Lavi Ido, Bhattacharya Supriya, Awase Ankita, Orgil Ola, Avital Nir, Journo Guy, Gurevich Vyacheslav, Shamay Meir
Daniella Lee Casper Laboratory in Viral Oncology, Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel.
PLoS Pathog. 2025 Mar 10;21(3):e1012972. doi: 10.1371/journal.ppat.1012972. eCollection 2025 Mar.
Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is associated with several human malignancies. During latency, the viral genomes reside in the nucleus of infected cells as large non-integrated plasmids, known as episomes. To ensure episome maintenance, the latency protein LANA tethers the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes is critical for the proper function of many nuclear processes. To test for recruitment directionality between LANA and cellular proteins, we directed LANA via catalytically inactive Cas9 (dCas9) to a repeat sequence to obtain easily detectable dots. Then, the recruitment of nuclear proteins to these dots can be evaluated. We termed this assay CRISPR-PITA for Protein Interaction and Telomere Recruitment Assay. Using this protein recruitment assay, we found that LANA recruits its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) and the methyl-CpG-binding domain (MBD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1), which interacts with the N-terminal of MeCP2, can recruit MeCP2. We propose that available interacting domains force this recruitment directionality. We hypothesized that the tandem repeats in the SunTag may force MeCP2 dimerization and mimic the form of DNA-bound MeCP2. Indeed, providing only the tandem epitopes of SunTag allows LANA to recruit MeCP2 in infected cells. Therefore, CRISPR-PITA revealed the rules of unidirectional recruitment and allowed us to break this directionality.
卡波西肉瘤相关疱疹病毒（KSHV，HHV - 8）与多种人类恶性肿瘤相关。在潜伏期，病毒基因组以大型非整合质粒的形式存在于受感染细胞的细胞核中，这些质粒被称为附加体。为确保附加体的维持，潜伏期蛋白LANA在细胞分裂期间将病毒附加体与细胞染色体相连。蛋白质复合物的定向募集对于许多核过程的正常功能至关重要。为了测试LANA与细胞蛋白之间的募集方向性，我们通过催化失活的Cas9（dCas9）将LANA引导至一个重复序列以获得易于检测的点。然后，可以评估核蛋白向这些点的募集情况。我们将此检测方法称为用于蛋白质相互作用和端粒募集检测的CRISPR - PITA。使用这种蛋白质募集检测方法，我们发现LANA募集其已知的相互作用蛋白ORC2和SIN3A。有趣的是，LANA无法募集MeCP2，但MeCP2能募集LANA。LANA和组蛋白去乙酰化酶1（HDAC1）都与MeCP2的转录抑制结构域（TRD）和甲基化CpG结合结构域（MBD）相互作用。与LANA类似，HDAC1也无法募集MeCP2。而异染色质蛋白1（HP1）与MeCP2的N端相互作用，能够募集MeCP2。我们认为可用的相互作用结构域决定了这种募集方向性。我们假设SunTag中的串联重复序列可能促使MeCP2二聚化并模拟与DNA结合的MeCP2形式。事实上，仅提供SunTag的串联表位就能使LANA在受感染细胞中募集MeCP2。因此，CRISPR - PITA揭示了单向募集的规则，并使我们能够打破这种方向性。
