Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANAinteracting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Multifunctional Angiogenin To Utilize Its Antiapoptotic Functions

“…LANA-1 also modifies transcriptional activity by altering the subcellular distribution of glycogen synthase kinase 3␤ (GSK-3␤), a negative regulator of ␤-catenin (13). Proteomic approaches have identified several additional LANA-1-interacting host proteins (6,20,31).…”

Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication

“…I-DIRT analyses defined 4 of these proteins as having specific mLANA interactions, while 15 proteins were scored as having nonspecific interactions ( Table 1). Interactors that were both conserved and specific included Hsc70, histone H1.2, EF1A1, and ␤-actin (8,39,40). Together, these data reveal a set of cellular factors that interact with mLANA during lytic MHV68 infection, prioritize protein-protein interactions through I-DIRT, evaluate the conserved nature of their interaction with kLANA, and organize them into functional networks.…”

“…Of note, 19 mLANA-binding cellular proteins were previously reported as kLANA-interacting factors in other proteomics-based interaction screens (Table 1) (8,(39)(40)(41)(42)(43)64). I-DIRT analyses defined 4 of these proteins as having specific mLANA interactions, while 15 proteins were scored as having nonspecific interactions ( Table 1).…”

“…Also, kLANA interacts with annexin A2 and angiogenin to prevent apoptosis in TIVE-LTC cells (40). Although protein-protein interaction studies for kLANA have focused on elucidating interacting factors in latently infected cells or through peptide arrays utilizing various affinity purification techniques (39,42), the overlap with mLANA interactors observed during lytic replication suggests that LANA homologs may exploit the functions of such factors in both lytic and latent stages of infection. For example, hnRNPK and other hnRNP proteins, Hsc70, dead-box helicases DHX3 and DHX9, and histone H1 identified in this study (Table 1; see Tables S1 and S2 in the supplemental material) associate with KSHV terminal repeats (TRs) in latently infected cells (22,72).…”

“…Since interactions with viral and/or cellular factors are hypothesized to modulate LANA-regulated processes, recent studies have employed proteomics approaches to identify host and/or viral proteins that interact directly with kLANA (38)(39)(40)(41)(42)(43). These studies have focused on defining roles for such interactions in latent KSHV infection.…”