Unidirectional protein recruitment is essential for viral latency

Abstract

Kaposis 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. All KSHV infected cells express LANA, and LANA is essential for viral latency. LANA binding to the viral episomes is critical for both 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. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1) which interacts with the N-terminal of MeCP2, was able to recruit MeCP2. Forcing MeCP2 dimerization via the tandem epitopes of SunTag, allows LANA to recruit MeCP2 in infected cells. We propose that available interacting domains force this recruitment directionality. Knock-down of MeCP2 by shRNA, as well as cells derived from Rett syndrome and express a mutant MeCP2 (T158M), dramatically reduced the ability of LANA to support viral latency. Therefore, this unidirectional recruitment of LANA by MeCP2 identified MeCP2 as a critical factor for KSHV viral maintenance. Significance StatementUsing 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. We were able to break this unidirectional recruitment by forcing MeCP2 dimerization. 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.