Espera, J. M. | Search bioRxiv

bioRxiv2025

Studies on Gene Enhancer with KSHV mini-chromatin

Kaposis sarcoma-associated herpesvirus (KSHV) genome contains a terminal repeats (TR) sequence. Previous studies demonstrated that KSHV TR functions as a gene enhancer for inducible lytic gene promoters. Gene enhancers anchor bromodomain-containing protein 4 (BRD4) at specific genomic region, where BRD4 interacts flexibly with transcription-related proteins through its intrinsically disordered domain and exerts transcription regulatory function. Here, we generated recombinant KSHV with reduced TR copy numbers and studied BRD4 recruitment and its contributions to the inducible promoter activation. Reducing the TR copy numbers from 21 (TR21) to 5 (TR5) strongly attenuated viral gene expression during de novo infection and impaired reactivation. The EF1 promoter encoded in the KSHV BAC backbone also showed reduced promoter activity, suggesting a global attenuation of transcription activity within TR5 latent episomes. Isolation of reactivating cells confirmed that the reduced inducible gene transcription from TR-shortened DNA template and is mediated by decreased efficacies of BRD4 recruitment to viral gene promoters. Separating the reactivating iSLK cell population from non-responders showed that reactivatable iSLK cells harbored larger LANA nuclear bodies (NBs) compared to non-responders. The cells with larger LANA NBs, either due to prior transcription activation or TR copy number, supported KSHV reactivation more efficiently than those with smaller LANA NBs. With auxin-inducible LANA degradation, we confirmed that LANA is responsible for BRD4 occupancies on latent chromatin. Finally, with purified fluorescence-tagged proteins, we demonstrated that BRD4 is required for LANA to form liquid-liquid phase-separated dots. The inclusion of TR DNA fragments further facilitated the formation of larger BRD4-containing LLPS in the presence of LANA, similar to the "cellular enhancer dot" formed by transcription factor-DNA bindings. These results suggest that LANA binding to TR establishes an enhancer domain for infected KSHV episomes. The strength of this enhancer, regulated by TR length or transcription memory, determines the outcome of KSHV replication. ImportanceGene enhancers are genomic domains that regulate frequency and duration of transcription burst at gene promoters, with BRD4 playing a critical role in their enhancer functions. KSHV latent mini-chromosome also contains an enhancer domain made with multiple copies of 801 bp identical repeat DNA fragments, terminal repeats. Here, we utilized manipulable mini-scale chromatins with convenient inducible KSHV reactivation to systematically examine the association between enhancer strength and the outcome of inducible promoter activation. This study illustrated the amount of BRD4 recruitment at the enhancer associated with frequencies of BRD4 distribution to the inducible promoters during KSHV reactivation and, therefore, KSHV lytic replication. Recruitment of BRD4 to the TR is specifically regulated by KSHV latent protein, LANA. KSHV evolves clever enhancer elements designed to be regulated by the KSHV own latent protein, LANA.

microbiology↗

bioRxiv2025

Design, development, and evaluation of gene therapeutics specific to KSHV-associated diseases

Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposis sarcoma (KS) and two human lymphoproliferative diseases: primary effusion lymphoma and AIDS-related multicentric Castlemans disease. KSHV-encoded latency-associated nuclear antigen (LANA) is expressed in KSHV-infected cancer cells and is responsible for maintaining viral genomes in infected cells. Thus, LANA is an attractive target for therapeutic intervention for KSHV-associated diseases. Here, we devised a cancer gene therapy vector using the adeno-associated virus (AAV), which capitalizes the LANAs function to maintain terminal repeat (TR) containing circular genome in latently infected cells and the TRs enhancer function for KSHV inducible gene promoters. By including two TR copies with a lytic inducible gene promoter (TR2-OriP), we prepared an AAV vector, which expresses an engineered thymidine kinase (TK) selectively in KSHV-infected cells. Ganciclovir (GCV), an anti-herpesvirus drug, effectively eradicated multiple KSHV-infected cells that include iPSC-derived epithelial colony-forming cells, but not non-KSHV-infected counterparts in the presence of AAV8-TR2-OriP-TK. In addition, AAV8-TR2-OriP-TK prevents KSHV virion production from reactivated cells, spreading KSHV infections from reactivated cells. Anti-cancer drugs, known to reactivate KSHV, stimulated TK expression from the vector and, therefore, synergized with AAV8 TR2-OriP-TK to induce KSHV-infected cancer cell death. Finally, the AAV8-TR2-OriP-TK with GCV completely diminished KSHV-infected cancer cells in the xenograft tumor model. The new cancer gene therapeutics should augment the current clinical protocol for KS.

microbiology↗

bioRxiv2023

KSHV Terminal Repeat Regulates Inducible Lytic Gene Promoters

The Kaposis sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140 kb unique coding region flanked by multiple copies of 0.8 kb terminal repeat (TR) sequence. While TRs function in plasmid maintenance is well-established, TRs transcription regulatory roles have not been fully explored. Here, we show KSHV TR is a large transcription regulatory domain. A series of Cleavage Under Targets & Release Using Nuclease demonstrated that TR fragments are occupied by histone modifying enzymes that are known to interact with LANA in naturally infected cells, and the TR possessed characteristic enhancer histone modifications. The H3K4me3 and H3K27Ac modifications were conserved in unique region of the KSHV genome among naturally infected cells, and the KSHV Origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with TRs. In the Ori-Lyt region, the LANA complex colocalizes with H3K27Ac-modified nucleosome along with paused RNA polymerase II, and two K-Rta recruitment sites frank the nucleosome. The isolated reporter assays demonstrated that neighboring TR fragments enhanced viral lytic gene promoter activity independent of orientation in KSHV-infected and non-infected 293FT cells. K-Rta transactivation function was drastically enhanced with TR, while LANA acquired promoter repression function with TR. KSHV TR is, therefore a regulatory domain for KSHV inducible genes. However, in contrast to cellular enhancers that are bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body with TR. KSHV evolved a clever mechanism to tightly control the latency-lytic switch with the TR/LANA complex. ImportanceEnhancers are a crucial regulator of differential gene expression programs. Enhancer is the cis-regulatory sequences that determine target genes spatiotemporal and quantitative expression. Here, we show that KSHV terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. KSHV enhancer is occupied by LANA and its interacting proteins, such as CHD4, and CHD4 is known to restrict enhancers to access promoters for activation. This study thus proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates lytic gene transcription.

microbiology↗

bioRxiv2023

Kaposis Sarcoma-Associated Herpesvirus (KSHV) LANA Prevents KSHV Episomes from Degradation

Protein knock-down with an inducible degradation system is a powerful tool to study proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposis Sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-AID (mAID) tag at the LANA N-terminus with KSHV BAC16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid (5-Ph-IAA), a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate treatment. Decreased overall lytic gene induction seemed to associate with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by treatment with lysosomal inhibitor chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and Stimulator of Interferon Genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. These results suggest that LANA is actively protecting viral genomic DNA from sensing by cGAS-STING signaling axis, and add novel insights into the role of LANA in latency maintenance.

microbiology↗

bioRxiv2022

Auxin-inducible Degron (AID) to Dissect Kaposi's Sarcoma associated Herpesvirus (KSHV) LANA protein function

Protein knock-down with an inducible degradation system is a powerful tool to study proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposis Sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-AID (mAID) tag at the LANA N-terminus with KSHV BAC16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-tagged LANA. Incubation with 5-phenyl-indole-3-acetic acid (5-Ph-IAA), a derivative of natural auxin, rapidly degraded LANA protein within 1.5 hours. In contrast to our hypothesis, depletion of LANA not only failed to trigger lytic reactivation but rather decreased inducible lytic gene expression when we triggered reactivation with a combination of ORF50 protein expression and sodium butyrate treatment. Decreased overall lytic gene induction seemed to associate with a rapid loss of KSHV genomes in the absence of LANA. Furthermore, we found that small cell fractions harbor non-depletable LANA dots in the presence of 5-Ph-IAA. In the cell population containing degradation-resistant LANA, induction of lytic reactivation was strongly attenuated. These results suggest that (i) there are at least two populations of LANA dots in cells, (ii) local nuclear environment and its epigenetic effects on the episomes are heritable to daughter cells; this biological had substantial effects in degree of KSHV reactivation, and finally (iii) LANA may have an additional function in protecting KSHV episomes from degradation. IMPORTANCEKSHV LANA protein plays a wide variety of roles in latency maintenance and lytic gene expression. We adapted the inducible protein knockdown approach to examine its role directly, and revealed that there are cell populations that possess viral episomes insensitive to reactivation stimuli. Viral reactivation is known to be highly heterogenic, and our observations suggest that LANA tethering sites on host chromatin may play a critical role in determining diverse responsiveness to the stimuli. We also demonstrated that depletion of LANA leads to rapid reduction of viral genome, which suggests that LANA might be actively protecting latent viral genome from degradation. These results add novel insights into the role of LANA in latency maintenance and regulation of lytic reactivation.

microbiology↗