PT - JOURNAL ARTICLE AU - Ido Lavi AU - Supriya Bhattacharya AU - Ola Orgil AU - Ankita Awase AU - Nir Avital AU - Guy Journo AU - Vyacheslav Gurevich AU - Meir Shamay TI - Unidirectional recruitment is essential for viral latency AID - 10.1101/2021.10.10.463816 DP - 2022 Jan 01 TA - bioRxiv PG - 2021.10.10.463816 4099 - http://biorxiv.org/content/early/2022/07/25/2021.10.10.463816.short 4100 - http://biorxiv.org/content/early/2022/07/25/2021.10.10.463816.full AB - 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 viral episomes. All KSHV infected cells express LANA, and LANA is essential for viral latency. LANA binding to the viral episomes is critical both for replication of the viral genomes during latency, and for tethering the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes are critical for 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, recruitment of nuclear proteins to these dots can be evaluated. We found that LANA recruited its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Similarly, histone deacetylase 1 (HDAC1) that interact with the transcriptional-repression domain (TRD) of MeCP2, same as LANA, was unable to recruit MeCP2, but MeCP2 was able to recruit HDAC1. In contrast, HP1a that interacts with MeCP2 through a different domain, was able to recruit MeCP2. We propose that available interacting domains in DNA bound/dimerized form of MeCP2, forces this recruitment directionality. We found that cells derived from Rett syndrome and express a mutant MeCP2 (T158M), impaired in DNA binding, cannot support KSHV genome maintenance. Therefore, this unidirectional recruitment of LANA by MeCP2 identified MeCP2 as a critical factor for viral maintenance.Significance Statement Using a CRISPR/Cas9 recruitment assay, we show that some interacting proteins have a unidirectional recruitment property, where only one of the proteins can recruit its partner. We found unidirectional recruitment relations between the methylated DNA binding protein MeCP2 and KSHV encoded LANA. Where MeCP2 recruits LANA, but LANA is unable to recruit MeCP2. Similarly, MeCP2 recruits histone deacetylase 1 (HDAC1), but HDAC1 is unable to recruit MeCP2. We propose that this unidirectional recruitment is the result of available interacting domains. Furthermore, this unidirectional recruitment seems to be critical for viral latency, since LANA fails to maintain the viral genomes in MeCP2 mutant cells. Therefore, in this case unidirectional recruitment is a matter of survival or extinction.Competing Interest StatementThe authors have declared no competing interest.