SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 'highly transmissible respiratory pathogen, leading to severe multi-organ damage. However, knowledge regarding SARS-CoV-2-induced cellular alterations is limited. In this study, we report that SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics and activates the EGFR-mediated cell survival signal cascade during the early stage of viral infection. SARS-CoV-2 causes an increase in mitochondrial transmembrane potential via the SARS-CoV-2 RNA-nucleocapsid cluster, thereby abnormally promoting mitochondrial elongation and the OXPHOS process, followed by enhancing ATP production. Furthermore, SARS-CoV-2 activates the EGFR signal cascade and subsequently induces mitochondrial EGFR trafficking, contributing to abnormal OXPHOS process and viral propagation. Approved EGFR inhibitors remarkably reduce SARS-CoV-2 propagation, among which vandetanib exhibits the highest antiviral efficacy. Treatment of SARS-CoV-2-infected cells with vandetanib decreases SARS-CoV-2-induced EGFR trafficking to the mitochondria and restores SARS-CoV-2-induced aberrant elevation in OXPHOS process and ATP generation, thereby resulting in the reduction of SARS-CoV-2 propagation. Furthermore, oral administration of vandetanib to SARS-CoV-2-infected hACE2 transgenic mice reduces SARS-CoV-2 propagation in lung tissue and mitigates SARS-CoV-2-induced lung inflammation. Vandetanib also exhibits potent antiviral activity against various SARS-CoV-2 variants of concern, including alpha, beta, delta and omicron, in in vitro cell culture experiments. Taken together, our findings provide novel insight into SARS-CoV-2-induced alterations in mitochondrial dynamics and EGFR trafficking during the early stage of viral infection and their roles in robust SARS-CoV-2 propagation, suggesting that EGFR is an attractive host target for combating COVID-19.

RSV Induces Activation of Intracellular EGFR on the Mitochondrial Membrane for Virus Propagation.

Respiratory syncytial virus (RSV) infects people of all ages and is one of the most common causative agents of lower respiratory tract infections, such as pneumonia, especially in infants under one year of age. However, no direct treatment has been developed for RSV infections. Maintenance of mitochondrial homeostasis and epidermal growth factor receptor (EGFR) activity is important for human cell growth. This study reported that RSV infection maintained the total cellular ATP levels and promoted the intracellular activity of EGFR to replicate RSV. RSV activates the intracellular EGFR-mediated cell survival signaling cascade and maintains mitochondrial EGFR expression for viral production during early events after infection. The approved EGFR inhibitor, vandetanib, markedly reduces RSV propagation, suggesting that EGFR is an attractive host target for RSV therapeutics. Our results suggest that RSV infection maintains cellular ATP levels and promotes the activation of intracellular EGFR in the mitochondrial membrane, significantly contributing to robust RSV propagation.

Role of Virus-Induced EGFR Trafficking in Proviral Functions.

Since its discovery in the early 1980s, the epidermal growth factor receptor (EGFR) has emerged as a pivotal and multifaceted player in elucidating the intricate mechanisms underlying various human diseases and their associations with cell survival, proliferation, and cellular homeostasis. Recent advancements in research have underscored the profound and multifaceted role of EGFR in viral infections, highlighting its involvement in viral entry, replication, and the subversion of host immune responses. In this regard, the importance of EGFR trafficking has also been highlighted in recent studies. The dynamic relocation of EGFR to diverse intracellular organelles, including endosomes, lysosomes, mitochondria, and even the nucleus, is a central feature of its functionality in diverse contexts. This dynamic intracellular trafficking is not merely a passive process but an orchestrated symphony, facilitating EGFR involvement in various cellular pathways and interactions with viral components. Furthermore, EGFR, which is initially anchored on the plasma membrane, serves as a linchpin orchestrating viral entry processes, a crucial early step in the viral life cycle. The role of EGFR in this context is highly context-dependent and varies among viruses. Here, we present a comprehensive summary of the current state of knowledge regarding the intricate interactions between EGFR and viruses. These interactions are fundamental for successful propagation of a wide array of viral species and affect viral pathogenesis and host responses. Understanding EGFR significance in both normal cellular processes and viral infections may not only help develop innovative antiviral therapies but also provide a deeper understanding of the intricate roles of EGFR signaling in infectious diseases.

Randomized multicenter phase II trial of prophylactic irradiation of para-aortic lymph nodes in advanced cervical cancer according to tumor hypoxia: Korean Radiation Oncology Group (KROG 07-01) study.

We conducted a prospective phase II study on whether extended-field irradiation (EFI) confers survival benefits depending on hypoxic markers in locally advanced uterine cervical cancer (LAUCC). RNA-seq was performed to identify immune and hypoxic gene signatures. A total of 288 patients were randomized to either EFI or pelvic radiotherapy (PRT). All patients completed chemoradiotherapy. Overall, significantly higher 5-year para-aortic recurrence free survival (PARFS) rate occurred in EFI (97.6%) than in PRT group (87.2%), with marginal tendency to improve disease-free survival (DFS; 78% vs 70%, P = .066). Subgroup analyses were performed based on carbonic anhydrase 9 (CA9)-only positive, CA9/hypoxia-inducible factor (HIF) double positive and CA9 negative. In the CA9-only positive, EFI successfully increased 5-year PARFS (100% vs 76.4%, P = .010), resulting in significantly improved long-term DFS (85.7% vs 54.7%, P = .023) compared to the PRT, while there was no such benefit of EFI in the CA9/HIFs double positive. RNA-seq analysis identified distinct immunehigh subgroup with negative correlation with hypoxia gene signatures (R = -.37, P < .01), which showed a higher 5-year DFS than the immunelow (P = .032). Hypoxia-related genes were upregulated in the CA9/HIFs double positive compared to CA9 negative (P < .05). Only 17.4% of patients in CA9-negative group showed immunelow signatures, while 40.0% of patients in the double-positive group exhibited immunelow signatures. In conclusion, EFI improved PARFS significantly in all patients, but therapeutic efficacy of EFI in terms of improved DFS was solely observed in CA9-only positive LAUCC, and not in CA9/HIFs double-positive subgroup. RNA-seq analysis suggested that hypoxia-induced immunosuppression may be related to treatment resistance in LAUCC.

Structure-Based Virtual Screening: Identification of a Novel NS2B-NS3 Protease Inhibitor with Potent Antiviral Activity against Zika and Dengue Viruses.

Zika virus (ZIKV), which is associated with severe diseases in humans, has spread rapidly and globally since its emergence. ZIKV and dengue virus (DENV) are closely related, and antibody-dependent enhancement (ADE) of infection between cocirculating ZIKV and DENV may exacerbate disease. Despite these serious threats, there are currently no approved antiviral drugs against ZIKV and DENV. The NS2B-NS3 viral protease is an attractive antiviral target because it plays a pivotal role in polyprotein cleavage, which is required for viral replication. Thus, we sought to identify novel inhibitors of the NS2B-NS3 protease. To that aim, we performed structure-based virtual screening using 467,000 structurally diverse chemical compounds. Then, a fluorescence-based protease inhibition assay was used to test whether the selected candidates inhibited ZIKV protease activity. Among the 123 candidate inhibitors selected from virtual screening, compound 1 significantly inhibited ZIKV NS2B-NS3 protease activity in vitro. In addition, compound 1 effectively inhibited ZIKV and DENV infection of human cells. Molecular docking analysis suggested that compound 1 binds to the NS2B-NS3 protease of ZIKV and DENV. Thus, compound 1 could be used as a new therapeutic option for the development of more potent antiviral drugs against both ZIKV and DENV, reducing the risks of ADE.

A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique.

Genetic screens using CRISPR/Cas9 have been exploited to discover host-virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus-host interaction and identify a novel target for antiviral therapeutics.

Butyrate enhances the efficacy of radiotherapy via FOXO3A in colorectal cancer patient­derived organoids.

Enhancing the radioresponsiveness of colorectal cancer (CRC) is essential for local control and prognosis. However, consequent damage to surrounding healthy cells can lead to treatment failure. We hypothesized that short­chain fatty acids (SCFAs) could act as radiosensitizers for cancer cells, allowing the administration of a lower and safer dose of radiation. To test this hypothesis, the responses of three­dimensional­cultured organoids, derived from CRC patients, to radiotherapy, as well as the effects of combined radiotherapy with the SCFAs butyrate, propionate and acetate as candidate radiosensitizers, were evaluated via reverse transcription­quantitative polymerase chain reaction, immunohistochemistry and organoid viability assay. Of the three SCFAs tested, only butyrate suppressed the proliferation of the organoids. Moreover, butyrate significantly enhanced radiation­induced cell death and enhanced treatment effects compared with administration of radiation alone. The radiation­butyrate combination reduced the proportion of Ki­67 (proliferation marker)­positive cells and decreased the number of S phase cells via FOXO3A. Meanwhile, 3/8 CRC organoids were found to be non­responsive to butyrate with lower expression levels of FOXO3A compared with the responsive cases. Notably, butyrate did not increase radiation­induced cell death and improved regeneration capacity after irradiation in normal organoids. These results suggest that butyrate could enhance the efficacy of radiotherapy while protecting the normal mucosa, thus highlighting a potential strategy for minimizing the associated toxicity of radiotherapy.

The association of integration patterns of human papilloma virus and single nucleotide polymorphisms on immune- or DNA repair-related genes in cervical cancer patients.

The present study investigated the association between single nucleotide polymorphisms (SNPs) in immune- or DNA repair-related genes and the integration pattern of human papillomavirus (HPV), a promising prognostic marker in cervical cancer. The HPV integration patterns of cervical cancer patients were determined by polymerase chain reaction and in situ hybridization, and categorized as episomal (group A), single-copy or multi-copy tandem repetition integrated (group B), and undetectable HPV types (group C). After sample and SNP quality control, 166,505 SNPs in 161 samples (38, 111, and 12 patients in groups A, B, and C, respectively) were examined. None of the SNPs reached genome-wide significance, and several candidate SNPs for future study were selected, including rs10999435 on chromosome 10q22, rs1322054 on chromosome 9q32-33, and rs10902171 on chromosome 11p15. Luciferase assay identified rs1322054 as the primary functional variant to regulate gene expression in immune cell. Further studies are needed to determine the genetic background of different integration patterns of HPV in cervical cancer patients.

A herpesvirus transactivator and cellular POU proteins extensively regulate DNA binding of the host Notch signaling protein RBP-Jκ to the virus genome.

Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein Jκ recombination signal-binding protein (RBP-Jκ or CSL). RBP-Jκ normally binds DNA sequence-specifically to determine the transcriptional targets of the Notch-signaling pathway, yet Notch alone cannot reactivate KSHV. We previously showed that Rta stimulates RBP-Jκ DNA binding to the viral genome. On a model viral promoter, this function requires Rta to bind to multiple copies of an Rta DNA motif (called "CANT" or Rta-c) proximal to an RBP-Jκ motif. Here, high-resolution ChIP/deep sequencing from infected primary effusion lymphoma cells revealed that RBP-Jκ binds nearly exclusively to different sets of viral genome sites during latency and reactivation. RBP-Jκ bound DNA frequently, but not exclusively, proximal to Rta bound to single, but not multiple, Rta-c motifs. To discover additional regulators of RBP-Jκ DNA binding, we used bioinformatics to identify cellular DNA-binding protein motifs adjacent to either latent or reactivation-specific RBP-Jκ-binding sites. Many of these cellular factors, including POU class homeobox (POU) proteins, have known Notch or herpesvirus phenotypes. Among a set of Rta- and RBP-Jκ-bound promoters, Rta transactivated only those that also contained POU motifs in conserved positions. On some promoters, POU factors appeared to inhibit RBP-Jκ DNA binding unless Rta bound to a proximal Rta-c motif. Moreover, POU2F1/Oct-1 expression was induced during KSHV reactivation, and POU2F1 knockdown diminished infectious virus production. Our results suggest that Rta and POU proteins broadly regulate DNA binding of RBP-Jκ during KSHV reactivation.

Protective association of HLA-DRB1*13:02, HLA-DRB1*04:06, and HLA-DQB1*06:04 alleles with cervical cancer in a Korean population.

Human leukocyte antigen (HLA) class II alleles have been previously associated with cervical cancer. However, these associations vary widely across racial and ethnic groups. Therefore, we evaluated the effect of HLA class II alleles on cervical cancer in a Korean population. HLA-DRB1, HLA-DQB1, and HLA-DQA1 alleles were analyzed in 457 cervical cancer patients and compared to those of 926 control subjects. The odds ratio (OR) of each allele between the patients and controls was calculated using the logistic regression model. Patients, had significantly lower frequencies of HLA-DRB1 and HLA-DQB1 alleles than control subjects: HLA-DRB1*13:02:01 (4.4% vs 8.8%; OR 0.48, 95% confidence interval (CI) 0.27-0.84; p = 0.001), HLA-DRB1*04:06 (2.1% vs 4.7%; OR 0.44, 95% CI 0.20-0.97; p = 0.033), and HLA-DQB1*06:04:01 (2.3% vs 5.0%; OR 0.46, 95% CI 0.22-0.94; p = 0.021). No significant association was observed for HLA-DQA1. Protective associations between HLA-DRB1*13:02, HLA-DRB1*04:06, and HLA-DQB1*06:04 alleles and cervical cancer were found in the Korean population.

Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity.

Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.

Promoter methylation of cysteine dioxygenase type 1: gene silencing and tumorigenesis in hepatocellular carcinoma.

BACKGROUNDS/AIMS: Cysteine dioxygenase type 1 (CDO1) acts as a tumor suppressor and is silenced by promoter methylation in various malignancies. The relationship between the CDO1 methylation status and hepatocellular carcinoma (HCC) tumorigenesis was evaluated. METHODS: Using a HCC cell line (SNU423), an in vitro demethylation study was performed to confirm whether promoter methylation causes CDO1 down-regulation. The SNU423 cells transfected with the CDO1 cell function was compared to that of naïve cells. An in vivo study using immunohistochemical staining of HCC specimens that were collected from patients who underwent curative liver resection was also performed. RESULTS: CDO1 was activated after demethylation treatment in the HCC specimens. Moreover, tumor cell proliferation, colony-forming, migration, and invasion activities significantly decreased after CDO1 transfection (p<0.05). The percentage of tumors that were larger than 5 cm was higher in patients who had a lower expression of CDO1 (p=0.030). Vascular invasion and histological grade were independent prognostic factors for poor overall and recurrence-free survival. The degree of CDO1 expression was not an independent prognostic factor in this study's population. CONCLUSIONS: These results suggested that methylation down-regulated CDO1 expression in the HCC cells. CDO1 methylation may be a potentially valuable diagnostic biomarker for HCC.

Inhibition of collagenase and melanogenesis by ethanol extracts of Orostachys japonicus A. Berger: possible involvement of Erk and Akt signaling pathways in melanoma cells.

Orostachys japonicus is an herb that contains several functional components and has traditionally been used to treat various diseases in Asia. In this study, bioactive components from different parts of the O. japonicus plant were investigated, and the contents of the functional components in ethanol extracts of O. japonicus cultivated in Korea and China were compared. The antioxidant effects of O. japonicus ethanol extracts were investigated using Raw 264.7 cells. It was found that 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity was significantly decreased in the cells treated with the extracts. Moreover, the novel inhibitory functions of O. japonicus extracts on collagenase, elastase, and tyrosinase were established. We also found that O. japonicus extracts strongly inhibited melanin synthesis in B16F10 melanoma cells by decreasing MITF protein levels and activating the Erk and Akt signaling pathways. Thus, these findings would be useful for developing new cosmetic and pharmaceutical formulations based on O. japonicus extracts.

Integration Pattern of Human Papillomavirus Is a Strong Prognostic Factor for Disease-Free Survival After Radiation Therapy in Cervical Cancer Patients.

PURPOSE: The standard chemoradiation therapy currently used for locally advanced cervical cancer (LACC) patients does not reflect the biological heterogeneity of this disease, and there is an increasing need for the development of biomarkers that can help guide the individualized treatment regimens. The purpose of this study was to investigate the prognostic value of the integration pattern of human papillomavirus (HPV) in LACC patients. METHODS AND MATERIALS: The HPV integration pattern was determined by in situ hybridization and polymerase chain reaction, and the tumors were classified as the episomal pattern (group A), as the single-copy integrated or multicopy tandem repetition-integrated pattern (group B), or as undetectable HPV (group C). Ninety-eight LACC patients were included in a development dataset and 106 independent patients in a validation dataset. The multivariate Cox model was used to examine the effect of the HPV integration pattern on disease-free survival (DFS). The model was validated internally by the leave-one-out cross-validation method and externally by an independent dataset. RESULTS: After adjustment for significant prognostic factors (stage, histologic grade, histologic type, and tumor size), the HPV integration pattern was significantly associated with DFS in the development (P=.032) and validation (P=.023) datasets. Survival was worst in group C and best in group A. The multivariate model with HPV integration pattern as an explanatory variable showed good discrimination ability and could separate patients with different risk profiles. CONCLUSIONS: This study identified the HPV integration pattern, as determined by in situ hybridization and polymerase chain reaction, as a strong prognostic biomarker for DFS in LACC patients treated by chemoradiation therapy. This finding may open the possibility of personalized treatment of these patients.

Nucleotide Excision Repair Gene ERCC2 and ERCC5 Variants Increase Risk of Uterine Cervical Cancer.

PURPOSE: Defects in the DNA damage repair process can cause genomic instability and play an important role in cervical carcinogenesis. The purpose of this study was to analyze the association of 29 candidate single nucleotide polymorphisms (SNPs) in genes in the DNA repair pathway, TP53, and TP53BP1 with the risk of cervical cancer. MATERIALS AND METHODS: Twenty-nine SNPs in four genes in the DNA repair pathway (ERCC2, ERCC5, NBS1, and XRCC1), TP53, and TP53BP1 were genotyped for 478 cervical cancer patients and 922 healthy control subjects, and their effects on cervical carcinogenesis were analyzed. RESULTS: The most significant association was found for rs17655 in ERCC5, with an age-adjusted p-value < 0.0001, for which a strong additive effect of the risk allele C was observed (odds ratio, 2.01 for CC to GG). On the other hand, another significant polymorphism rs454421 in ERCC2 showed a dominant effect (odds ratio, 1.68 for GA+AA to GG) with an age-adjusted p-value of 0.0009. The association of these polymorphisms remained significant regardless of the age of onset. The significant result for rs17655 was also consistent for subgroups of patients defined by histology and human papillomavirus (HPV) types. However, for rs454421, the association was observed only in patients with squamous cell carcinoma and non-HPV 18 type. CONCLUSION: The results of this study show a novel association of cervical cancer and the genes involved in the nucleotide excision pathway in the Korean population.

A new hope in immunotherapy for malignant gliomas: adoptive T cell transfer therapy.

Immunotherapy emerged as a promising therapeutic approach to highly incurable malignant gliomas due to tumor-specific cytotoxicity, minimal side effect, and a durable antitumor effect by memory T cells. But, antitumor activities of endogenously activated T cells induced by immunotherapy such as vaccination are not sufficient to control tumors because tumor-specific antigens may be self-antigens and tumors have immune evasion mechanisms to avoid immune surveillance system of host. Although recent clinical results from vaccine strategy for malignant gliomas are encouraging, these trials have some limitations, particularly their failure to expand tumor antigen-specific T cells reproducibly and effectively. An alternative strategy to overcome these limitations is adoptive T cell transfer therapy, in which tumor-specific T cells are expanded ex vivo rapidly and then transferred to patients. Moreover, enhanced biologic functions of T cells generated by genetic engineering and modified immunosuppressive microenvironment of host by homeostatic T cell expansion and/or elimination of immunosuppressive cells and molecules can induce more potent antitumor T cell responses and make this strategy hold promise in promoting a patient response for malignant glioma treatment. Here we will review the past and current progresses and discuss a new hope in adoptive T cell therapy for malignant gliomas.

Physical status of human papillomavirus integration in cervical cancer is associated with treatment outcome of the patients treated with radiotherapy.

Integration of human papillomavirus (HPV) DNA into the host genome is a critical aetiological event in the progression from normal cervix to intraepithelial neoplasm, and finally to invasive cervical cancer. However, there has been little work on how HPV integration status relates to treatment outcome for cervical carcinomas. In the current study, HPV E2 and E6 gene copy numbers were measured in 111 cervical cancer tissues using real-time QPCR. Integration patterns were divided into four groups: single copy-integrated with episomal components (group 1), single copy-integrated without episomal components (group 2), multicopy tandem repetition-integrated (group 3), and low HPV (group 4) groups. A relapse-predicting model was constructed using multivariable Cox proportional hazards model to classify patients into different risk groups for disease-free survival (DFS). The model was internally validated using bootstrap resampling. Oligonucleotide microarray analysis was performed to evaluate gene expression patterns in relation to the different integration groups. DFS rate was inferior in the order of the patients in group 4, group 2/3, and group 1. Multivariate analysis showed that histologic grade, clinical stage group, and integration pattern were significant prognostic factors for poor DFS. The current prognostic model accurately predicted the risk of relapse, with an area under the receiver operating characteristic curve (AUC) of 0.74 (bootstrap corrected, 0.71). In conclusion, these data suggest that HPV integration pattern is a potent prognostic factor for tailored treatment of cervical cancer.

Histone deacetylase classes I and II regulate Kaposi's sarcoma-associated herpesvirus reactivation.

In primary effusion lymphoma (PEL) cells infected with latent Kaposi's sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta's promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.