Viral Lytic Gene Research Articles

LKB1 suppresses KSHV reactivation and promotes primary effusion lymphoma progression.

Viruses normally reprogram the host cell metabolic pathways as well as metabolic sensors to facilitate their persistence. The serine-threonine liver kinase B1 (LKB1) is a master upstream kinase of 5'-AMP-activated protein kinase (AMPK) that senses the energy status and therefore regulates the intracellular metabolic homeostasis. Previous studies showed that AMPK restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in endothelial cells during primary infection and promotes primary effusion lymphoma (PEL) cell survival. However, the role of LKB1 in KSHV lytic reactivation and KSHV-associated malignancies is unclear. In this study, we found that LKB1 is phosphorylated or activated in KSHV-positive PEL cells. Mechanistically, KSHV-encoded vCyclin mediated LKB1 activation in PEL cells, as vCyclin knockout ablated, while vCyclin overexpression enhanced LKB1 activation. Furthermore, knockdown of LKB1 inactivated AMPK and induced KSHV reactivation, as indicated by the increased expression of viral lytic genes and the increased virions in supernatants. Accordingly, AMPK inhibition by functional knockdown or a pharmacologic inhibitor, Compound C, promoted KSHV reactivation in PEL cells. Furthermore, inhibition of either LKB1 or AMPKα1 efficiently induced cell death by apoptosis of PEL cells both in vitro and in vivo. Together, these results identify LKB1 as a vulnerable target for PEL, which could be potentially exploited for treating other virus-associated diseases.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human cancers, such as primary effusion lymphoma (PEL). Here, we showed that serine-threonine liver kinase B1 (LKB1), upstream of 5' AMP-activated protein kinase (AMPK), is activated by KSHV-encoded vCyclin and maintains KSHV latency in PEL cells. Inhibition of either LKB1 or AMPK enhances KSHV lytic replication from latency, which at least partially accounts for PEL cell death by apoptosis. Compound C, a potent AMPK inhibitor, induced KSHV reactivation and efficiently inhibited PEL progression in vivo. Thus, our work revealed that LKB1 is a potential therapeutic target for KSHV-associated cancers.

Co-option of mitochondrial nucleic acid sensing pathways by HSV-1 UL12.5 for reactivation from latent Infection.

Although viruses subvert innate immune pathways for their replication, there is evidence they can also co-opt anti-viral responses for their benefit. The ubiquitous human pathogen, Herpes Simplex Virus-1 (HSV-1), encodes a protein (UL12.5) that induces the release of mitochondrial nucleic acid into the cytosol, which activates immune sensing pathways and reduces productive replication in non-neuronal cells. HSV-1 establishes latency in neurons and can reactivate to cause disease. We found that UL12.5 is required for HSV-1 reactivation in neurons and acts to directly promote viral lytic gene expression during initial exit from latency. Further, the direct activation of innate immune sensing pathways triggered HSV reactivation and compensated for a lack of UL12.5. Finally, we found that the induction of HSV-1 lytic genes during reactivation required intact RNA and DNA sensing pathways, demonstrating that HSV-1 can both respond to and active antiviral nucleic acid sensing pathways to reactivate from a latent infection.

Lactylation of NAT10 promotes N4-acetylcytidine modification on tRNASer-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation.

N4-acetylcytidine (ac4C), a conserved but recently rediscovered RNA modification on tRNAs, rRNAs and mRNAs, is catalyzed by N-acetyltransferase 10 (NAT10). Lysine acylation is a ubiquitous protein modification that controls protein functions. Our latest study demonstrates a NAT10-dependent ac4C modification, which occurs on the polyadenylated nuclear RNA (PAN) encoded by oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), can induce KSHV reactivation from latency and activate inflammasome. However, it remains unclear whether a novel lysine acylation occurs in NAT10 during KSHV reactivation and how this acylation of NAT10 regulates tRNAs ac4C modification. Here, we showed that NAT10 was lactylated by α-tubulin acetyltransferase 1 (ATAT1), as a writer at the critical domain, to exert RNA acetyltransferase function and thus increase the ac4C level of tRNASer-CGA-1-1. Mutagenesis at the ac4C site in tRNASer-CGA-1-1 inhibited its ac4C modifications, translation efficiency of viral lytic genes, and virion production. Mechanistically, KSHV PAN orchestrated NAT10 and ATAT1 to enhance NAT10 lactylation, resulting in tRNASer-CGA-1-1 ac4C modification, eventually boosting KSHV reactivation. Our findings reveal a novel post-translational modification in NAT10, as well as expand the understanding about tRNA-related ac4C modification during KSHV replication, which may be exploited to design therapeutic strategies for KSHV-related diseases.

Neuronal miR-9 promotes HSV-1 epigenetic silencing and latency by repressing Oct-1 and Onecut family genes

Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.

Kaposi's sarcoma-associated herpesvirus (KSHV) LANA prevents KSHV episomes from degradation.

Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone 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. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.

Kaposi's sarcoma herpesvirus latency-associated nuclear antigen broadly regulates viral gene expression and is essential for lytic infection.

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.

Hypoxia and HIF-1α promote lytic de novo KSHV infection.

The current view is that the default pathway of Kaposi's sarcoma-associated herpesvirus (KSHV) infection is the establishment of latency, which is a prerequisite for lifelong infection and viral oncogenesis. This view about KSHV infection is supported by the observations that KSHV latently infects most of the cell lines cultured in vitro in the absence of any environmental stresses that may occur in vivo. The goal of this study was to determine the effect of hypoxia, a natural stress stimulus, on primary KSHV infection. Our data indicate that hypoxia promotes euchromatin formation on the KSHV genome following infection and supports lytic de novo KSHV infection. We also discovered that hypoxia-inducible factor-1α is required and sufficient for allowing lytic KSHV infection. Based on our results, we propose that hypoxia promotes lytic de novo infection in cells that otherwise support latent infection under normoxia; that is, the environmental conditions can determine the outcome of KSHV primary infection.

KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing human herpesvirus that establishes a persistent infection in humans. The lytic viral cycle plays a crucial part in lifelong infection as it is involved in the viral dissemination. The master regulator of the KSHV lytic replication cycle is the viral replication and transcription activator (RTA) protein, which is necessary and sufficient to push the virus from latency into the lytic phase. Thus, the identification of host factors utilized by RTA for controlling the lytic cycle can help to find novel targets that could be used for the development of antiviral therapies against KSHV. Using a proteomics approach, we have identified a novel interaction between RTA and the cellular E3 ubiquitin ligase complex RNF20/40, which we have shown to be necessary for promoting RTA-induced KSHV lytic cycle.

Ostreid herpesvirus 1 latent infection and reactivation in adult Pacific oysters, Crassostrea gigas

Ostreid herpesvirus 1 (OsHV-1) is one of the most economically important pathogens of Pacific oysters. Understanding the pathogenesis of this virus is critical to developing tools to control outbreaks on shellfish farms. OsHV-1 is genetically related to vertebrate herpesviruses, which have a lytic and a latent stage, with the latent stage capable of being reactivated to the lytic stage. Here, OsHV-1 latency in Pacific oysters was investigated in experimentally and naturally infected oysters. Lytic infection in one-year-old oysters injected with the Tomales Bay strain of OsHV-1 was detectable between 1 and 4 days post-injection (dpi) but was not detectable after 5 dpi. The injected oysters shed 1 × 102 to 1 × 104 DNA copies/ml into the water during the 4-day acute phase. Lytic shedding was not detectable in two-year-old oysters injected similarly with the same strain of OsHV-1; however, the OsHV-1 genome was detectable by qPCR in the adductor muscle, gill, mantle, and hemocytes within the first 3 dpi, after which it became undetectable. No OsHV-1 was detectable in the adductor muscle, gill, or mantle from experimentally infected oysters on days 15 and 21 post-injection or from oysters sampled 9 months after surviving an OsHV-1 mortality event; however, OsHV-1 DNA could be detected in hemocytes of both experimentally infected oysters at 21 dpi and naturally infected oysters using nested PCR. In addition, lytic viral gene transcription was detectable in hemocytes of experimentally infected oysters between 1 and 21 dpi and in hemocytes of naturally infected oysters. Furthermore, OsHV-1 reactivation from latency was induced in experimentally infected oysters at 21 dpi and in naturally infected oysters 12 months after an OsHV-1 outbreak.

NAT10-dependent N4‐acetylcytidine modification mediates PAN RNA stability, KSHV reactivation, and IFI16-related inflammasome activation

N-acetyltransferase 10 (NAT10) is an N4‐acetylcytidine (ac4C) writer that catalyzes RNA acetylation at cytidine N4 position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac4C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac4C addition to the polyadenylated nuclear RNA (PAN), a long non-coding RNA encoded by the oncogenic DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV), triggering viral lytic reactivation from latency. Mutagenesis of ac4C sites in PAN RNA in the context of KSHV infection abolishes PAN ac4C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac4C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac4C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac4C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac4C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity.

Transcriptional landscape of Kaposi sarcoma tumors identifies unique immunologic signatures and key determinants of angiogenesis

BackgroundKaposi sarcoma (KS) is a multicentric tumor caused by Kaposi sarcoma herpesvirus (KSHV) that leads to morbidity and mortality among people with HIV worldwide. KS commonly involves the skin but can occur in the gastrointestinal tract (GI) in severe cases.MethodsRNA sequencing was used to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions in 44 paired samples from 19 participants with KS alone or with concurrent KSHV-associated diseases. Analyses of KSHV expression from KS lesions identified transcriptionally active areas of the viral genome.ResultsThe transcript of an essential viral lytic gene, ORF75, was detected in 91% of KS lesions. Analyses of host genes identified 370 differentially expressed genes (DEGs) unique to skin KS and 58 DEGs unique to GI KS lesions as compared to normal tissue. Interleukin (IL)-6 and IL-10 gene expression were higher in skin lesions as compared to normal skin but not in GI KS lesions. Twenty-six cellular genes were differentially expressed in both skin and GI KS tissues: these included Fms-related tyrosine kinase 4 (FLT4), encoding an angiogenic receptor, and Stanniocalcin 1 (STC1), a secreted glycoprotein. FLT4 and STC1 were further investigated in functional studies using primary lymphatic endothelial cells (LECs). In these models, KSHV infection of LECs led to increased tubule formation that was impaired upon knock-down of STC1 or FLT4.ConclusionsThis study of transcriptional profiling of KS tissue provides novel insights into the characteristics and pathogenesis of this unique virus-driven neoplasm.

Inhibition of SIRT2 promotes death of human cytomegalovirus-infected peripheral blood monocytes via apoptosis and necroptosis

Peripheral blood monocytes are the cells predominantly responsible for systemic dissemination of human cytomegalovirus (HCMV) and a significant cause of morbidity and mortality in immunocompromised patients. HCMV establishes a silent/quiescent infection in monocytes, which is defined by the lack of viral replication and lytic gene expression. The absence of replication shields the virus within infected monocytes from the current available antiviral drugs that are designed to suppress active replication. Our previous work has shown that HCMV stimulates a noncanonical phosphorylation of Akt and the subsequent upregulation of a distinct subset of prosurvival proteins in normally short-lived monocytes. In this study, we found that SIRT2 activity is required for the unique activation profile of Akt induced within HCMV-infected monocytes. Importantly, both therapeutic and prophylactic treatment with a novel SIRT2 inhibitor, FLS-379, promoted death of infected monocytes via both the apoptotic and necroptotic cell death pathways. Mechanistically, SIRT2 inhibition reduced expression of Mcl-1, an Akt-dependent antiapoptotic Bcl-2 family member, and enhanced activation of MLKL, the executioner kinase of necroptosis. We have previously reported HCMV to block necroptosis by stimulating cellular autophagy. Here, we additionally demonstrate that inhibition of SIRT2 suppressed Akt-dependent HCMV-induced autophagy leading to necroptosis of infected monocytes. Overall, our data show that SIRT2 inhibition can simultaneously promote death of quiescently infected monocytes by two distinct death pathways, apoptosis and necroptosis, which may be vital for limiting viral dissemination to peripheral organs in immunosuppressed patients.

Expression of EBV-encoded genes in children with asymptomatic infection detected by sensitive methods

Epstein–Barr virus (EBV) is an usually harmless virus whose oncogenic properties in vitro are related to its ability to transform lymphoid cells, and, in consequence, it can be associated with lymphomas. Since a few studies detected EBV presence in supposedly EBV-negative lymphomas, our aim was to evaluate EBV presence by sensitive gene expression assays in the tonsils from healthy pediatric donors from a region with high incidence of EBV-associated lymphomas. EBERs transcripts were detected by View RNA ISH in all cases, even in cases assessed negative by widely used in situ hybridization. The presence of LMP1 transcripts was proved in 93% of cases, co-expressed with EBNA2 in 30%. In this study, evidence for the expression of different latent and lytic viral genes in a population of young age of primary infection, detected with more sensitive methods, in particular at the germinal center, where most EBV-associated lymphomas originate, was provided.

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

In silico interrogation of the miRNAome of infected hematopoietic cells to predict processes important for human cytomegalovirus latent infection

Human cytomegalovirus (HCMV) latency in CD34+ progenitor cells is the outcome of a complex and continued interaction of virus and host that is initiated during very early stages of infection and reflects pro- and anti-viral activity. We hypothesized that a key event during early infection could involve changes to host miRNAs, allowing for rapid modulation of the host proteome. Here, we identify 72 significantly upregulated miRNAs and three that were downregulated by 6hpi of infection of CD34+ cells which were then subject to multiple in silico analyses to identify potential genes and pathways important for viral infection. The analyses focused on the upregulated miRNAs and were used to predict potential gene hubs or common mRNA targets of multiple miRNAs. Constitutive deletion of one target, the transcriptional regulator JDP2, resulted in a defect in latent infection of myeloid cells; interestingly, transient knockdown in differentiated dendritic cells resulted in increased viral lytic IE gene expression, arguing for subtle differences in the role of JDP2 during latency establishment and reactivation of HCMV. Finally, in silico predictions identified clusters of genes with related functions (such as calcium signaling, ubiquitination, and chromatin modification), suggesting potential importance in latency and reactivation. Consistent with this hypothesis, we demonstrate that viral IE gene expression is sensitive to calcium channel inhibition in reactivating dendritic cells. In conclusion, we demonstrate HCMV alters the miRNAome rapidly upon infection and that in silico interrogation of these changes reveals new insight into mechanisms controlling viral gene expression during HCMV latency and, intriguingly, reactivation.

Dendrosomal nanocurcumin prevents EBV-associated cell transformation by targeting the lytic cycle genes of the Epstein-Barr virus in the generation of lymphoblastoid cell line.

Targeting the lytic cycle of the Epstein-Barr virus (EBV) has been considered a new treatment strategy for malignancies caused by this virus. This study aimed to investigate the effect of Dendrosomal NanoCurcumin (DNC) to prevent cell transformation and inhibit the expression of viral lytic gene expression in the generation of lymphoblastoid cell line (LCL). Cell viability of LCLs and PBMCs was performed by MTT assay, and flow cytometry (Annexin/PI) was used for evaluation of apoptosis. CD markers on the surface of generated LCL (CD19) cells were examined for cell validation. The effect of DNC on transformation was evaluated by examining cell morphology and determining the expression level of lytic genes BZLF1, Zta, BHRF1, and BRLF1 of EBV using Real-time PCR. Student's t-test was used for statistical analysis. The MTT assay showed that DNC can inhibit the proliferation of LCL in a dose-dependent manner. The 50% cytotoxic concentration (CC50) of DNC and curcumin for LCL was determined 38.8 µg/ml and 75 µg/ml, respectively after 72 hr. Also, Real-time PCR data analysis showed that DNC in 30 µg/ml concentration significantly inhibited cell transformation in the LCL and significantly reduced viral lytic genes such as BZLF1, Zta, BHRF1, and BRLF1expression compared to control. Overall, these findings show that DNC reduces the expression of the viral lytic cycle genes and also the induction of cell apoptosis and finally prevents the generation of LCL.

Stress Triggers Expression of Bovine Herpesvirus 1 Infected Cell Protein 4 (bICP4) RNA during Early Stages of Reactivation from Latency in Pharyngeal Tonsil.

Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, establishes lifelong latency in sensory neurons within trigeminal ganglia (TG) after acute infection. The BoHV-1 latency-reactivation cycle, like other alphaherpesvirinae subfamily members, is essential for viral persistence and transmission. Notably, cells within pharyngeal tonsil (PT) also support a quiescent or latent BoHV-1 infection. The synthetic corticosteroid dexamethasone, which mimics the effects of stress, consistently induces BoHV-1 reactivation from latency allowing early stages of viral reactivation to be examined in the natural host. Based on previous studies, we hypothesized that stress-induced cellular factors trigger expression of key viral transcriptional regulatory genes. To explore this hypothesis, RNA-sequencing studies compared viral gene expression in PT during early stages of dexamethasone-induced reactivation from latency. Strikingly, RNA encoding infected cell protein 4 (bICP4), which is translated into an essential viral transcriptional regulatory protein, was detected 30 min after dexamethasone treatment. Ninety minutes after dexamethasone treatment bICP4 and, to a lesser extent, bICP0 RNA were detected in PT. All lytic cycle viral transcripts were detected within 3 h after dexamethasone treatment. Surprisingly, the latency related (LR) gene, the only viral gene abundantly expressed in latently infected TG neurons, was not detected in PT during latency. In TG neurons, bICP0 and the viral tegument protein VP16 are expressed before bICP4 during reactivation, suggesting distinct viral regulatory genes mediate reactivation from latency in PT versus TG neurons. Finally, these studies confirm PT is a biologically relevant site for BoHV-1 latency, reactivation from latency, and virus transmission. IMPORTANCE BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. RNA-sequencing studies revealed the viral infected cell protein 4 (bICP4) RNA was expressed in PT of latently infected calves within 30 min after dexamethasone was used to initiate reactivation. As expected, bICP4 RNA was not detected during latency. All lytic cycle viral genes were expressed within 3 h after dexamethasone treatment. Conversely, bICP0 and the viral tegument protein VP16 are expressed prior to bICP4 in trigeminal ganglionic neurons during reactivation. The viral latency related gene, which is abundantly expressed in latently infected neurons, was not abundantly expressed in PT during latency. These studies provide new evidence PT is a biologically relevant site for BoHV-1 latency and reactivation. Finally, we predict other alphaherpesvirinae subfamily members utilize PT as a site for latency and reactivation.

Mitochondrial protein, TBRG4, modulates KSHV and EBV reactivation from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr (EBV) are gammaherpesviruses associated with multiple human malignancies. KSHV is the etiological agent of Kaposi's Sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). EBV is associated with Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). KSHV and EBV establish life-long latency in the human host with intermittent periods of lytic reactivation. Here, we identified a cellular factor named transforming growth factor-beta regulator 4 (TBRG4) that plays a role in the gammaherpesvirus lifecycle. We find that TBRG4, a protein that is localized to the mitochondria, can regulate lytic reactivation from latency of both KSHV and EBV. Knockdown of TBRG4 in cells latently infected with KSHV or EBV induced viral lytic gene transcription and replication. TBRG4 deficiency causes mitochondrial stress and increases reactive oxygen species (ROS) production. Treatment with a ROS scavenger decreased viral reactivation from latency in TBRG4-depleted cells. These data suggest that TBRG4 serves as a cellular repressor of KSHV and EBV reactivation through the regulation of ROS production.

Plasma EBV DNA Methylation: Evidence of EBV Lytic Infection in HIV(+) EBV(+) Plasmablastic Lymphoma

Background: Among people living with HIV, plasmablastic lymphoma (PBL) and classical Hodgkin lymphoma (HL) are almost uniformly associated with Epstein-Barr virus (EBV). Immunohistochemical studies have demonstrated high levels of lytic gene expression in PL,1 whereas EBV(+) HL shows exclusively latent viral gene expression. EBV virion DNA is never CpG methylated, but tumor-derived plasma EBV is CpG methylated as previously shown by our group in EBV(+) HL and nasopharyngeal carcinoma (NPC).2 Others have also presented evidence that patterns of CpG methylation of plasma EBV DNA varies between people with NPC and non-malignant EBV(+) conditions.3 To better characterize the nature of plasma EBV in patients with lymphoma, we investigated EBV DNA copy number and EBV DNA methylation in HIV-associated PBL and HL. Methods: With appropriate approvals from the Human Research Ethics Committee of the University of the Witwatersrand and the Johns Hopkins Institutional Review Boards, we collected plasma from newly diagnosed untreated patients with HIV and PBL or HL in Johannesburg, South Africa. Diagnostic biopsy specimens were reviewed in Baltimore by a hematopathologist to confirm the diagnosis and Epstein-Barr encoding region (EBER) in situ hybridization was performed to determine EBV association. Plasma EBV copies were measured by standard qPCR targeting the BamW repeats. qPCR using the same primers was also performed after capture with methyl-DNA binding protein 2 (MBD2) magnetic beads, where the captured fraction represents methylated DNA and the flow-through represents unmethylated DNA. Results: We studied plasma from 7 patients with PBL and 12 patients with HL. Four of five PBL tumors were EBV (+) and eight of 10 HL tumors were EBV (+) by tissue EBER in situ hybridization. Analysis of plasma EBV DNA copy number showed overlapping levels of EBV DNA (median 2.7 Log10 copy/mL in PBL and 3.2 Log10 copy/mL in HL) (Fig. 1). Patients with at least 1.5 Log10 copy/mL of EBV underwent CpG methylation evaluation, which showed very different EBV CpG methylation indices. The EBV methylation index was highly variable in PBL (median of 51%) and was uniformly high in HL (median of 97%). Conclusions: Although plasma from plasmablastic lymphoma patients show high EBV copy number, the characteristic of that EBV DNA is very different from the plasma EBV detected in Hodgkin lymphoma patients. This difference is hypothesized to be related to evidence of viral lytic gene expression seen in PBL. The degree of plasma EBV DNA methylation may have broad implications for lymphoma diagnosis and subtyping.

The Human Cytomegalovirus β2.7 Long Non-Coding RNA Prevents Induction of Reactive Oxygen Species to Maintain Viral Gene Silencing during Latency.

Human cytomegalovirus (HCMV) is a significant source of disease for the immunosuppressed and immunonaive. The treatment of HCMV is made more problematic by viral latency, a lifecycle stage in which the virus reduces its own gene expression and produces no infectious virus. The most highly expressed viral gene during HCMV latency is the viral β2.7 long non-coding RNA. Although we have recently shown that the β2.7 lncRNA lowers levels of reactive oxygen species (ROS) during infection in monocytes, how this impacts latency is unclear. We now show that β2.7 is important for establishing and maintaining HCMV latency by aiding the suppression of viral lytic gene expression and that this is directly related to its ability to quench reactive oxygen species (ROS). Consistent with this, we also find that exogenous inducers of ROS cause reactivation of latent HCMV. These effects can be compensated by treatment with an antioxidant to lower ROS levels. Finally, we show that ROS-mediated reactivation is independent of myeloid differentiation, but instead relies on NF-κB activation. Altogether, these results reveal a novel factor that is central to the complex process that underpins HCMV latency. These findings may be of particular relevance in the transplant setting, in which transplanted tissue/organs are subject to very high ROS levels, and HCMV reactivation poses a significant threat.