Lytic Cycle Research Articles

Contribution of Epstein–Barr Virus Lytic Proteins to Cancer Hallmarks and Implications from Other Oncoviruses

Epstein-Barr virus (EBV) is a prevalent human gamma-herpesvirus that infects the majority of the adult population worldwide and is associated with several lymphoid and epithelial malignancies. EBV displays a biphasic life cycle, namely, latent and lytic replication cycles, expressing a diversity of viral proteins. Among the EBV proteins being expressed during both latent and lytic cycles, the oncogenic roles of EBV lytic proteins are largely uncharacterized. In this review, the established contributions of EBV lytic proteins in tumorigenesis are summarized according to the cancer hallmarks displayed. We further postulate the oncogenic properties of several EBV lytic proteins by comparing the evolutionary conserved oncogenic mechanisms in other herpesviruses and oncoviruses.

ICTV Virus Taxonomy Profile: Sphaerolipoviridae 2023.

Members of the family Sphaerolipoviridae have non-enveloped tailless icosahedral virions with a protein-rich internal lipid membrane. The genome is a linear double-stranded DNA of about 30 kbp with inverted terminal repeats and terminal proteins. The capsid has a pseudo triangulation T=28 dextro symmetry and is built of two major capsid protein types. Spike complexes decorate fivefold vertices. Sphaerolipoviruses have a narrow host range and a lytic life cycle, infecting haloarchaea in the class Halobacteria (phylum Euryarchaeota). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Sphaerolipoviridae, which is available at ictv.global/report/sphaerolipoviridae.

WGS analysis of two Staphylococcus aureus bacteriophages from sewage in China provides insights into the genetic feature of highly efficient lytic phages

The study of bacteriophages is experiencing a resurgence with the increasing development of antimicrobial resistance in Staphylococcus aureus. Nonetheless, the genetic features of highly efficient lytic S. aureus phage remain to be explored. In this study, two lytic S. aureus phages, SapYZU11 and SapYZU15, were isolated from sewage samples from Yangzhou, China. The phage morphology, one-step growth, host spectrum and lytic activity of these phages were examined, and their whole-genome sequences were analysed and compared with 280 published genomes of staphylococcal phages. The structural organisation and genetic contents of SapYZU11 and SapYZU15 were investigated. The Podoviridae phage SapYZU11 and Herelleviridae phage SapYZU15 effectively lysed all of the 53 S. aureus strains isolated from various sources. However, SapYZU15 exhibited a shorter latent period, larger burst size and stronger bactericidal ability with an anti-bacterial rate of approximately 99.9999% for 24 h. Phylogenetic analysis revealed that Herelleviridae phages formed the most ancestral clades and the S. aureus Podoviridae phages were clustered in the staphylococcal Siphoviridae phage clade. Moreover, phages in different morphology families contain distinct types of genes associated with host cell lysis, DNA packaging and lysogeny. Notably, SapYZU15 harboured 13 DNA metabolism-related genes, 5 lysin genes, 1 holin gene and 1 DNA packaging gene. The data suggest that S. aureus Podoviridae and Siphoviridae phages originated from staphylococcal Herelleviridae phages, and the module exchange of S. aureus phages occurred in the same morphology family. Moreover, the extraordinary lytic capacity of SapYZU15 was likely due to the presence of specific genes associated with DNA replication, DNA packaging and the lytic cycle.

The Broad-Spectrum Endolysin LySP2 Improves Chick Survival after Salmonella Pullorum Infection.

Salmonella pullorum causes typical "Bacillary White Diarrhea" and loss of appetite in chicks, which leads to the death of chicks in severe cases; thus, it is still a critical issue in China. Antibiotics are conventional medicines used for Salmonella infections; however, due to the extensive long-term use and even abuse of antibiotics, drug resistance becomes increasingly severe, making treating pullorum disease more difficult. Most of the endolysins are hydrolytic enzymes produced by bacteriophages to cleave the host's cell wall during the final stage of the lytic cycle. A virulent bacteriophage, YSP2, of Salmonella was isolated in a previous study. A Pichia pastoris expression strain that can express the Salmonella bacteriophage endolysin was constructed efficiently, and the Gram-negative bacteriophage endolysin, LySP2, was obtained in this study. Compared with the parental phage YSP2, which can only lyse Salmonella, LySP2 can lyse Salmonella and Escherichia. The survival rate of Salmonella-infected chicks treated with LySP2 can reach up to 70% and reduce Salmonella abundance in the liver and intestine. The treatment group showed that LySP2 significantly improved the health of infected chicks and alleviated organ damage caused by Salmonella infection. In this study, the Salmonella bacteriophage endolysin was expressed efficiently by Pichia pastoris, and the endolysin LySP2 showed good potential for the treatment of pullorum disease caused by Salmonella pullorum.

Synthesis vs. salvage of ester- and ether-linked phosphatidylethanolamine in the intracellular protozoan pathogen Toxoplasma gondii

Toxoplasma gondii is a prevalent zoonotic pathogen infecting livestock as well as humans. The exceptional ability of this parasite to reproduce in several types of nucleated host cells necessitates a coordinated usage of endogenous and host-derived nutritional resources for membrane biogenesis. Phosphatidylethanolamine is the second most common glycerophospholipid in T. gondii, but how its requirement in the acutely-infectious fast-dividing tachyzoite stage is satisfied remains enigmatic. This work reveals that the parasite deploys de novo synthesis and salvage pathways to meet its demand for ester- and ether-linked PtdEtn. Auxin-mediated depletion of the phosphoethanolamine cytidylyltransferase (ECT) caused a lethal phenotype in tachyzoites due to impaired invasion and cell division, disclosing a vital role of the CDP-ethanolamine pathway during the lytic cycle. In accord, the inner membrane complex appeared disrupted concurrent with a decline in its length, parasite width and major phospholipids. Integrated lipidomics and isotope analyses of the TgECT mutant unveiled the endogenous synthesis of ester-PtdEtn, and salvage of ether-linked lipids from host cells. In brief, this study demonstrates how T. gondii operates various means to produce distinct forms of PtdEtn while featuring the therapeutic relevance of its de novo synthesis.

Tinospora crispa Ethanolic Extract Downregulates Protein Kinase Genes Expression and Activity during Toxoplasma gondii Infection: A Prospective Drug Target for Lytic Cycle Inhibition

Infection with Toxoplasma gondii remains widespread among humans and animals as water, soil and food continue to serve as the major carriers of the sporulated oocyst. The infection is poorly controlled due to lack of a potent vaccine against the parasite, and the current medication presents severe side effects on the host, less efficacy on the parasite and is accompanied by the potential development of resistance by the parasite. The aim of this study was to evaluate the in vitro activities of ethanolic extract of Tinospora crispa (EETC) on protein kinases involved in the lytic cycle of T. gondii infection in Vero cell line. The EETC was obtained through the maceration of dried stem powder. Vero cells infected with the RH strain of T. gondii were used to evaluate the inhibitory effect of EETC against T. gondii calcium dependent protein kinase (CDPK) genes and microneme proteins (MIC) that are essential for host cell invasion and intracellular replication of the tachyzoite. Gene expression profiling of CDPK genes was determined through quantitative real-time PCR (qPCR) after 24 h of treatment. The expression of microneme protein was determined through western blot technique. The RT-qPCR revealed downregulation of most protein kinase (PK) genes after treatment with EETC. The expression of CDPK1, PKG, CDPK3, CDPK6, CDPK7 genes that participate in the lytic cycle of T. gondii infection was downregulated. The expression of the TgMIC1 and TgMIC2 proteins were observed to have decreased in both 4 and 24 h post-infection treatment models. This study showed that EETC contains promising drug candidates effective against T. gondii that can target the protein kinase genes involved in the lytic cycle of the parasite to prevent disease progression. HIGHLIGHTS This study explored the possible mechanism of action of phytochemicals on Toxoplasma gondii parasite-host cell invasion and intracellular replication The study evaluated the effects of ethanolic extract of Tinospora crispa Miers on protein kinase genes that are involved in host cell invasion The target protein kinase genes were downregulated which prevents the secretion of microneme proteins Inhibition of microneme secretion prevents host cell invasion and intracellular replication by Toxoplasma gondi GRAPHICAL ABSTRACT

Epstein–Barr virus envelope glycoprotein 110 inhibits NF-κB activation by interacting with NF-κB subunit p65

Epstein-Barr virus (EBV) is a member of the lymphotropic virus family and is highly correlated with some human malignant tumors. It has been reported that envelope glycoprotein 110 (gp110) plays an essential role in viral fusion, DNA replication, and nucleocapsid assembly of EBV. However, it has not been established whether gp110 is involved in regulating the host's innate immunity. In this study, we found that gp110 inhibits tumor necrosis factor α-mediated NF- κB promoter activity and the downstream production of NF- κB-regulated cytokines under physiological conditions. Using dual-luciferase reporter assays, we showed that gp110 might impede the NF-κB promoter activation downstream of NF-κB transactivational subunit p65. Subsequently, we used coimmunoprecipitation assays to demonstrate that gp110 interacts with p65 during EBV lytic infection, and that the C-terminal cytoplasmic region of gp110 is the key interaction domain with p65. Furthermore, we determined that gp110 can bind to the N-terminal Rel homologous and C-terminal domains of p65. Alternatively, gp110 might not disturb the association of p65 with nontransactivational subunit p50, but we showed it restrains activational phosphorylation (at Ser536) and nuclear translocation of p65, which we also found to be executed by the C-terminal cytoplasmic region of gp110. Altogether, these data suggest that the surface protein gp110 may be a vital component for EBV to antagonize the host's innate immune response, which is also helpful for revealing the infectivity and pathogenesis of EBV.

Protein Degradation by Gammaherpesvirus RTAs: More Than Just Viral Transactivators.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a member of the Gammaherpesvirus subfamily that encodes several viral proteins with intrinsic E3 ubiquitin ligase activity or the ability to hijack host E3 ubiquitin ligases to modulate the host's immune response and to support the viral life cycle. This review focuses specifically on how the immediate-early KSHV protein RTA (replication and transcription activator) hijacks the host's ubiquitin-proteasome pathway (UPP) to target cellular and viral factors for protein degradation to allow for robust lytic reactivation. Notably, RTA's targets are either potent transcription repressors or they are activators of the innate and adaptive immune response, which block the lytic cycle of the virus. This review mainly focuses on what is currently known about the role of the E3 ubiquitin ligase activity of KSHV RTA in the regulation of the KSHV life cycle, but we will also discuss the potential role of other gammaherpesviral RTA homologs in UPP-mediated protein degradation.

Sequencing the genomes of LPP-1, the first isolated cyanophage, and its relative LPP-2 reveal different integration mechanisms in closely related phages

In the early 1960s, the first cyanophage was isolated. The description of this phage, named LPP-1, led to the extensive investigation of various cyanophages and to the study of their interactions with their cyanobacterial hosts towards controlling blooms. Here, the genomes of LPP-1 and its putative relative, LPP-2 were sequenced. Sequencing these genomes revealed that LPP-1 and LPP-2 are members of a group of short-tailed cyanophages, which are distantly related to the T7-like cyanophages. Most of the phages in this group have the ability to lysogenize their hosts. Their ability to switch between lytic and lysogenic infection may explain the formation of cyanobacterial blooms despite the persistence of their phages. This lysogenic capacity of the LPP-1-like phages occurs despite the lack of an obvious integrase gene within their genomes. Interestingly, we show that LPP-2 integrates into the host genome through an integration site in high proximity to a recombination endonuclease that may have integrase activity. Further understanding of cyanobacterial-phage relationships may provide insight into their population dynamics and suggest novel approaches for control of destructive cyanobacterial blooms.

Polio virotherapy targets the malignant glioma myeloid infiltrate with diffuse microglia activation engulfing the CNS.

Malignant gliomas commandeer dense inflammatory infiltrates with glioma-associated macrophages and microglia (GAMM) promoting immune suppression, evasion, and tumor progression. Like all cells in the mononuclear phagocytic system, GAMM constitutively express the poliovirus receptor, CD155. Besides myeloid cells, CD155 is widely upregulated in the neoplastic compartment of malignant gliomas. Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, yielded long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). This scenario raises questions about the contributions of myeloid versus neoplastic cells to polio virotherapy of malignant gliomas. We investigated PVSRIPO immunotherapy in immunocompetent mouse brain tumor models with blinded, board-certified neuropathologist review, a range of neuropathological, immunohistochemical, and immunofluorescence analyses, and RNAseq of the tumor region. PVSRIPO treatment caused intense engagement of the GAMM infiltrate associated with substantial, but transient tumor regression. This was accompanied by marked microglia activation and proliferation in normal brain surrounding the tumor, in the ipsilateral hemisphere and extending into the contralateral hemisphere. There was no evidence for lytic infection of malignant cells. PVSRIPO-instigated microglia activation occurred against a backdrop of sustained innate antiviral inflammation, associated with induction of the Programmed Cell Death Ligand 1 (PD-L1) immune checkpoint on GAMM. Combining PVSRIPO with PD1/PD-L1 blockade led to durable remissions. Our work implicates GAMM as active drivers of PVSRIPO-induced antitumor inflammation and reveals profound and widespread neuroinflammatory activation of the brain-resident myeloid compartment by PVSRIPO.

Diversity, evolution and life strategies of CbK-like phages.

Caulobacter phage CbK has been extensively studied as a model system in virology and bacteriology. Lysogeny-related genes have been found in each CbK-like isolate, suggesting a life strategy of both lytic and lysogenic cycles. However, whether CbK-related phages can enter lysogeny is still undetermined. This study identified new CbK-like sequences and expanded the collection of CbK-related phages. A common ancestry with a temperate lifestyle was predicted for the group, however, which subsequently evolved into two clades of different genome sizes and host associations. Through the examination of phage recombinase genes, alignment of attachment sites on the phage and bacterial genomes (attP-attB pairing), and the experimental validation, different lifestyles were found among the different members. A majority of clade II members retain a lysogenic lifestyle, whereas all clade I members have evolved into an obligate lytic lifestyle via a loss of the gene encoding Cre-like recombinase and the coupled attP fragment. We postulated that the loss of lysogeny may be a by-product of the increase in phage genome size, and vice versa. Clade I is likely to overcome the costs through maintaining more auxiliary metabolic genes (AMGs), particularly for those involved in protein metabolism, to strengthen host takeover and further benefit virion production.

Analysis of the Interactome of the Toxoplasma gondii Tgj1 HSP40 Chaperone.

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein-protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.

Elevated iNOS and 3′-nitrotyrosine in Kaposi's Sarcoma tumors and mouse model

Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Here, we show that KS lesions express iNOS/NOS2 broadly throughout KS lesions, with enrichment in LANA positive spindle cells. The iNOS byproduct 3-nitrotyrosine is also enriched in LANA positive tumor cells and colocalizes with a fraction of LANA-nuclear bodies. We show that iNOS is highly expressed in the L1T3/mSLK tumor model of KS. iNOS expression correlated with KSHV lytic cycle gene expression, which was elevated in late-stage tumors (>4 weeks) but to a lesser degree in early stage (1 week) xenografts. Further, we show that L1T3/mSLK tumor growth is sensitive to an inhibitor of nitric oxide, L-NMMA. L-NMMA treatment reduced KSHV gene expression and perturbed cellular gene pathways relating to oxidative phosphorylation and mitochondrial dysfunction. These finding suggest that iNOS is expressed in KSHV infected endothelial-transformed tumor cells in KS, that iNOS expression depends on tumor microenvironment stress conditions, and that iNOS enzymatic activity contributes to KS tumor growth.

NFκB and Cyclic AMP Response Element Sites Mediate the Valproic Acid and UL138 Responsiveness of the Human Cytomegalovirus Major Immediate Early Enhancer and Promoter.

The major immediate early enhancer and promoter (MIEP) of human cytomegalovirus (HCMV) drives the transcription of the immediate early one (IE1) and IE2 genes, whose encoded proteins stimulate productive, lytic replication. The MIEP is activated by the virally encoded and tegument-delivered pp71 protein at the start of de novo lytic infections of fully differentiated cells. Conversely, the MIEP is silenced at the start of de novo latent infections within incompletely differentiated myeloid cells in part because tegument-delivered pp71 is sequestered in the cytoplasm in these cells, but also by viral factors that repress transcription from this locus, including the UL138 protein. During both modes of infection, MIEP activity can be increased by the histone deacetylase inhibitor valproic acid (VPA); however, UL138 inhibits the VPA-responsiveness of the MIEP. Here, we show that two families of cellular transcription factors, NF-κB and cAMP response element-binding protein (CREB), together control the VPA-mediated activation and UL138-mediated repression of the HCMV MIEP. IMPORTANCE Artificial regulation of the HCMV MIEP, either activation or repression, is an attractive potential means to target the latent reservoirs of virus for which there is currently no available intervention. The MIEP could be repressed to prevent latency reactivation or induced to drive the virus into the lytic stage that is visible to the immune system and inhibited by multiple small-molecule antiviral drugs. Understanding how the MIEP is regulated is a critical part of designing and implementing either strategy. Our revelation here that NF-κB and CREB control the responsiveness of the MIEP to the viral UL138 protein and the FDA-approved drug VPA could help in the formulation and execution of promoter regulatory strategies against latent HCMV.

Exposure of Shewanella oneidensis MR-1 to Sublethal Doses of Ionizing Radiation Triggers Short-Term SOS Activation and Longer-Term Prophage Activation.

Currently, there is a lack of bacterial biomarkers indicative of exposure to ionizing radiation (IR). IR biomarkers have applications for medical treatment planning, population exposure surveillance, and IR sensitivity studies. In this study, we compared the utility of signals originating from prophages and the SOS regulon as biomarkers of IR exposure in the radiosensitive bacterium Shewanella oneidensis. Using RNA sequencing, we demonstrated that 60 min after exposure to acute doses of IR (40, 1, 0.5, and 0.25 Gy), the transcriptional activation of the SOS regulon and the lytic cycle of the T-even lysogenic prophage So Lambda are comparable. Using quantitative PCR (qPCR), we showed that 300 min after exposure to doses as low as 0.25 Gy, the fold change of transcriptional activation of the So Lambda lytic cycle surpassed that of the SOS regulon. We observed an increase in cell size (a phenotype of SOS activation) and plaque production (a phenotype of prophage maturation) 300 min after doses as low as 1 Gy. While the transcriptional responses of the SOS and So Lambda regulons have been examined in S. oneidensis after lethal IR exposures, the potential of these (and other transcriptome-wide) responses as biomarkers of sublethal levels of IR (<10 Gy) and the longer-term activity of these two regulons have not been investigated. A major finding is that after exposure to sublethal doses of IR, the most upregulated transcripts are associated with a prophage regulon and not with a DNA damage response. Our findings suggest that prophage lytic cycle genes are a promising source of biomarkers of sublethal DNA damage. IMPORTANCE The bacterial minimum threshold of sensitivity to ionizing radiation (IR) is poorly understood, which hinders our understanding of how living systems recover from the doses of IR experienced in medical, industrial, and off-world environments. Using a transcriptome-wide approach, we studied how in the highly radiosensitive bacterium S. oneidensis, genes (including the SOS regulon and the So Lambda prophage) are activated after exposure to low doses of IR. We found that 300 min after exposure to doses as low as 0.25 Gy, genes within the So Lambda regulon remained upregulated. As this is the first transcriptome-wide study of how bacteria respond to acute sublethal doses of IR, these findings serve as a benchmark for future bacterial IR sensitivity studies. This is the first work to highlight the utility of prophages as biomarkers of exposure to very low (i.e., sublethal) doses of IR and to examine the longer-term impacts of sublethal IR exposure on bacteria.

Cytomegalovirus at the crossroads of immunosenescence and oncogenesis

Human cytomegalovirus (HCMV), whose genome is around 235 kb, is a ubiquitous human herpesvirus that infects between 40% and 95% of the population. Though HCMV infection is commonly asymptomatic and leads to subtle clinical symptoms, it can promote robust immune responses and establish lifelong latency. In addition, in immunocompromised hosts, including individuals with acquired immunodeficiency syndrome (AIDS), transplant recipients, and developing fetuses it can lead to severe diseases. Immunosenescence, well-defined as the alterations in the immune system, is linked mainly to aging and has been recently gathering considerable attention. Senescence was characterized by an elevated inflammation and hence considered a powerful contributor to “inflammaging” that is measured mainly by tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) levels as well as latent viral infections, for instance, cytomegalovirus (CMV). Inflammaging resulted in a senescence-associated secretory phenotype (SASP). HCMV is markedly associated with accelerated aging of the immune system as well as several age-associated diseases that accumulate and subsequently deteriorate the immune responses, thus have been linked to mortality, declined vaccine efficacy, serious diseases, and tumors in the elderly. HCMV triggers or exacerbates immunosenescence; on the other hand, the weakened immune responses and inflammaging favor viral reactivation and highlight the role of HCMV in aging as well as viral-associated tumors. HCMV reactivation resulting in sequential lytic and latent viral cycles could contribute to HCMV genomic variability. Besides the oncomodulatory role and transforming capacities of HCMV, the immune-privileged tumor microenvironment has been considered the main element in tumor progression and aggressiveness. Therefore, the interplay between HCMV, immunosenescence, and cancer will aid in discovering new therapeutic approaches that target HCMV and act as immune response boosters mainly to fight cancers of poor prognosis, particularly in the elderly population.

Kaposi's Sarcoma-Associated Herpesvirus Immediate Early Proteins Trigger FOXQ1 Expression in Oral Epithelial Cells, Engaging in a Novel Lytic Cycle-Sustaining Positive Feedback Loop.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that can replicate in oral epithelial cells to promote viral transmission via saliva. To identify novel regulators of KSHV oral infection, we performed a transcriptome analysis of KSHV-infected primary human gingival epithelial (HGEP) cells, which identified the gene coding for the host transcription factor FOXQ1 as the top induced host gene. FOXQ1 is nearly undetectable in uninfected HGEP and telomerase-immortalized gingival keratinocytes (TIGK) cells but is highly expressed within hours of KSHV infection. We found that while the FOXQ1 promoter lacks activating histone acetylation marks in uninfected oral epithelial cells, these marks accumulate in the FOXQ1 promoter in infected cells, revealing a rapid epigenetic reprogramming event. To evaluate FOXQ1 function, we depleted FOXQ1 in KSHV-infected TIGK cells, which resulted in reduced accumulation of KSHV lytic proteins and viral DNA over the course of 4 days of infection, uncovering a novel lytic cycle-sustaining role of FOXQ1. A screen of KSHV lytic proteins demonstrated that the immediate early proteins ORF45 and replication and transcription activator (RTA) were both sufficient for FOXQ1 induction in oral epithelial cells, indicating active involvement of incoming and rapidly expressed factors in altering host gene expression. ORF45 is known to sustain extracellular signal-regulated kinase (ERK) p90 ribosomal s6 kinase (RSK) pathway activity to promote lytic infection. We found that an ORF45 mutant lacking RSK activation function failed to induce FOXQ1 in TIGK cells, revealing that ORF45 uses a shared mechanism to rapidly induce both host and viral genes to sustain lytic infection in oral epithelial cells. IMPORTANCE The oral cavity is a primary site of initial contact and entry for many viruses. Viral replication in the oral epithelium promotes viral shedding in saliva, allowing interpersonal transmission, as well as spread to other cell types, where chronic infection can be established. Understanding the regulation of KSHV infection in the oral epithelium would allow for the design of universal strategies to target the first stage of viral infection, thereby halting systemic viral pathogenesis. Overall, we uncover a novel positive feedback loop in which immediate early KSHV factors drive rapid host reprogramming of oral epithelial cells to sustain the lytic cycle in the oral cavity.

Transcriptomics-Driven Characterization of LUZ100, a T7-like Pseudomonas Phage with Temperate Features.

Autographiviridae is a diverse yet distinct family of bacterial viruses marked by a strictly lytic lifestyle and a generally conserved genome organization. Here, we characterized Pseudomonas aeruginosa phage LUZ100, a distant relative of type phage T7. LUZ100 is a podovirus with a limited host range which likely uses lipopolysaccharide (LPS) as a phage receptor. Interestingly, infection dynamics of LUZ100 indicated moderate adsorption rates and low virulence, hinting at temperate characteristics. This hypothesis was supported by genomic analysis, which showed that LUZ100 shares the conventional T7-like genome organization yet carries key genes associated with a temperate lifestyle. To unravel the peculiar characteristics of LUZ100, ONT-cappable-seq transcriptomics analysis was performed. These data provided a bird's-eye view of the LUZ100 transcriptome and enabled the discovery of key regulatory elements, antisense RNA, and transcriptional unit structures. The transcriptional map of LUZ100 also allowed us to identify new RNA polymerase (RNAP)-promoter pairs that can form the basis for biotechnological parts and tools for new synthetic transcription regulation circuitry. The ONT-cappable-seq data revealed that the LUZ100 integrase and a MarR-like regulator (proposed to be involved in the lytic/lysogeny decision) are actively cotranscribed in an operon. In addition, the presence of a phage-specific promoter transcribing the phage-encoded RNA polymerase raises questions on the regulation of this polymerase and suggests that it is interwoven with the MarR-based regulation. This transcriptomics-driven characterization of LUZ100 supports recent evidence that T7-like phages should not automatically be assumed to have a strictly lytic life cycle. IMPORTANCE Bacteriophage T7, considered the "model phage" of the Autographiviridae family, is marked by a strictly lytic life cycle and conserved genome organization. Recently, novel phages within this clade have emerged which display characteristics associated with a temperate life cycle. Screening for temperate behavior is of utmost importance in fields like phage therapy, where strictly lytic phages are generally required for therapeutic applications. In this study, we applied an omics-driven approach to characterize the T7-like Pseudomonas aeruginosa phage LUZ100. These results led to the identification of actively transcribed lysogeny-associated genes in the phage genome, pointing out that temperate T7-like phages are emerging more frequent than initially thought. In short, the combination of genomics and transcriptomics allowed us to obtain a better understanding of the biology of nonmodel Autographiviridae phages, which can be used to optimize the implementation of phages and their regulatory elements in phage therapy and biotechnological applications, respectively.

Pseudomonas Phage ZCPS1 Endolysin as a Potential Therapeutic Agent.

The challenge of antibiotic resistance has gained much attention in recent years due to the rapid emergence of resistant bacteria infecting humans and risking industries. Thus, alternatives to antibiotics are being actively searched for. In this regard, bacteriophages and their enzymes, such as endolysins, are a very attractive alternative. Endolysins are the lytic enzymes, which are produced during the late phase of the lytic bacteriophage replication cycle to target the bacterial cell walls for progeny release. Here, we cloned, expressed, and purified LysZC1 endolysin from Pseudomonas phage ZCPS1. The structural alignment, molecular dynamic simulation, and CD studies suggested LysZC1 to be majorly helical, which is highly similar to various phage-encoded lysozymes with glycoside hydrolase activity. Our endpoint turbidity reduction assay displayed the lytic activity against various Gram-positive and Gram-negative pathogens. Although in synergism with EDTA, LysZC1 demonstrated significant activity against Gram-negative pathogens, it demonstrated the highest activity against Bacillus cereus. Moreover, LysZC1 was able to reduce the numbers of logarithmic-phase B. cereus by more than 2 log10 CFU/mL in 1 h and also acted on the stationary-phase culture. Remarkably, LysZC1 presented exceptional thermal stability, pH tolerance, and storage conditions, as it maintained the antibacterial activity against its host after nearly one year of storage at 4 °C and after being heated at temperatures as high as 100 °C for 10 min. Our data suggest that LysZC1 is a potential candidate as a therapeutic agent against bacterial infection and an antibacterial bio-control tool in food preservation technology.

Therapeutic targeting of HCMV-encoded chemokine receptor US28: Progress and challenges.

The pervasive human cytomegalovirus (HCMV) causes significant morbidity in immunocompromised individuals. Treatment using the current standard-of-care (SOC) is limited by severe toxic adverse effects and anti-viral resistance development. Furthermore, they only affect HCMV in its lytic phase, meaning viral disease is not preventable as latent infection cannot be treated and the viral reservoirs persist. The viral chemokine receptor (vCKR) US28 encoded by HCMV has received much attention in recent years. This broad-spectrum receptor has proven to be a desirable target for development of novel therapeutics through exploitation of its ability to internalize and its role in maintaining latency. Importantly, it is expressed on the surface of infected cells during both lytic and latent infection. US28-targeting small molecules, single-domain antibodies, and fusion toxin proteins have been developed for different treatment strategies, e.g. forcing reactivation of latent virus or using internalization of US28 as a toxin shuttle to kill infected cells. These strategies show promise for providing ways to eliminate latent viral reservoirs and prevent HCMV disease in vulnerable patients. Here, we discuss the progress and challenges of targeting US28 to treat HCMV infection and its associated diseases.