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Espera, J. M.

Publications and source records attributed to Espera, J. M..

3 recordsLinked to original sources
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↗