KSHV and the Role of Notch Receptor Dysregulation in Disease Progression

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of two human cancers, Kaposi's Sarcoma (KS) and primary effusion lymphoma (PEL), and a lymphoproliferation, Multicentric Castleman's Disease (MCD). Progression to tumor development in KS is dependent upon the reactivation of the virus from its latent state. We, and others, have shown that the Replication and transcriptional activator (Rta) protein is the only viral gene product that is necessary and sufficient for viral reactivation. To induce the reactivation and transcription of viral genes, Rta forms a complex with the cellular DNA binding component of the canonical Notch signaling pathway, recombination signal binding protein for Jk (RBP-Jk). Formation of this Rta:RBP-Jk complex is necessary for viral reactivation to occur. Expression of activated Notch has been shown to be dysregulated in KSHV infected cells and to be necessary for cell growth and disease progression. Studies into the involvement of activated Notch in viral reactivation have yielded varied results. In this paper, we review the current literature regarding Notch dysregulation by KSHV and its role in viral infection and cellular pathogenesis.

Keywords: HHV8; KSHV; RBP-Jk; Rta; kaposi’s sarcoma; multicentric castleman’s disease; notch; primary effusion lymphoma.

Figure 1

Mature Kaposi’s sarcoma-associated herpesvirus (KSHV) virion structure. The 160–170 kDa linear double stranded DNA genome is surrounded by an icosahedral capsid. This capsid is composed of viral proteins ORF62, 26, 65, and decorated with ORF25 proteins (gold ovals). The capsid is connected to the envelope (blue) via the tegument layer, which contains the viral proteins ORF 11, 21, 33, 45, 50, 52, 63, 75, (green ovals) and 64 (green rectangles). The envelope consists of a lipid bilayer decorated with viral glycoproteins (gM, gN, gL, gH, gB, K8.1 (blue lollipops)) used for the binding and entry of target cells.

Figure 2

KSHV infection of target cells. Glycoproteins on the surface of the virion bind to heparan sulfate, dendritic cell specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and integrin receptors on the surface of host cells. This interaction between the virus and the host induces a signaling cascade that recruits proteins, such as myosin and dynein (blue lines), that are required for endocytosis and the transport of viral DNA to the nucleus. Once delivered to the nucleus, viral and host cell genes are expressed to help subvert host immunity and establish latent infection.

Figure 3

KSHV egress. Viral DNA is packaged into preformed capsid. At the inner nuclear membrane (brown), the virus acquires a primary envelope which is lost during fusion with the outer nuclear membrane (dark purple). As the encapsidated viral DNA moves through the cytoplasm it acquires tegument proteins which allow for entry into the Golgi apparatus (green). The virus acquires its final envelope by budding into Golgi-derived vesicles. These vesicles fuse with the plasma membrane, releasing the mature virion into the extracellular space.

Figure 4

During the latent phase of the KSHV life cycle, a subset of viral genes (pink box) are expressed which maintain latency and allow the virus to subvert the host immune system. Environmental triggers induce reactivation, or the transition from the latent to lytic phase. The lytic phase is induced by cellular transcription factors (TFs, purple circle), which contribute to transcription of viral immediately early genes (maroon box), such as replication and transcriptional activator (Rta, green circle). Rta then induces the transcription of viral delayed early genes (light blue box) known to contribute to viral oncogenesis and viral DNA replication. Expression of the delayed early genes induces viral replication (grey box), which, once complete, triggers the expression of late genes (dark blue box). These late genes are involved in viral packaging. One of these late genes, K8.1, is a glycoprotein that is often used as a marker of viral reactivation. Mature virions are then released from the cell via egress as described previously (Figure 3). IE, immediate early; DE, delayed early.

Figure 5

Protein structure of replication and transcriptional activator (Rta). The Rta protein is 691 amino acids and includes two nuclear localization signals (black arrows/gray boxes), a serine/threonine rich region (green rectangle), a basic amino acid rich region (blue), a leucine heptapeptide repeat domain (yellow), and a region of hydrophobic and acid amino acid repeats (pink). Figure and legend modified with permission from [157].

Figure 6

Model of viral transcription and reactivation by Rta. In the current model of viral reactivation, Rta (green) forms a tetramer and then complexes with cellular RBP-Jk (orange oval). This complex then recognizes RBP-Jk binding sites (orange outlined boxes) located within viral promoters through binding to CANT DNA repeats (blue outlined boxes). Once bound, the Rta:RBP-Jk complex activates the transcription of viral genes, thus leading to viral reactivation. Adapted with permission from [154].

Figure 7

The canonical Notch signaling pathway. Jagged- and Delta-like ligands (dark blue) expressed on the surface of neighboring cells induce the proteolytic cleavage of the Notch extracellular domain (teal) from the intracellular domain (purple) by A Disintegrin and Metalloprotease (ADAM) and γ-secretase (lightning bolts). Following cleavage, the intracellular domain translocates to the nucleus where it associates with RBP-Jk (orange circle) to release the transcriptional co-repressors (red hexagon) and recruit co-activators (green hexagon and circle). These co-activators change the epigenetic landscape, allowing transcription to occur. NICD, Notch intracellular domain.

Figure 8

Summary of Notch’s secondary structure. The four Notch isoforms each consist of an extracellular and an intracellular domain (indicated by black bars). The extracellular domain comprises epidermal growth factor (EGF)-like repeats (pink box), lin-12 Notch repeats (light pink square), and the heterodimerization domain (light green square). While these domains are highly conserved, the four Notch isoforms differ in the number of EGF repeats (29–36). The components of the extracellular domain all play a role in ligand recognition and Notch activation. The intracellular domain of Notch consists of the transmembrane domain (yellow box) which spans the cellular membrane (indicated by orange lines), RBP-Jk association module (RAM) (purple box), ankyryn (magenta box), and proline/glutamic acid/serine/threonine-rich (PEST) (green box) domains. The RAM and ankyryn domains play a key role in interacting with the transcriptional repressor, RBP-Jk, and recruiting other coactivators to the protein complex, while the PEST domain regulates Notch stability. The NICD also contains a nuclear localization signal (gray box), which directs NICD for transport into the nucleus. NICD 1 and 2 both contain a transactivation domain (TAD) that may allow other pathways to regulate Notch activity.