Human Immunodeficiency Virus Type 1 Induces Lytic Cycle Replication of Kaposi's-Sarcoma-Associated Herpesvirus: Role of Ras/c-Raf/MEK1/2, PI3K/AKT, and NF-κB Signaling Pathways

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection significantly increases the risk and development of Kaposi's sarcoma (KS) in individuals infected with KS-associated herpesvirus (KSHV). Previously, we reported that HIV-1 Tat protein induced KSHV replication by modulating the Janus kinase/signal transducers and activators of transcription signaling pathway. Here, we further investigated the possible signaling pathways involved in HIV-1-induced reactivation of KSHV. We showed that HIV-1 infection of primary effusion lymphoma cell lines triggered the reactivation of KSHV, as demonstrated by the expression of KSHV replication and transcription activator, the early viral lytic protein vIL-6 and ORF59 and the production of progeny virions. By utilizing microarray gene expression analyses, transfecting a series of dominant negative mutants, and adding pharmacologic inhibitors, we identified a group of diverse cellular signaling proteins and found that HIV-1 infection of BCBL-1 cells activated phosphatidylinositol 3-kinase/AKT (also called protein kinase B, PKB) pathway and inactivated phosphatase and tensin homolog deleted on chromosome ten and glycogen synthase kinase-3β, which partially modulated HIV-1-induced KSHV reactivation. Furthermore, activation of Ras/c-Raf/MAPK/ERK kinase1/2 pathway contributed to HIV-1-induced KSHV replication. Finally, we discovered that HIV-1 infection activated nuclear factor κB signaling, which exhibits an inhibitory effect on KSHV reactivation in BCBL-1 cells. Collectively, our data demonstrated that HIV-1 infection stimulated these cell signaling pathways that, in turn, contributed to KSHV reactivation, which may be of therapeutic value in acquired immunodeficiency syndrome-related KS patients.

Introduction

Kaposi's-sarcoma-associated herpesvirus (KSHV) is a human γ₂-herpesvirus that is causally linked to the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and some forms of multicentric Castleman's disease.¹ All herpesviruses, including KSHV, have two distinct phases in their life cycle: latency and lytic replication. In latent infection, the viral genome persists extrachromosomally as a circular episome, with viral gene expression severely restricted, and viral progeny is not produced. During reactivation, a variety of KSHV lytic cycle genes are expressed and viral progeny are produced, which ultimately results in host cell lysis.2, 3 Previous studies have shown that some latency-associated viral genes, including latency-associated nuclear antigen and viral Fas-associated protein with death-domain-like interleukin-1β-converting enzyme/caspase-8-inhibitory protein (vFLIP), contributed to the oncogenesis of KSHV.4, 5 However, lytic replication was also known to play a crucial role in KS pathogenesis. It was likely that lytic cycle replication led to efficient viral transmission and altered the balance between clearance of infected cells by the host immune system and infection of new cells by virus, which accelerated the spread and infection of KSHV. However, the mechanism of the reactivation of KSHV remains to be demonstrated.

We and others showed that several agents, such as human herpesvirus 6, herpes simplex virus type 1, human immunodeficiency virus type 1 (HIV-1), and human cytomegalovirus, which are commonly found in immunocompromised individuals, have been considered as cofactors that reactivate KSHV.6, 7, 8, 9, 10, 11 Among these cofactors, HIV-1 was a potentially important cofactor. A few soluble viral proteins and virions of HIV-1 could influence KSHV lytic replication and tumorigenesis. For instance, our recent studies indicated that HIV-1 Tat not only induced KSHV replication by modulating Janus kinase/signal transducers and activators of transcription pathway but also accelerated KSHV Kaposin-A-mediated tumorigenesis of transformed fibroblasts in vitro and in vivo.12, 13 Although pseudotyped and wild-type HIV-1 infection of BC-3 cells also triggered KSHV replication,7, 8, 14 the possible mechanisms for viral replication are not well defined.

Cellular signaling pathways are critical in regulating the balance between latency and lytic replication of KSHV. For example, induction of KSHV reactivation by 12-O-tetradecanoyl-phorbol-13-acetate was partially mediated by calcineurin signaling and protein kinase C pathway.15, 16 Inhibition of the AKT (also called protein kinase B, PKB) pathway facilitated the reactivation of KSHV from latency in BC-3 cells.¹⁷ Although the role of HIV-1 in KSHV reactivation has been extensively studied, the signaling pathways involved in the process remain unclear. To address this issue, in this study, we used vesicular stomatitis virus glycoprotein (VSV-GP)-pseudotyped HIV-1 to introduce the virus genome into the PEL cell lines and observed HIV-1-induced KSHV replication. We showed that complicated modulation of multiple signaling pathways played an important role in HIV-1-induced reactivation of KSHV.