scholarly journals Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Xiaojuan Li ◽  
Lu Huang ◽  
Yunjun Xiao ◽  
Xiangyang Yao ◽  
Xubing Long ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Inhibitory Effect ◽  
Lytic Replication ◽  
Binding Region ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Virion Production ◽  
Infected Cells

ABSTRACT The lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) requires sustained extracellular signal-regulated kinase (ERK)-p90 ribosomal S6 kinase (RSK) activation, which is induced by an immediate early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single-point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease in late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56 to 76 amino acid (aa) region and the HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis. IMPORTANCE ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.

scholarly journals HMGB1 knockout decreases Kaposi's sarcoma-associated herpesvirus virion production in iSLK BAC16 cells by attenuating viral gene expression

2021 ◽  
Author(s):  
Su-Kyung Kang ◽  
Yun Hee Kang ◽  
Seung-Min Yoo ◽  
Changhoon Park ◽  
Hong Seok Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
High Mobility ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Virion Production ◽  
Viral Genes ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Multiple host proteins affect the gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) during latent and lytic replication. The high mobility group box 1 (HMGB1) serves as a highly conserved chromosomal protein inside the cell and a prototypical damage-associated molecular pattern molecule outside the cell. HMGB1 has been shown to play a pathogenic role in viral infectious diseases and to regulate the lytic replication of KSHV. However, its functional effects on the KSHV life cycle in KSHV-infected cells have not been fully elucidated. Here, we explored the role of the intracellular and extracellular HMGB1 in KSHV virion production by employing CRISPR/Cas9-mediated HMGB1 knockout in the KSHV-producing iSLK BAC16 cell line. Intracellular HMGB1 formed complexes with various proteins, and the abundance of HMGB1-interacting proteins changed during latent and lytic replication. Moreover, extracellular HMGB1 was found to enhance lytic replication by phosphorylating JNK. Of note, the expression of viral genes was attenuated during lytic replication in HMGB1- knockout iSLK BAC16 cells, with significantly decreased production of infectious virions compared to that in wild-type cells. Collectively, our results demonstrate that HMGB1 is an important cellular cofactor that affects the generation of infectious KSHV progeny during lytic replication. Author Summary The high mobility group box 1 protein ( HMGB1 ) has many intra- and extracellular biological functions with an intricate role in various diseases. In certain viral infections, HMGB1 affects the viral life cycle and pathogenesis. In this study, we explored the effects of HMGB1 knockout on the production of Kaposi’s sarcoma-associated herpesvirus (KSHV). HMGB1 knockout decreased virion production in KSHV-producing cells by decreasing the expression of viral genes. The processes by which HMGB1 affects KSHV production may occur inside or outside of infected cells. For instance, several cellular and viral proteins interacted with intracellular HMGB1 in a nucleosomal complex; whereas extracellular HMGB1 induced JNK phosphorylation, thus enhancing lytic replication. Our results suggest that both intracellular and extracellular HMGB1 are necessary for efficient KSHV replication. Thus, HMGB1 may represent an effective therapeutic target for the regulation of KSHV production.

scholarly journals Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication

2015 ◽  
Vol 89 (10) ◽  
pp. 5308-5317 ◽  
Author(s):  
Qiming Liang ◽  
Brian Chang ◽  
Patrick Lee ◽  
Kevin F. Brulois ◽  
Jianning Ge ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Dramatic Reduction ◽  
Viral Dna ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle.IMPORTANCEThe present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.

scholarly journals Activation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression during Epithelial Differentiation

2005 ◽  
Vol 79 (21) ◽  
pp. 13769-13777 ◽  
Author(s):  
Andrew S. Johnson ◽  
Nicole Maronian ◽  
Jeffrey Vieira
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Fluorescent Protein ◽  
Kaposi’S Sarcoma ◽  
Epithelial Differentiation ◽  
Lytic Replication ◽  
Oral Epithelium ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1α promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.

scholarly journals The Kaposi’s Sarcoma-Associated Herpesvirus ORF34 Protein Interacts and Stabilizes HIF-2α via Binding to the HIF-2α bHLH and PAS Domains

2019 ◽  
Vol 93 (17) ◽  
Author(s):  
Muzammel Haque ◽  
K. G. Kousoulas
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Proteasome Pathway ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTHypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposi’s sarcoma (KS) and other AIDS-related malignancies. Kaposi’s sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1α and HIF-2α. HIF-1α and HIF-2α have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1α and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1α-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2α in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2α and that both bHLH and PAS domains of HIF-2α interacted with ORF34. Unlike HIF-1α, dose-dependent coexpression of ORF34 stabilized the HIF-2α protein, ensuring HIF-2α-dependent transcriptional activity. The ORF34 protein enhanced HIF-2α ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1α and HIF-2α proteins.IMPORTANCEHypoxia inducible factor 1α (HIF-1α) and HIF-2α are transcription factors which play important roles in the Kaposi’s sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2α, in contrast to its role in binding HIF-1α and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1α and HIF-2α expression.

A Pilot Trial of Valproic Acid in Patients with Kaposi’s Sarcoma: A Multi-Center Trial of the AIDS Malignancy Consortium.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2279-2279 ◽  
Author(s):  
Mary Jo Lechowicz ◽  
Jeannette Lee ◽  
Dirk Dittmer ◽  
Susan Krown ◽  
Jonathan Said ◽  
...  
Keyword(s):  
Gene Expression ◽  
Valproic Acid ◽  
Copy Number ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Viral Rna ◽  
Short Term Treatment ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Viral Copy Number

Abstract PURPOSE: Although the use of highly active antiretroviral therapy (HAART) has led to improvements in the management of AIDS-associated Kaposi’s sarcoma (KS), KS may persist or progress despite HAART. It has been hypothesized that activation of KS-associated herpesvirus (KSHV, HHV8) lytic expression might render tumor cells susceptible to immune surveillance by cytotoxic T cells. Alternatively, it has been suggested that lytic induction could lead to tumor progression. In vitro, valproic acid (VA) and other histone deacetylase inhibitors induce KSHV lytic gene expression in primary effusion lymphoma cell lines. We investigated VA in AIDS/KS patients to assess its safety and its impact on lytic viral gene expression in tumor and viral copy number in blood. PATIENTS AND METHODS: VA was given orally to patients with AIDS and cutaneous KS on stable antiviral regimens; the dose was titrated to maintain trough concentrations between 50 and 100 mcg/mL. VA was given daily for 28 days followed by a rapid taper and patients were then followed for 6 months. Quantitative real time PCR was used to assess viral DNA in plasma and PBMC, and viral RNA in tumor specimens. Immunohistochemistry was used to assess viral antigen expression in tumor specimens. RESULTS: 18 patients were treated. 15/18 patients completed therapy; 3 patients discontinued therapy early, one secondary to grade 2 toxicity and 2 to patient preference. One patient showed a partial response and 17 showed stable disease at the completion of therapy. No patients progressed during treatment. There were no differences between KSHV copy number in plasma or PBMC before, during, or after therapy. Similarly, although serial biopsies in some patients showed an increase in lytic gene expression, these changes did not achieve statistical significance. However, in multivariate analyses, viral lytic RNA increased in tumor biopsies on day 8 as a function of VA level. There was no change in HIV viral load with VA treatment. CONCLUSION : VA was well tolerated in AIDS patients, was not associated with accelerated disease progression, but rarely induced tumor regression after short-term treatment. In patients who achieved the highest serum VA levels there was increased lytic viral RNA expression. These findings support investigation of more potent HDAC inhibitors over longer treatment courses in patients with AIDS-associated KS.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus G-Protein-Coupled Receptor Prevents AU-Rich-Element-Mediated mRNA Decay

2012 ◽  
Vol 86 (16) ◽  
pp. 8859-8871 ◽  
Author(s):  
Jennifer A. Corcoran ◽  
Denys A. Khaperskyy ◽  
Benjamin P. Johnston ◽  
Christine A. King ◽  
David P. Cyr ◽  
...  
Keyword(s):  
Gene Expression ◽  
G Protein ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Mrna Degradation ◽  
Lytic Replication ◽  
G Protein Coupled Receptor ◽  
Infected Cells ◽  
Host Shutoff ◽  
G Protein Coupled

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these containcis-acting AU-rich elements (AREs) in their 3′ untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene ORF57 Is Essential for Infectious Virion Production

2006 ◽  
Vol 80 (11) ◽  
pp. 5251-5260 ◽  
Author(s):  
Zhao Han ◽  
Sankar Swaminathan
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Gene ◽  
Barr Virus ◽  
Virion Production ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The ORF57 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a nuclear protein expressed during the lytic phase of KSHV replication. An ORF57 homolog is present in all known human herpesviruses and many animal herpesviruses. Many of these proteins have been demonstrated to have essential transcriptional and posttranscriptional regulatory functions. ORF57 enhances expression of reporter genes posttranscriptionally in vitro and may synergize with transcription factors to enhance gene transcription. However, the biologic role of ORF57 in KSHV replication has not been established. In this study, we demonstrate that ORF57 is essential for productive KSHV lytic replication by constructing a recombinant KSHV in which ORF57 expression has been specifically inactivated. The ORF57-null KSHV recombinant was unable to produce virion progeny or fully express several other lytic KSHV genes except when ORF57 was provided in trans. The Epstein-Barr virus (EBV) homolog of ORF57, SM, was unable to rescue lytic KSHV virion production, although EBV SM does enhance KSHV lytic gene expression from the ORF57-null mutant. Conversely, ORF57 did not rescue an SM-null recombinant EBV, indicating the existence of virus-specific functions for the ORF57 family of genes.

scholarly journals Functional co-operation between the Kaposi's sarcoma-associated herpesvirus ORF57 and ORF50 regulatory proteins

2004 ◽  
Vol 85 (8) ◽  
pp. 2155-2166 ◽  
Author(s):  
Poonam Malik ◽  
David J. Blackbourn ◽  
Ming Fei Cheng ◽  
Gary S. Hayward ◽  
J. Barklie Clements
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Regulatory Proteins ◽  
Viral Gene ◽  
Transcriptional Level ◽  
Physical Interaction ◽  
Infected Cells

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) proteins ORF57 (also known as MTA) and ORF50 (also known as RTA) act post-transcriptionally and transcriptionally to regulate viral lytic gene expression and synergistically activate certain early and late KSHV promoters. When ORF57 and ORF50 were co-expressed, they co-operatively stimulated expression from the promoter of the immediate-early ORF50 gene itself. Co-immunoprecipitations with extracts of KSHV-infected cells showed that ORF57 and ORF50 proteins were present in the same complex. Using the pull-down assay with extracts of KSHV-infected cells, ORF50 protein was shown to interact with a glutathione S-transferase–ORF57 fusion protein. A chromatin immunoprecipitation assay showed that ORF50 promoter sequences were preferentially associated with immunoprecipitated chromatin using both anti-ORF50 and anti-ORF57 antibodies consistent with both an in vivo physical association between ORF57 and ORF50 and a potential role for ORF57 at the transcriptional level. This is the first demonstration of an interaction between these two lytic regulatory proteins in a gammaherpesvirus. Expression of ORF50 protein is sufficient to induce lytic replication in latently infected cells and may determine viral host range, spread and KS pathogenesis in vivo. A new insight into the co-ordinated activities of these two key regulatory proteins is provided in which upregulation of the ORF50 promoter with augmentation of ORF50 activity by ORF57 protein, and vice versa, would facilitate the cascade of lytic viral gene expression, thereby breaking latency. A functional and physical interaction between these two gammaherpesvirus regulatory protein counterparts could be a general feature of the herpesviruses.

scholarly journals Global Changes in Kaposi's Sarcoma-Associated Virus Gene Expression Patterns following Expression of a Tetracycline-Inducible Rta Transactivator

2003 ◽  
Vol 77 (7) ◽  
pp. 4205-4220 ◽  
Author(s):  
Hiroyuki Nakamura ◽  
Michael Lu ◽  
Yousang Gwack ◽  
John Souvlis ◽  
Steven L. Zeichner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Expression System ◽  
Kaposi’S Sarcoma ◽  
Lytic Gene ◽  
Gene Expression System ◽  
Lytic Gene Expression ◽  
Lytic Reactivation ◽  
Kshv Gene

ABSTRACT An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. In order to study the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), we developed a gene expression system in KSHV-infected primary effusion lymphoma cells. This system uses Flp-mediated efficient recombination and tetracycline-inducible expression. The Rta transcriptional activator, which acts as a molecular switch for lytic reactivation of KSHV, was efficiently integrated downstream of the Flp recombination target site, and its expression was tightly controlled by tetracycline. Like stimulation with tetradecanoyl phorbol acetate (TPA), the ectopic expression of Rta efficiently induced a complete cycle of viral replication, including a well-ordered program of KSHV gene expression and production of infectious viral progeny. A striking feature of Rta-mediated lytic gene expression was that Rta induced KSHV gene expression in a more powerful and efficient manner than TPA stimulation, indicating that Rta plays a central, leading role in KSHV lytic gene expression. Thus, our streamlined gene expression system provides a novel means not only to study the effects of viral gene products on overall KSHV gene expression and replication, but also to understand the natural viral reactivation process.

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