LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection

Abstract

One of the hallmarks of the latent phase of Kaposi's sarcoma-associated herpesvirus (KSHV) infection is the global repression of lytic viral gene expression. Following de novo KSHV infection, the establishment of latency involves the chromatinization of the incoming viral genomes and recruitment of the host Polycomb repressive complexes (PRC1 and PRC2) to the promoters of lytic genes, which is accompanied by the inhibition of lytic genes. However, the mechanism of how PRCs are recruited to the KSHV episome is still unknown. Utilizing a genetic screen of latent genes in the context of KSHV genome, we identified the latency-associated nuclear antigen (LANA) to be responsible for the genome-wide recruitment of PRCs onto the lytic promoters following infection. We found that LANA initially bound to the KSHV genome right after infection and subsequently recruited PRCs onto the viral lytic promoters, thereby repressing lytic gene expression. Furthermore, both the DNA and chromatin binding activities of LANA were required for the binding of LANA to the KSHV promoters, which was necessary for the recruitment of PRC2 to the lytic promoters during de novo KSHV infection. Consequently, the LANA-knockout KSHV could not recruit PRCs to its viral genome upon de novo infection, resulting in aberrant lytic gene expression and dysregulation of expression of host genes involved in cell cycle and proliferation pathways. In this report, we demonstrate that KSHV LANA recruits host PRCs onto the lytic promoters to suppress lytic gene expression following de novo infection.

Fig 1. LANA is required for the recruitment of Polycomb proteins to lytic promoters during de novo infection.

(A) Schematic of the KSHV latency locus encoding latent genes. (B) ChIP assays testing the recruitment of PRC2 factor EZH2 and PRC1 factor RING1B onto viral lytic promoters in SLK cells 72 hours after infection with WT or the indicated KSHV mutants. (C and D) ChIP assays for the repressive (H3K27me3) and activating (H3K4me3) histone marks on viral and cellular promoters at 72 hpi. The LANA and actin (ACT) promoters devoid of H3K27me3 and the PRC2-regulated MYT1 promoter served as controls. (E) Immunoblots showing LANA protein levels in mock and KSHV-infected SLK cells at 72 hpi. *Non-specific. (F) ChIP assays for EZH2 and RING1B associations with viral promoters at 72 hpi.

Fig 2. LANA binds to the entire KSHV genome and is required for the recruitment of EZH2 onto the viral episome during de novo infection.

(A) Immunoblot analysis of LANA expression in BAC16-3xF-LANA infected SLK cells at 72 hpi using anti-LANA and anti-FLAG antibodies. (B and C) FLAG and LANA ChIP assays for testing LANA-binding to viral promoters in BAC16 and BAC16-3xF-LANA KSHV-infected SLK cells at 72 hpi. Cellular MYT1 promoter was used as a control. (D) ChIP-on-chip analysis of the genome-wide binding of LANA and EZH2 to the KSHV genome in WT and LANA KO KSHV-infected cells at 72 hpi.

Fig 3. Deregulated viral and cellular gene expression in LANA KO KSHV-infected cells.

(A) Analysis of viral gene expression in LANA KO and LANA/RTA KO KSHV-infected cells compared to WT KSHV-infected cells by NanoString technology. (B) Heat map of differential gene expression between mock and KSHV-infected cells. Rows A and B, C and D, E and F represent two biological replicates of mock-, WT, and LANA KO KSHV-infected SLK cells, respectively. Also see S3 Table. (C) Biological pathways significantly affected by the lack of LANA in KSHV-infected cells were determined by gene set enrichment analysis. Also see S4 Table.

Fig 4. Additional LANA expression is not sufficient for the early recruitment of EZH2 onto the KSHV genome.

(A) Time course analysis of LANA protein expression during de novo infection. (B) Time course ChIP analysis of LANA-binding to viral promoters during de novo infection. (C) Immunoblot analysis of LANA expression in empty lentiviral vector and 3xFLAG-LANA expressing lentivirus infected cells. (D) ChIP assays for binding of LANA and EZH2 to viral promoters in LANA-overexpressing cells during de novo KSHV infection.

Fig 5. Co-localization and focal enrichment of polycomb proteins with LANA in KSHV-infected cells.

KSHV-infected iSLK cells (iSLKBAC16-3xF-LANA) were subjected to confocal microscopic analysis for LANA (Green, false color) and polycomb proteins (Red). SPT5 and CYCT1 served as controls. FLAG antibody was used to detect the 3xF-LANA. Representative LANA puncta were connected by white lines and the co-localization of LANA with the indicated cellular proteins was measured by the image processing program ImageJ.

Fig 6. Interaction of LANA with PRC2.

(A) Gel filtration chromatography of nuclear extracts derived from iSLK and iSLKBAC16 cells. Fractions from 28 to 72 were tested by immunoblot analysis for LANA and polycomb proteins. Molecular weight markers are indicated. (B) Co-immunoprecipitation of LANA-V5 (using V5 antibody) from 293T cells expressing LANA-V5 and the indicated 3xFLAG-tagged PRC2 (EZH2, SUZ12 and EED) or PRC1 (RING1B and BMI-1) factors. Immunoprecipitation and input blots were probed with V5 and FLAG antibodies. (C) Cell lysates derived from de novo KSHV infected iSLK cells (72 hpi) were used for immunoprecipitation with anti-EZH2 rabbit antibody (αEZH2) or normal rabbit IgG (αIgG), followed by immunoblotting with αLANA and αEZH2.

Fig 7. LANA-binding to the lytic KSHV promoters is required for the EZH2 recruitment during de novo infection.

(A) Immunoblot analysis of SLK cells infected with lentiviruses expressing 3xFLAG-tagged WT or mutant LANA using anti-FLAG antibody. (B) ChIP assays for the recruitment of LANA (using αLANA antibody) and EZH2 on viral promoters in RTA KO and LANA/RTA dKO KSHV-infected SLK cells as well as in LANA/RTA dKO KSHV-infected SLK cells expressing WT or mutant LANA. (C) Total lysates of 293T cells expressing either 3xF-LANA or 3xF-GMRRL mutant LANA were subjected to FLAG immunoprecipitation followed by immunoblotting with anti-FLAG and anti-EZH2 antibodies. Input lysates were included as controls. (D) ChIP assays for LANA and EZH2 occupancy on the KSHV promoters during de novo LANA/RTA dKO KSHV infection of WT- or GMRRL mutant LANA-expressing SLK cells. (E) Model showing LANA-mediated recruitment PRCs onto the KSHV genome during de novo infection.