Is Essential for of and Is a Target of an Immediate Early Protein during Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation.

Bivalent histone modiﬁcations are deﬁned as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression, while the H3K4me3 mark prevents the region from be-coming permanently silenced and prepares the domain for activation when needed. Speciﬁc regions of Kaposi’s sarcoma-associated herpesvirus (KSHV) latent episomes are poised to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function; thus, we hypothesized that identiﬁcation of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication. Here we show that a zinc ﬁnger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquiti-nate ZIC2. ZIC2 localized at immediate early and early gene cluster regions of the KSHV genome and contributed to tethering of polycomb repressive complex 2 through physical interaction, thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted the balance of bivalent histone modiﬁcations toward more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication. IMPORTANCE Posttranslational histone modiﬁcations regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reacti-vatable by K-Rta remains unclear. We found that ZIC2, a transcriptional factor essential for stem cell pluripotency, plays a role in maintaining KSHV latent infection in naturally infected cells. We found that ZIC2 degradation by K-Rta shifts bivalent histone marks to a more active conﬁguration, leading to KSHV reactivation. ZIC2 interacts with and maintains polycomb repressor complex 2 at the K-Rta promoter. Our ﬁndings uncover (i) a mechanism utilized by KSHV to maintain latent infection, (ii) a latency-lytic cycle switch operated by K-Rta, and (iii) a molecular mechanism of ZIC2-mediated local histone modiﬁcation. cofactors to maximize lytic gene expression. puromycin, 1200 (cid:3) g/ml hygromycin B, and 800 (cid:3) g/ml G418. Jung (University of Southern California) the TREx-F3H3-K-Rta BCBL-1 cell line and Don Ganem (Novartis Institutes for Biomedical Research) for the BCBL-1 and iSLK.219 cell lines. 293 (ATCC) and 293T (ATCC) cells were grown in DMEM containing 10% FBS. Plasmids. Plasmids expressing the K-Rta wild type and mutants were described previously (20). For construction of pcDNA3.1-ZIC2, a codon-optimized and shRNA-resistant ZIC2 cDNA was synthesized (Integrated DNA Technologies) and cloned into pcDNA3.1 with FLAG or HA epitope tags. For construc- tion of the ZIC2-expressing lentivirus vector, the same cDNA was cloned into the pLenti-C-Myc-DDK vector (Origene) at the MluI and SgfI sites. For protein expression in insect cells, the cDNAs of interest were cloned into the pFastBac vector (Invitrogen). of TREx-K-Rta His-ubiquitin 293 cells. The inducible pcDNA5 FRT/TO FLAG (cid:7) 3-HA (cid:7) 3- K-Rta expression construct was ﬁrst generated by cloning of 3 (cid:7) FLAG and 3 (cid:7) HA tandem epitope tags fused to the N-terminal region of K-Rta. Inducible 293 cells were subsequently generated by cotrans- fection of pcDNA5 FRT/TO FLAG (cid:7) 3-HA (cid:7) 3-K-Rta and pOG44 (Flp recombinase) into Flp-In TREx-293 cells following the manufacturer’s protocol (Invitrogen). The K-Rta-inducible 293 cells were then used to create cells stably expressing His-ubiquitin by transfection of pcDNA-His-ubiquitin. Stable clones were selected in the presence of zeocin for 3 weeks. Several clones were picked, and His-ubiquitin expression in the presence or absence of MG132 (10 (cid:3) g/ml) was conﬁrmed. Multiple clones were then pooled and used for these studies. and HA-ubiquitin were obtained from a commer- cial source (Boston Biochem). FLAG-ZIC2 was used as a substrate. Ubiquitin conjugation reactions were performed using a ﬁnal protein concentration of E1 of 60 nM, E2 of 300 nM, E3 (K-Rta) of 100 nM, and ubiquitin of 10 (cid:3) M in ubiquitin conjugation reaction buffer supplemented with an energy regeneration solution (Boston Biochem) for 2.5 h at 37°C. Statistical analysis. Results standard deviations from at least three independent experiments. Data unpaired