Post-Translational Modifications of Kaposi's Sarcoma-Associated Herpesvirus Regulatory Proteins - SUMO and KSHV

Abstract

KSHV latency can be envisioned as an outcome that is balanced between factors that promote viral gene expression and lytic replication against those that facilitate gene silencing and establish or maintain latency. A large body of work has focused on the activities of the key viral regulatory proteins involved in KSHV latent or lytic states. Moreover, recent studies have also begun to document the importance of epigenetic landscape evolution of the KSHV viral genome during latency and reactivation. However, one area of KSHV molecular virology that remains largely unanswered is the precise role of post-translational modifications on the activities of viral factors that function during latency and reactivation. In this review, we will summarize the post-translational modifications associated with three viral factors whose activities contribute to the viral state. The viral proteins discussed are the two major KSHV encoded transcription factors, K-Rta (KSHV replication and transcriptional activator) and K-bZIP (KSHV basic leucine zipper) and the viral latency-associated nuclear antigen (LANA). A special emphasis will be placed on the role of the sumoylation pathway in the modulation of the KSHV lifecycle. Newly uncovered small ubiquitin-like modifier (SUMO)-associated properties of LANA and K-Rta will also be presented, namely LANA histone targeting SUMO E3 ligase activity and K-Rta SUMO-targeted ubiquitin ligase function.

Keywords: K-Rta; K-bZIP; KSHV; LANA; SUMO; post-translational modification; transcription.

Figure 1

Schematic representation of KSHV K-bZIP. K-bZIP protein and its post-translational modification sites as discussed in the text are depicted. Phosphorylation (Phospho), acetylation (Ac), and sumoylation (SUMO) sites are shown. The K-bZIP SUMO interaction motif (SIM) and nuclear localization signal (NLS) are also indicated. Numbers indicate amino acid position.

Figure 2

Schematic representation of KSHV K-Rta. K-Rta protein and its post-translational modification sites as discussed in the text are depicted. Phosphorylation (Phospho) site numbering and PARylation (PAR) site localization of Gwack et al. (2003) is utilized. The K-Rta nuclear localization signals (NLS-1 and NLS-2), ring-finger like domain, SUMO interaction motifs (SIMs), and serine/threonine (S/T) rich regions are also indicated. Numbers indicate amino acid position.

Figure 3

SUMO-targeting ubiquitin ligase activity of KSHV K-Rta. (A) 293T cells were co-transfected with his-tagged SUMO-2 and the indicated K-Rta expression vector. Forty-eight hours later cellular lysates were prepared and analyzed by immunoblotting using the indicated antibodies. (H145L, ring-finger like domain mutant; ΔSIM, SIM deletion mutant). (B) In vitro ubiquitin conjugation reactions were reconstituted using purified components (E1, Ube1, 25 nM; E2, Ubc H5a, 50 nM; E3, K-Rta, 150 nM). Reaction products were probed by immunoblotting. In the presence of K-Rta, GST–SUMO was conjugated with ubiquitin. (C) KSHV promoter reporter assay. 293T cells were co-transfected with K-Rta wt or mutant and the indicated KSHV promoter luciferase reporter. Forty eight hour later cellular lysates were prepared and assayed for luciferase activity. Values represent the mean fold induction (mean ± SD, n = 3 determinations) relative to the value derived from reporter + empty vector set as = 1 (K-Rta wt, blue; H145L, red; ΔSIM, yellow).

Figure 4

Schematic representation of KSHV LANA. LANA protein and its post-translational modification sites as discussed in the text are depicted. Phosphorylation (Phospho) sites and the arginine methylation (Me) site are shown. The N-terminal histone binding domain, nuclear localization signal (NLS) and SUMO interaction motif (SIM) are also indicated. Numbers indicate amino acid position.

Figure 5

Latency-associated nuclear antigen enhances histone sumoylation in vitro and in vivo. (A) LANA enhances histone H2A and H2B SUMO-modification in vitro. The in vitro SUMO conjugation reaction was performed with purified histone octamer (5 μg; Roche) as a substrate. SAE1/2 (E1, 50 nM), Ubc9 (E2, 50 nM), and HA-SUMO-2 (50 μM) were incubated in reaction buffer containing 1 mM ATP and MgCl₂ with or without a catalytic amount of LANA (50 nM). In the presence of LANA, H2A, and H2B SUMO-modification is enhanced. The arrows indicate SUMO-modified histones, which were increased in SUMO-reaction mixtures supplemented with LANA (three lanes from left). Full-length LANA Wt and mutant proteins were prepared with recombinant baculoviruses (B) and used as an E3 SUMO-ligase for in vitro SUMO conjugation reactions. CBB, Coomassie brilliant blue. (C) Histone binding domain and SUMO-interacting motif of LANA are necessary for its SUMO-ligase function. LANA Wt but neither a histone binding mutant (HB Mut) or SUMO-binding mutant (SIM Mut) can catalyze SUMO-modification of histones. The corresponding number of SUMO-chains is indicated on the right (20 kDa increments). LANA ring-finger like domain is dispensable for LANA-mediated SUMO conjugation of histones (Ring Mut). (D) Correlation between LANA recruitment sites and SUMO-enriched loci on the latent KSHV genome. ChIP analyses were performed with anti-LANA or anti-SUMO-2 antibodies using latent BCBL-1 cells. ChIP with LANA antibody was hybridized with custom-made KSHV tiling arrays and SUMO-ChIP was directly analyzed by sequencing (ChIP-seq). Enrichment over input DNA is shown (upper panel). Counts and position of sequence reads of SUMO-ChIP are shown in the bottom panel. SUMO-2 is enriched where LANA is recruited. Annotation of the KSHV genome is shown in the middle panel. The KSHV terminal repeat region is located at the extreme right of the annotated genome. (E) cDNA microarray analyses. RNA was prepared from indicated LANA stable cells or vector control stable cells and hybridized to Affymetrix human whole genome U133 plus 2.0 arrays. Raw data was subjected to baseline transformation and analyzed by hierarchical clustering using GeneSpring GX11 software. Genes highly repressed by LANA wt but not mutants are indicated by blue vertical lines to the right.

Figure 6

KSHV SUMO-dependent balance. The outcome of the KSHV life cycle is depicted as a balance between the functions of the viral factors LANA, K-Rta, and K-bZIP which are mediated through their SUMO-dependent activities. The SUMO E3 ligase activities of K-bZIP and LANA and the STUbL activity of K-Rta are shown (see text for details).