 and beta1, and is a major mediator of virus entry into target cells. To test if it was integrin ligation that induces the production of ROS, we treated DMECs with fibronectin or laminin, the respective natural ligands for alpha3 and beta1 integrins. We observed a similar induction of intracellular ROS in DMECs by either matrix protein. These results indicated that the HHV-8-induced production of ROS was, at least in part, mediated by stimulation of integrins through the RGD-containing viral gB protein. ROS have recently been shown to function as second messengers in cellular signaling. To assess at which steps of cell signaling ROS may be functioning, we studied the signaling cascade in DMECs activated by the HHV-8 gB protein. Previous studies have shown that HHV-8, through gB/integrin interaction, induces cytoskeletal rearrangement and activates focal adhesion kinase (FAK), Src kinase and Akt, which are critical for virus entry into the target cells. We found that the activation of FAK, c-Src or Akt by this viral protein was inhibited by pretreatment with N-acetyl-L-cysteine (NAC), a potent thiol antioxidant. These results suggested that generation of ROS was involved in HHV-8-triggered signaling. We next examined if a change in ROS production modulated HHV-8 virus entry. We used green fluorescent protein (GFP)-labeled HHV-8 at a multiplicity of infection of 5–6, and quantitated the infection by fluorescence analysis of the DMECs. Short term exposure to low concentrations of H₂O₂ enhanced HHV-8 infection in DMECs, while treatment with NAC significantly decreased infection. These data indicated that ROS generation participated in HHV-8-mediated signaling and entry into target cells. Our study demonstrates a novel role of ROS in virus pathogenesis and provides a framework for the development of novel antioxidant strategies in AIDS-KS treatment.