Nakajima, K.-i. | Search bioRxiv

bioRxiv2026

Nerve Injury-Induced Protein 2 Is Necessary for the Lysosome Membrane Integrity and Protects Cells from Ferroptosis

Nerve injury-induced protein 1 (NINJ1), a cell adhesion molecule, is oligomerized during lytic cell death and mediates plasma membrane rupture to release large intracellular molecules that propagate the inflammatory response. We and others previously showed that NINJ2, a close relative of NINJ1, does not promote plasma membrane rupture to spread inflammation. Here, we identify that NINJ2 is necessary for the lysosome membrane integrity to protect cells from ferroptosis. Specifically, we found that NINJ2 localizes to lysosomes and interacts with LAMP1, an anchor glycoprotein of the lysosome membranes and a sensor of stressed lysosomes. We also found that loss of NINJ2 exacerbates lysosomal membrane permeabilization (LMP), which allows for selective leakage of lysosomal contents, such as labile iron, into the cytosol. Accordingly, loss of NINJ2 elevates cellular labile iron accumulation and decreases expression of ferritins, the primary intracellular iron storage protein complexes. Mechanistically, we found that loss of NINJ2 promotes ferritin FTH degradation in lysosomes, which can be reversed by knockdown of LAMP1. Moreover, we found that loss of NINJ2 sensitizes cells to ferroptosis induced by RSL3 and Erastin, consistent with a recent study that loss of Ninj2 predisposes mice to chronic inflammation. Together, these findings uncover a previously unrecognized activity of NINJ2 from lysosome homeostasis to ferroptosis, which can be explored as a cancer therapeutic strategy especially considering that NINJ2 and ferritins are found to be overexpressed and positively associated with iron-addicted cancers.

cancer biology↗

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

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↗

bioRxiv2020

Proximity Biotin Labeling Reveals KSHV Interferon Regulatory Factor Networks

Studies on "HIT&RUN" effects by viral protein are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled study of both static and dynamic protein-protein interactions. Here we applied PL method with recombinant Kaposis sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) in the genome, and we identified KSHV vIRF-1 and vIRF-4 interacting proteins during reactivation. Fusion of mini-TurboID with vIRF-1 or vIRF-4 did not interfere with KSHV gene expression, DNA replication, or de novo infections. PL identified 213 and 70 proteins for vIRF-1 and vIRF-4 respectively, which possibly interact during KSHV reactivation, and 47 of those were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological functions such as mRNA processing and transcriptional regulation by TP53. Involvement of commonly interacting 44 cellular proteins in innate immune regulation were examined by siRNAs, and we identified that splicing factor 3B (SF3B) family proteins were clearly involved in interferons transcription and suppressed KSHV reactivation. We propose that recombinant TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors may have a function beyond connecting two exon sequences to regulate innate immune responses. ImportanceViral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to use of affinity-precipitation approaches, the majority of our studies focused on how the virus takes advantage of the newly-formed protein interactions for its own replication. Proximity labeling (PL) however, can also highlight the transient and negative effects - those interactions which lead to dissociation from the existing protein complex. Here we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

microbiology↗