Lytic Cycle Research Articles

A prophage competition element protects Salmonella from lysis.

Most bacteria are polylysogens that carry multiple prophages integrated into the chromosome. These prophages confer advantages to their bacterial host, yetalso pose a lethal threat as they can reactivate and enter a lytic cycle. DNA damage of the bacterial host is a common trigger of prophage lytic cycles. Because DNA damage is frequently experienced by bacterial pathogens exposed to host immune defenses, prophage activation may be common during infection. Investigating the consequences of prophage induction in Salmonella, we discover a prophage competition element in the Gifsy-1 prophage that we name ribonuclease effector module with ATPase, inhibitor, and nuclease (RemAIN) because it blocks the lytic cycles and release of viral particles of co-resident prophages. Intramacrophage Salmonella persisters, a subpopulation that incurs DNA damage, experience prophage reactivation and subsequent RemAIN activation, which influences Salmonella persisters and macrophage response to infection. Our findings reveal a multi-layered host-pathogen arms race in which prophage-prophage competition influences bacterial persistence and the mammalian immune response.

Curcumin derivative C210 induces Epstein–Barr virus lytic cycle and inhibits virion production by disrupting Hsp90 function

Lytic induction therapy was devised to selectively combat malignancies associated with Epstein–Barr virus (EBV) by triggering viral reactivation from latency. At present, the major challenges of lytic induction therapy are to maximize reactivating efficiencies and meanwhile minimize infectious virion production. C210, a novel curcumin derivative with potent Hsp90 inhibitory activity, was explored for EBV-reactivating and virion-producing effects in EBV-positive nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC) cell lines. And the molecular mechanisms underlying these effects were determined. Follow C210 treatment, EBV lytic RNAs and proteins were upregulated, but infectious virions were not produced. Knockdown of heat shock protein 90 (Hsp90) induced expression of lytic RNAs and proteins, and diminished C210-driven EBV lytic induction. Pretreatment with an X box binding protein 1 (XBP1) inhibitor reduced C210-induced EBV lytic RNA. Furthermore, we demonstrated that C210 inhibited the binding of Hsp90 with its clients, signal transducer and activator of transcription 3 (STAT3) and xeroderma pigmentosum group B-complementing protein (XPB), which subsequently promoted their proteasomal degradation. Degradation of STAT3 by C210 enhanced the EBV-reactivating and anticancer capacity of suberoylanilide hydroxamic acid (SAHA). Depletion of XPB blocked SAHA-induced expression of late viral genes and production of infectious virions. These results elucidate a novel Hsp90 inhibitor targeting EBV lytic phase and extend the research on lytic induction strategy, which may offer reference value in the treatment of EBV-positive malignancies.

Autoregulation ensures vertical transmission of the linear prophage GIL01

Betatectiviruses are prophages consisting of linear extrachromosomal genomes without obvious plasmid modules. It remains unclear how betatectiviruses are maintained in low-copy numbers in host cells and how they are vertically transmitted. Phage GIL01 is a model betatectivirus that infects the mosquito pathogen Bacillus thuringiensis serovar israelensis. Previous studies identified two closely spaced promoters, P1 and P2, responsible for the expression of GIL01 genes required for prophage replication and the switch from the lysogenic to lytic cycle. Here, we report that the GIL01-encoded 58-amino acid long gp1 protein forms a large nucleoprotein complex that represses its transcription from the strong promoter P2. Notably, ectopic expression of gp1 resulted in the loss of GIL01 in exponential cultures and immunized cells against infection with GIL01, indicating that gp1 plays a repressive role in the phage cycle. This finding is consistent with mutations in gp1 committing GIL01 to the lytic cycle and we show that maintenance of this phage variant in the bacterial population is contingent on the accumulation of deletions in the P1-P2 region. The fact that gp1 is conserved across most sequenced betatectiviruses suggests that the regulatory mechanism of gp1 that controls prophage maintenance is widespread among these bacteriophages.

Epstein-Barr virus reactivation induces divergent abortive, reprogrammed, and host shutoff states by lytic progression.

Viral infection leads to heterogeneous cellular outcomes ranging from refractory to abortive and fully productive states. Single cell transcriptomics enables a high resolution view of these distinct post-infection states. Here, we have interrogated the host-pathogen dynamics following reactivation of Epstein-Barr virus (EBV). While benign in most people, EBV is responsible for infectious mononucleosis, up to 2% of human cancers, and is a trigger for the development of multiple sclerosis. Following latency establishment in B cells, EBV reactivates and is shed in saliva to enable infection of new hosts. Beyond its importance for transmission, the lytic cycle is also implicated in EBV-associated oncogenesis. Conversely, induction of lytic reactivation in latent EBV-positive tumors presents a novel therapeutic opportunity. Therefore, defining the dynamics and heterogeneity of EBV lytic reactivation is a high priority to better understand pathogenesis and therapeutic potential. In this study, we applied single-cell techniques to analyze diverse fate trajectories during lytic reactivation in three B cell models. Consistent with prior work, we find that cell cycle and MYC expression correlate with cells refractory to lytic reactivation. We further found that lytic induction yields a continuum from abortive to complete reactivation. Abortive lytic cells upregulate NFκB and IRF3 pathway target genes, while cells that proceed through the full lytic cycle exhibit unexpected expression of genes associated with cellular reprogramming. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.

Metagenomic characterization of viruses and mobile genetic elements associated with the DPANN archaeal superphylum.

The archaeal superphylum DPANN (an acronym formed from the initials of the first five phyla discovered: Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota and Nanoarchaeota) is a group of ultrasmall symbionts able to survive in extreme ecosystems. The diversity and dynamics between DPANN archaea and their virome remain largely unknown. Here we use a metagenomic clustered regularly interspaced short palindromic repeats (CRISPR) screening approach to identify 97 globally distributed, non-redundant viruses and unclassified mobile genetic elements predicted to infect hosts across 8 DPANN phyla, including 7 viral groups not previously characterized. Genomic analysis suggests a diversity of viral morphologies including head-tailed, tailless icosahedral and spindle-shaped viruses with the potential to establish lytic, chronic or lysogenic infections. We also find evidence of a virally encoded Cas12f1 protein (probably originating from uncultured DPANN archaea) and a mini-CRISPR array, which could play a role in modulating host metabolism. Many metagenomes have virus-to-host ratios >10, indicating that DPANN viruses play an important role in controlling host populations. Overall, our study illuminates the underexplored diversity, functional repertoires and host interactions of the DPANN virome.

Epstein-Barr virus infection induces tissue-resident memory T cells in mucosal lymphoid tissues.

EBV contributes to around 2% of all tumors worldwide. Simultaneously, more than 90% of healthy human adults persistently carry EBV without clinical symptoms. In most EBV carriers, it is thought that virus-induced tumorigenesis is prevented by cell-mediated immunity. Specifically, memory CD8+ T cells recognize EBV-infected cells during latent and lytic infection. Using a symptomatic primary infection model, similar to infectious mononucleosis (IM), we found EBV-induced CD8+ tissue resident memory T cells (TRMs) in mice with a humanized immune system. These human TRMs were preferentially established after intranasal EBV infection in nasal-associated lymphoid tissues (NALT), equivalent to tonsils, the primary site of EBV infection in humans. They expressed canonical TRM markers, including CD69, CD103, and BLIMP-1, as well as granzyme B, CD107a, and CCL5. Despite cytotoxic activity and cytokine production ex vivo, these TRMs demonstrated reduced CD27 expression and proliferation and failed to control EBV viral loads in the NALT during infection, although effector memory T cells (TEMs) controlled viral titers in spleen and blood. Overall, TRMs are established in mucosal lymphoid tissues by EBV infection, but primarily, systemic CD8+ T cell expansion seems to control viral loads in the context of IM-like infection.

DeepPL: A deep-learning-based tool for the prediction of bacteriophage lifecycle.

Bacteriophages (phages) are viruses that infect bacteria and can be classified into two different lifecycles. Virulent phages (or lytic phages) have a lytic cycle that can lyse the bacteria host after their infection. Temperate phages (or lysogenic phages) can integrate their phage genomes into bacterial chromosomes and replicate with bacterial hosts via the lysogenic cycle. Identifying phage lifecycles is a crucial step in developing suitable applications for phages. Compared to the complicated traditional biological experiments, several tools have been designed for predicting phage lifecycle using different algorithms, such as random forest (RF), linear support-vector classifier (SVC), and convolutional neural network (CNN). In this study, we developed a natural language processing (NLP)-based tool-DeepPL-for predicting phage lifecycles via nucleotide sequences. The test results showed that our DeepPL had an accuracy of 94.65% with a sensitivity of 92.24% and a specificity of 95.91%. Moreover, DeepPL had 100% accuracy in lifecycle prediction on the phages we isolated and biologically verified previously in the lab. Additionally, a mock phage community metagenomic dataset was used to test the potential usage of DeepPL in viral metagenomic research. DeepPL displayed a 100% accuracy for individual phage complete genomes and high accuracies ranging from 71.14% to 100% on phage contigs produced by various next-generation sequencing technologies. Overall, our study indicates that DeepPL has a reliable performance on phage lifecycle prediction using the most fundamental nucleotide sequences and can be applied to future phage and metagenomic research.

Evaluating STING Agonist Impact on Kaposi’s Sarcoma-Associated Virus-related Cancers

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiological agent of multiple malignancies associated with compromised immunity, such as Kaposi’s sarcoma (KS) and Primary Effusion Lymphoma (PEL). KSHV infection is lifelong with no available treatment or vaccines and poses a high risk to immunocompromised individuals. The virus is transmitted via the oral and sexual route, and has a biphasic life cycle with a short lytic replication cycle followed by long-term latency, with the expression of few genes making detection difficult. Reactivation from latency results in a cascade of viral gene expression and pathogenesis. Critical to controlling KSHV infection is the cGAS-STING DNA-sensing pathway (STING), known to activate the type I interferon response and induce cancer cell death or growth inhibition. STING exhibits both anti-viral and anti-tumor responses, showing promise as a protein of interest to study for the development of a therapeutic treatment. By examining the in vitro effectiveness of STING agonists in inhibiting KSHV oncogenesis in PEL, the study aimed to provide the foundation for a potential novel therapeutic approach. Six patient-derived PEL cell lines were treated with the STING agonist diaminobenzamidazole (diABZI) in a dose-dependent manner. Four of the PEL cell lines exhibited decreased cell growth, viability, and colony formation, while two PEL cell lines were resistant to treatment, correlating with the presence or absence of STING expression. RNA-sequencing was performed and discovered upregulation of multiple interferon-stimulated genes (ISG) in responsive cell lines. Further investigations will explore STING agonists’ inhibition of PEL oncogenesis in vitro via colony formation and in vivo using a PEL xenograft model with NSG mice. Successful completion of this project will offer insight into potential clinical treatments for targeting KSHV infection and reactivation.

Isolation and preliminary characterization of a novel bacteriophage vB_KquU_φKuK6 that infects the multidrug-resistant pathogen Klebsiella quasipneumoniae.

Klebsiella quasipneumoniae (previously known as K. pneumoniae K6) strains are among the multidrug-resistant hypervirulent bacterial pathogens. Phage therapy can help treat infections caused by such pathogens. Here we report some aspects of virology and therapeutic potentials of vB_KquU_φKuK6, a bacteriophage that infects Klebsiella quasipneumoniae. K. quasipneumoniae (ATCC 700603) was used to screen wastewater lytic phages. The isolate vB_KquU_φKuK6 that consistently created large clear plaques was characterized using standard virological and molecular methods. vB_KquU_φKuK6 has a complex capsid with an icosahedral head (~60 nm) and a slender tail (~140 nm × 10 nm). The phage has a 51% AT-rich linear dsDNA genome (51,251 bp) containing 121 open reading frames. The genome contains genes encoding spanin, endolysin, and holin proteins necessary for lytic infection and a recombinase gene possibly involved in lysogenic infection. vB_KquU_φKuK6 is stable at -80 to +67°C, pH 4-9, and brief exposure to one volume percent of chloroform. vB_KquU_φKuK6 has a narrow host range. Its lytic infection cycle involves a latency of 20 min and a burst size of 435 plaque-forming units. The phage can cause lysogenic infection, and the resulting lysogens are resistant to lytic infection by vB_KquU_φKuK6. vB_KquU_φKuK6 reduces the host cells' ability to form biofilm but fails to eliminate that ability. vB_KquU_φKuK6 demonstrates phage-antibiotic synergy and reduces the minimum inhibitory concentration of chloramphenicol and neomycin sulfate by about 8 folds. vB_KquU_φKuK6 cannot be directly used for phage therapy because it is a temperate bacteriophage. However, genetically modified strains of vB_KquU_φKuK6 alone or combined with antibiotics or other lytic Klebsiella phages can have therapeutic utilities in treating K. quasipneumoniae infections.

Immune Modulation by Epstein-Barr Virus Lytic Cycle: Relevance and Implication in Oncogenesis.

EBV infects more than 90% of people globally, causing lifelong infection. The phases of the EBV life cycle encompass primary infection, latency, and subsequent reactivation or lytic phase. The primary infection usually happens without noticeable symptoms, commonly in early life stages. If it manifests after childhood, it could culminate in infectious mononucleosis. Regarding potential late consequences, EBV is associated with multiple sclerosis, rheumatoid arthritis, chronic active EBV infection, lymphomas, and carcinomas. Previous reports that the lytic phase plays a negligible or merely secondary role in the oncogenesis of EBV-related tumors are steadily losing credibility. The right mechanisms through which the lytic cycle contributes to carcinogenesis are still unclear, but it is now recognized that lytic genes are expressed to some degree in different cancer-type cells, implicating their role here. The lytic infection is a persistent aspect of virus activity, continuously stimulating the immune system. EBV shows different strategies to modulate and avoid the immune system, which is thought to be a key factor in its ability to cause cancer. So, the principal goal of our review is to explore the EBV's lytic phase contribution to oncogenesis.

The HSV-1 encoded CCCTC-binding factor, CTRL2, impacts the nature of viral chromatin during HSV-1 lytic infection.

HSV-1 genomes are rapidly heterochromatinized following entry by host cells to limit viral gene expression. Efficient HSV-1 genome replication requires mechanisms that de-repress chromatin associated with the viral genome. CCCTC-binding factors, or CTCF insulators play both silencing and activating roles in cellular transcriptional regulation. Importantly, the HSV-1 genome encodes several CTCF insulators that flank IE genes, implying that individual HSV-1 encoded CTCF insulators regulate IE transcription during all stages of the HSV-1 life cycle. We previously reported that the HSV-1 encoded CTCF insulator located downstream of the LAT (CTRL2) controlled IE gene silencing during latency. To further characterize the role of this insulator during the lytic infection we leveraged a ΔCTRL2 recombinant virus to show that there was a genome replication defect that stemmed from decreased IE gene expression in fibroblasts and epithelial cells at early times following initiation of infection. Further experiments indicated that the defect in gene expression resulted from chromatin inaccessibility in the absence of the insulator. To elucidate how chromatin accessibility was altered in the absence of the CTRL2 insulator, we showed that enrichment of Alpha-thalassemia/mental retardation, X-linked chromatin remodeler (ATRX), and the histone variant H3.3, both of which are known for their roles in maintaining repressive histone markers on the HSV-1 viral genome were increased on IE regions of HSV-1. Finally, both H3K27me3 and H3K9me3 repressive histone marks remained enriched by 4 hours post infection in the absence of the CTRL2 insulator, confirming that the CTRL2 insulator is required for de-repression of IE genes of viral genomes. To our knowledge these are the first data that show that a specific CTCF insulator in the HSV-1 genome (CTRL2) regulates chromatin accessibility during the lytic infection.

Regulation of voltage-gated sodium channels by TNF-α during herpes simplex virus latency establishment.

During lytic or latent infection of sensory neurons with herpes simplex virus type 1 (HSV-1) there are significant changes in the expression of voltage-gated Na+ channels, which may disrupt the transmission of pain information. HSV-1 infection can also evoke the secretion of various pro-inflammatory cytokines, including TNF-α and IL-6. In this work, we hypothesized that TNF-α regulates the expression of Na+ channels during HSV-1 latency establishment in ND7/23 sensory-like neurons. Latency establishment was mimicked by culturing HSV-1 infected ND7/23 cells in the presence of acyclovir (ACV) for 3 days. Changes in the functional expression of voltage-gated Na+ channels were assessed by whole-cell recordings. Our results demonstrate that infection of ND7/23 cells with the HSV-1 strain McKrae with GFP expression (M-GFP) causes a significant decrease in sodium currents during latency establishment. Exposure of ND7/23 cells to TNF-α during latency establishment reverses the effect of HSV-1, resulting in a significant increase in sodium current density. However, Na+ currents were not restored by 3day-treatment with IL-6. There were no changes in the pharmacological and biophysical properties of sodium currents promoted by TNF-α, including sensitivity to tetrodotoxin and the current-voltage relationship. TNF-α stimulation of ND7/23 cells increases p38 signaling. Inhibition of p38 signaling with SB203580 or SB202190 eliminates the stimulatory effect of TNF-α on sodium currents. These results indicate that TNF-α signaling in sensory neurons during latency establishment upregulates the expression of voltage-gated Na+ channels in order to maintain the transmission of pain information.

Motility-dependent processes in Toxoplasma gondii tachyzoites and bradyzoites: same same but different.

The tachyzoite stage of the apicomplexan parasite Toxoplasma gondii utilizes motility for multiple purposes during its lytic cycle, including host cell invasion, egress from infected cells, and migration to new uninfected host cells to repeat the process. Bradyzoite stage parasites, which establish a new infection in a naïve host, must also use motility to escape from the cysts that are ingested by the new host and then migrate to the gut wall, where they either invade cells of the intestinal epithelium or squeeze between these cells to infect the underlying connective tissue. We know very little about the motility of bradyzoites, which we analyze in detail here and compare to the well-characterized motility and motility-dependent processes of tachyzoites. Unexpectedly, bradyzoites were found to be as motile as tachyzoites in a 3D model extracellular matrix, and they showed increased invasion into and transmigration across certain cell types, consistent with their need to establish the infection in the gut. The motility of the two stages was inhibited to the same extent by cytochalasin D and KNX-002, compounds known to target the parasite's actomyosin-based motor. In contrast, other compounds that impact tachyzoite motility (tachyplegin and enhancer 5) have less of an effect on bradyzoites, and rapid bradyzoite egress from infected cells is not triggered by treatment with calcium ionophores, as it is with tachyzoites. The similarities and differences between these two life cycle stages highlight the need to characterize both tachyzoites and bradyzoites for a more complete understanding of the role of motility in the parasite life cycle and the effect that potential therapeutics targeting parasite motility will have on disease establishment and progression.

Isolation and Characterization of Two Novel Lytic Bacteriophages against Salmonella typhimurium and Their Biocontrol Potential in Food Products.

Foodborne pathogens, such as Salmonella, are major factors that pose significant threats to global food safety and public health. Salmonella typhimurium is a prominent serotype contributing to non-typhoidal salmonellosis, which is a prevalent foodborne illness affecting humans and animals. Bacteriophages are considered one of the most environmentally friendly biocontrol agents, particularly in the food industry, owing to their high specificity and high safety. However, the emergency of phage-resistant mutants limits the biocontrol effect of phage treatment, leading to the requirement for a high diversity of lytic phages. Therefore, the study isolated and characterized two novel lytic Salmonella bacteriophages (SPYS_1 and SPYS_2) targeting S. typhimurium ATCC14028 and evaluated their effectiveness in reducing the contamination rates for milk and chicken tenders. Morphological and genomic analyses indicated that Salmonella phages SPYS_1 and SPYS_2 are novel species classified under the genus Skatevirus and the genus Berlinvirus, respectively. Both phages exhibited high stability across a broad range of thermal and pH conditions. The one-step growth curve result suggested that both phages had a short adsorption time and a large burst size in a single lytic cycle. The phage SPYS_1 demonstrated a noteworthy inhibition effect on the growth of S. typhimurium ATCC14028 in milk, resulting in a ~2-log reduction within the 2 to 4 h range. Overall, both phages have shown significant potential for application in food safety in the future.

The essential kinase TgGSK regulates centrosome division and endodyogeny in Toxoplasma gondii.

Intracellular replication is crucial for the success of apicomplexan parasites, including Toxoplasma gondii. Therefore, essential players in parasite replication present potential targets for drug development. In this study, we have characterized TgGSK, a glycogen synthase kinase homolog that plays an important role in Toxoplasma endodyogeny. We have shown that TgGSK has a dynamic localization that is concurrent with the cell cycle. In non-dividing parasites, this kinase is highly concentrated in the nucleus. However, during division, TgGSK displays a cytosolic localization, with concentration foci at the centrosomes, a key organelle involved in parasite division, and the basal end. Conditional knockdown of TgGSK determined that it is essential for the completion of the lytic cycle and proper parasite division. Parasites lacking endogenous protein levels of TgGSK exhibited defects in division synchronicity and the segregation of the nucleus and apicoplast into forming daughter cells. These phenotypes are associated with defects in centrosome duplication and fission. Global phosphoproteomic analysis determined TgGSK-dependent phosphorylation of RNA-processing, basal end, and centrosome proteins. Consistent with the putative regulation of RNA-processing proteins, global transcriptomic analysis suggests that TgGSK is needed for proper splicing. Finally, we show that TgGSK interacts with GCN5b, a well-characterized acetyltransferase with roles in transcriptional control. Conversely, GCN5b chemical inhibition results in specific degradation of TgGSK. Thus, these studies reveal the involvement of TgGSK in various crucial processes, including endodyogeny and splicing, and identify acetylation as a possible mechanism by which this essential kinase is regulated.

Single-cell virology: On-chip, quantitative characterization of the dynamics of virus spread from one single cell to another.

Virus spread at the single-cell level is largely uncharacterized. We have designed and constructed a microfluidic device in which each nanowell contained a single, infected cell (donor) and a single, uninfected cell (recipient). Using a GFP-expressing poliovirus as our model, we observed both lytic and non-lytic spread. Donor cells supporting lytic spread established infection earlier than those supporting non-lytic spread. However, non-lytic spread established infections in recipient cells substantially faster than lytic spread and yielded higher rates of genome replication. While lytic spread was sensitive to the presence of capsid entry/uncoating inhibitors, non-lytic spread was not. Consistent with emerging models for non-lytic spread of enteroviruses using autophagy, reduction of LC3 levels in cells impaired non-lytic spread and elevated the fraction of virus in donor cells spreading lytically. The ability to distinguish lytic and non-lytic spread unambiguously will enable discovery of viral and host factors and host pathways used for non-lytic spread of enteroviruses and other viruses as well.

Ubiquitin-Mediated Effects on Oncogenesis during EBV and KSHV Infection.

The Herpesviridae include the Epstein-Barr Virus (EBV) and the Kaposi Sarcoma-associated Herpesvirus (KSHV), both of which are oncogenic gamma-herpesviruses. These viruses manipulate host cellular mechanisms, including through ubiquitin-mediated pathways, to promote viral replication and oncogenesis. Ubiquitin, a regulatory protein which tags substrates for degradation or alters their function, is manipulated by both EBV and KSHV to facilitate viral persistence and cancer development. EBV infects approximately 90% of the global population and is implicated in malignancies including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), post-transplant lymphoproliferative disorder (PTLD), and nasopharyngeal carcinoma. EBV latency proteins, notably LMP1 and EBNA3C, use ubiquitin-mediated mechanisms to inhibit apoptosis, promote cell proliferation, and interfere with DNA repair, contributing to tumorigenesis. EBV's lytic proteins, including BZLF1 and BPLF1, further disrupt cellular processes to favor oncogenesis. Similarly, KSHV, a causative agent of Kaposi's Sarcoma and lymphoproliferative disorders, has a latency-associated nuclear antigen (LANA) and other latency proteins that manipulate ubiquitin pathways to degrade tumor suppressors, stabilize oncogenic proteins, and evade immune responses. KSHV's lytic cycle proteins, such as RTA and Orf64, also use ubiquitin-mediated strategies to impair immune functions and promote oncogenesis. This review explores the ubiquitin-mediated interactions of EBV and KSHV proteins, elucidating their roles in viral oncogenesis. Understanding these mechanisms offers insights into the similarities between the viruses, as well as provoking thought about potential therapeutic targets for herpesvirus-associated cancers.

Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7.

Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses.IMPORTANCECells rapidly induce changes in the transcription of RNA in response to stress and pathogens. Herpes simplex virus (HSV) disrupts many processes of host mRNA transcription, and it is necessary to separate the actions of viral proteins from cellular responses. Here, we demonstrate that viral proteins inhibit two key phosphorylation patterns on the C-terminal domain (CTD) of cellular RNA polymerase II and that this is separate from the degradation of polymerases later in infection. Furthermore, we show that viral genes do not require the full "CTD code." Together, these data distinguish multiple steps in the remodeling of RNA polymerase during infection and suggest that shared transcriptional phenotypes during stress responses do not revolve around a core disruption of CTD modifications.

EZH2 Inhibition by DS3201 Triggers the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle and Potentiates the Effects Induced by SAHA in Primary Effusion Lymphoma Cells.

Primary Effusion Lymphoma (PEL) cells carry Kaposi's sarcoma-associated herpesvirus (KSHV) in a latent state, except for a small number of cells in which the virus replicates to ensure its persistence into the infected host. However, the lytic cycle can be reactivated in vitro by exposing these lymphoma cells to various treatments, leading to cell lysis. To restrict viral antigen expression, KSHV induces repressive epigenetic changes, including DNA methylation and histone modifications. Among the latter, histone deacetylation and tri-methylation of Histone H3 lisyne-27 (H3K27me3) have been reported to play a role. Here, we found that the inhibition of H3K27 tri-methylation by valemetostat DS3201 (DS), a small molecule that inhibits Enhancer of Zeste Homolog 2 (EZH2) methyltransferase, induced the KSHV lytic cycle in PEL cells, and that this effect involved the activation of the wtp53-p21 axis and autophagic dysregulation. DS also potentiated the lytic cycle activation mediated by the Histone deacetylases (HDAC) inhibitor Suberoylanilide hydroxamic acid (SAHA) and reinforced its cytotoxic effect, suggesting that such a combination could be used to unbalance the latent/lytic cycle and further impair the survival of PEL cells.

The Epstein–Barr virus small capsid protein BFRF3 disrupts the NF‐кB signaling pathway by inhibiting p65 activity

AbstractEpstein–Barr virus (EBV), a common gamma herpesvirus, establishes a life‐long latent infection in the host to defend against innate immune recognition, which is closely related to a variety of malignant tumors, but its specific mechanism is unclear. BFRF3, an EBV‐encoded small capsid protein, is mainly involved in the assembly of the viral capsid structure and the maintenance of its stability. Here, we showed that BFRF3 can inhibit TNF‐α‐mediated NF‐кB promoter activation. Moreover, BFRF3 downregulates NF‐кB‐mediated promoter activation and transcription of inflammatory cytokines, including IL‐6 and IL‐8. Dual‐luciferase reporter assay demonstrated that BFRF3 restrains NF‐кB promoter activity at or below the p65 level, and coimmunoprecipitation analysis revealed that BFRF3 not only interacts with p65 but also binds to its critical truncated Rel homology domain (RHD) and transcriptional activation domain (TAD). However, BFRF3 does not affect the dimerization of p65‐p50, but overexpression of BFRF3 reduces the nuclear accumulation of p65, and the phosphorylation of p65 (Ser536) is repressed during BFRF3 transfection and EBV lytic infection, which promotes the proliferation of EBV. Overall, our study suggested that BFRF3 may play a crucial role in antiviral immunity to defend against EBV infection by inhibiting NF‐κB activity.