Lytic Cycle Research Articles

HHV-6B detection and host gene expression implicate HHV-6B as pulmonary pathogen after hematopoietic cell transplant

Limited understanding of the immunopathogenesis of human herpesvirus 6B (HHV-6B) has prevented its acceptance as a pulmonary pathogen after hematopoietic cell transplant (HCT). In this prospective multicenter study of patients undergoing bronchoalveolar lavage (BAL) for pneumonia after allogeneic HCT, we test blood and BAL fluid (BALF) for HHV-6B DNA and mRNA transcripts associated with lytic infection and perform RNA-seq on paired blood. Among 116 participants, HHV-6B DNA is detected in 37% of BALs, 49% of which also have HHV-6B mRNA detection. We establish HHV-6B DNA viral load thresholds in BALF that are highly predictive of HHV-6B mRNA detection and associated with increased risk for overall mortality and death from respiratory failure. Participants with HHV-6B DNA in BALF exhibit distinct host gene expression signatures, notable for enriched interferon signaling pathways in participants clinically diagnosed with idiopathic pneumonia. These data implicate HHV-6B as a pulmonary pathogen after allogeneic HCT.

Update of Natural Products and Their Derivatives Targeting Epstein-Barr Infection.

Epstein-Barr (EBV) is a human γ-herpesvirus that undergoes both a productive (lytic) cycle and a non-productive (latent) phase. The virus establishes enduring latent infection in B lymphocytes and productive infection in the oral mucosal epithelium. Like other herpesviruses, EBV expresses its genes in a coordinated pattern during acute infection. Unlike others, it replicates its DNA during latency to maintain the viral genome in an expanding pool of B lymphocytes, which are stimulated to divide upon infection. The reactivation from the latent state is associated with a productive gene expression pattern mediated by virus-encoded transcriptional activators BZLF-1 and BRLF-1. EBV is a highly transforming virus that contributes to the development of human lymphomas. Though viral vectors and mRNA platforms have been used to develop an EBV prophylactic vaccine, currently, there are no vaccines or antiviral drugs for the prophylaxis or treatment of EBV infection and EBV-associated cancers. Natural products and bioactive compounds are widely studied for their antiviral potential and capability to modulate intracellular signaling pathways. This review was intended to collect information on plant-derived products showing their antiviral activity against EBV and evaluate their feasibility as an alternative or adjuvant therapy against EBV infections and correlated oncogenesis in humans.

Essential contribution of the JAK/STAT pathway to carcinogenesis, lytic infection of herpesviruses and pathogenesis of COVID‑19 (Review).

The intracellular pathway of Janus kinase/signal transducer and activator of transcription (JAK/STAT) and modification of nucleosome histone marks regulate the expression of proinflammatory mediators, playing an essential role in carcinogenesis, antiviral immunity and the interaction of host proteins with Herpesviral particles. The pathway has also been suggested to play a vital role in the clinical course of the acute infection caused by severe acute respiratory syndrome coronavirus type2 (SARS‑CoV‑2; known as coronavirus infection‑2019), a novel human coronavirus initially identified in the central Chinese city Wuhan towards the end of 2019, which evolved into a pandemic affecting nearly two million people worldwide. The infection mainly manifests as fever, cough, myalgia and pulmonary involvement, while it also attacks multiple viscera, such as the liver. The pathogenesis is characterized by a cytokine storm, with an overproduction of proinflammatory mediators. Innate and adaptive host immunity against the viral pathogen is exerted by various effectors and is regulated by different signaling pathways notably the JAK/STAT. The elucidation of the underlying mechanism of the regulation of mediating factors expressed in the viral infection would assist diagnosis and antiviral targeting therapy, which will help overcome the infection caused by SARS‑CoV‑2.

Unveiling the mechanism of bactericidal activity of a cecropin A-fused endolysin LNT113

Endolysins are lytic enzymes produced by bacteriophages at the end of their lytic cycle and degrade the peptidoglycan layer of the bacterial cell wall. Thus, they have been extensively explored as a promising antibacterial agent to replace or supplement current antibiotics. Gram-negative bacteria, however, are prone to resist exogenous endolysins owing to their protective outer membrane. We previously engineered endolysin EC340, encoded by the Escherichia coli phage PBEC131, by substituting its seven amino acids and fusing an antimicrobial peptide cecropin A at its N-terminus. The engineered endolysin LNT113 exerted superior activity to its intrinsic form. This study investigated how cecropin A fusion facilitated the bactericidal activity of LNT113 toward Gram-negative bacteria. Cecropin A of LNT113 markedly increased the interaction with lipopolysaccharides, while the E. coli defective in the core oligosaccharide was less susceptible to endolysins, implicating the interaction between the core oligosaccharide and endolysins. In fact, E. coli with compromised lipid A construction was more vulnerable to LNT113 treatment, suggesting that the integrity of the lipid A layer was important to resist the internalization of LNT113 across the outer membrane. Cecropin A fusion further accelerated the inner membrane destabilization, thereby enabling LNT113 to deconstruct it promptly. Owing to the increased membrane permeability, LNT113 could inactivate some Gram-positive bacteria as well. This study demonstrates that cecropin A fusion is a feasible method to improve the membrane permeability of endolysins in both Gram-negative and Gram-positive bacteria.

EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation.

Epstein-Barr virus (EBV) is associated with several types of human cancer including nasopharyngeal carcinoma (NPC). The activation of EBV to the lytic cycle has been observed in advanced NPC and is believed to contribute to late-stage NPC development. However, how EBV lytic cycle promotes NPC progression remains elusive. Analysis of clinical NPC samples indicated that EBV reactivation and immunosuppression were found in advanced NPC samples, as well as abnormal angiogenesis and invasiveness. To investigate the role of the EBV lytic cycle in tumor development, we established a system that consists of two NPC cell lines, respectively, in EBV abortive lytic cycle and latency. In a comparative analysis using this system, we found that the NPC cell line in EBV abortive lytic cycle exhibited the superior chemotactic capacity to recruit monocytes and polarized their differentiation toward tumor-associated macrophage (TAM)-like phenotype and away from DCs, compared to EBV-negative or EBV-latency NPC cells. EBV-encoded transcription activator ZTA is responsible for regulating monocyte chemotaxis and TAM phenotype by up-regulating the expression of GM-CSF, IL-8, and GRO-α. As a result, TAM induced by EBV abortive lytic cycle promotes NPC angiogenesis, invasion, and migration. Overall, this study elucidated the role of the EBV lytic life cycle in the late development of NPC and revealed a mechanism underlying the ZTA-mediated establishment of the tumor microenvironment (TME) that promotes NPC late-stage progression.

Mammalian orthoreovirus can exit cells in extracellular vesicles.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.

Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

Active prophages in coral-associated Halomonas capable of lateral transduction.

Temperate phages can interact with bacterial hosts through lytic and lysogenic cycles via different mechanisms. Lysogeny has been identified as the major form of bacteria-phage interaction in the coral-associated microbiome. However, the lysogenic-to-lytic switch of temperate phages in ecologically important coral-associated bacteria and its ecological impact have not been extensively investigated. By studying the prophages in coral-associated Halomonas meridiana, we found that two prophages, Phm1 and Phm3, are inducible by the DNA-damaging agent mitomycin C and that Phm3 is spontaneously activated under normal cultivation conditions. Furthermore, Phm3 undergoes an atypical lytic pathway that can amplify and package adjacent host DNA, potentially resulting in lateral transduction. The induction of Phm3 triggered a process of cell lysis accompanied by the formation of outer membrane vesicles (OMVs) and Phm3 attached to OMVs. This unique cell-lysis process was controlled by a four-gene lytic module within Phm3. Further analysis of the Tara Ocean dataset revealed that Phm3 represents a new group of temperate phages that are widely distributed and transcriptionally active in the ocean. Therefore, the combination of lateral transduction mediated by temperate phages and OMV transmission offers a versatile strategy for host-phage coevolution in marine ecosystems.

Coral mucus as a reservoir of bacteriophages targeting Vibrio pathogens.

The increasing trend in sea surface temperature promotes the spread of Vibrio species, which are known to cause diseases in a wide range of marine organisms. Among these pathogens, Vibrio mediterranei has emerged as a significant threat, leading to bleaching in the coral species Oculina patagonica. Bacteriophages, or phages, are viruses that infect bacteria, thereby regulating microbial communities and playing a crucial role in the coral's defense against pathogens. However, our understanding of phages that infect V. mediterranei is limited. In this study, we identified two phage species capable of infecting V. mediterranei by utilizing a combination of cultivation and metagenomic approaches. These phages are low-abundance specialists within the coral mucus layer that exhibit rapid proliferation in the presence of their hosts, suggesting a potential role in coral defense. Additionally, one of these phages possesses a conserved domain of a leucine-rich repeat protein, similar to those harbored in the coral genome, that plays a key role in pathogen recognition, hinting at potential coral-phage coevolution. Furthermore, our research suggests that lytic Vibrio infections could trigger prophage induction, which may disseminate genetic elements, including virulence factors, in the coral mucus layer. Overall, our findings underscore the importance of historical coral-phage interactions as a form of coral immunity against invasive Vibrio pathogens.

Transcriptome analysis of Burkitt lymphoma cells treated with anti-convulsant drugs that are inhibitors of Epstein-Barr virus lytic reactivation.

Herpesviruses have two distinct life cycle stages, latency and lytic replication. Epstein-Barr virus (EBV), a gamma-herpesvirus, establishes latency in vivo and in cultured cells. Cell lines harboring latent EBV can be induced into the lytic cycle by treatment with chemical inducing agents. In the Burkitt lymphoma cell line HH514-16 the viral lytic cycle is triggered by butyrate, a histone deacetylase (HDAC) inhibitor. Butyrate also alters expression of thousands of cellular genes. However, valproic acid (VPA), another HDAC inhibitor with global effects on cellular gene expression blocks EBV lytic gene expression in Burkitt lymphoma cell lines. Valpromide (VPM), an amide derivative of VPA, is not an HDAC inhibitor, but like VPA blocks induction of the EBV lytic cycle. VPA and VPM are the first examples of inhibitors of initial stages of lytic reactivation. We compared the effects of VPA and VPM, alone and in combination with butyrate, on host cellular gene expression using whole transcriptome analysis (RNA-seq). Gene expression was analyzed 6 h after addition of the compounds, a time before the first EBV lytic transcripts are detected. The results address two alternative, yet possibly complementary, mechanisms for regulation of EBV lytic reactivation. First, cellular genes that were up- or down-regulated by butyrate, but no longer altered in the presence of VPA or VPM, represent genes that correlated with EBV lytic reactivation. Second, genes regulated similarly by VPA and VPM in the absence and presence of butyrate are candidates for suppressors of EBV reactivation. Two genes upregulated by the lytic cycle inhibitors, CHAC1 and SLC7A11, are related to redox status and the iron-dependent cell death pathway ferroptosis. This study generates new hypotheses for control of the latency to lytic cycle switch of EBV and provides the first description of effects of the anti-convulsant drug VPM on global human cellular gene expression.

Isolation and characterization of bacteriophages for controlling Rhizobium radiobacter - causing stem and crown gall of highbush blueberry.

Stem and crown gall disease caused by the plant pathogen Rhizobium radiobacter has a significant impact on highbush blueberry (Vaccinium corymbosum) production. Current methods for controlling the bacterium are limited. Lytic phages, which can specifically target host bacteria, have been widely gained interest in agriculture. In this study, 76 bacteriophages were recovered from sewage influent and screened for their inhibitory effect against Rhizobium spp. The phages were genetically characterized through whole-genome sequencing, and their lytic cycle was confirmed. Five potential candidate phages (isolates IC12, IG49, AN01, LG08, and LG11) with the ability to lyse a broad range of hosts were chosen and assessed for their morphology, environmental stability, latent period, and burst size. The morphology of these selected phages revealed a long contractile tail under transmission electron microscopy. Single-step growth curves displayed that these phages had a latent period of 80-110 min and a burst size ranging from 8 to 33 phages per infected cell. None of these phages contained any antimicrobial resistance or virulence genes in their genomes. Subsequently, a combination of two-, three- and four-phage cocktails were formulated and tested for their efficacy in a broth system. A three-phage cocktail composed of the isolates IC12, IG49 and LG08 showed promising results in controlling a large number of R. radiobacter strains in vitro. In a soil/peat-based model, the three-phage cocktail was tested against R. radiobacter PL17, resulting in a significant reduction (p < 0.05) of 2.9 and 1.3 log10 CFU/g after 24 and 48 h of incubation, respectively. These findings suggest that the three-phage cocktail (IC12, IG49 and LG08) has the potential to serve as a proactive antimicrobial solution for controlling R. radiobacter on blueberry.

Comparative proteomic analysis of PK-15 cells infected with wild-type strain and its EP0 gene-deleted mutant strain of pseudorabies virus.

As one of the main etiologic agents of infectious diseases in pigs, pseudorabies virus (PRV) infections have caused enormous economic losses worldwide. EP0, one of the PRV early proteins (EP) plays a vital role in PRV infections, but the mechanisms are unclear. This study examined the function of EP0 to provide a direction for its in-depth analysis. In this study, the EP0-deleted PRV mutant was obtained, and Tandem Mass Tag-based proteomic analysis was used to screen the differentially expressed proteins (DEPs) quantitatively in EP0-deleted PRV- or wild-type PRV-infected porcine kidney 15 cells. This study identified 7,391 DEPs, including 120 and 21 up-regulated and down-regulated DEPs, respectively. Western blot analysis confirmed the changes in the expression of the selected proteins, such as speckled protein 100. Comprehensive analysis revealed 141 DEPs involved in various biological processes and molecular functions, such as transcription regulator activity, biological regulation, and localization. These results holistically outlined the functions of EP0 during a PRV infection and might provide a direction for more detailed function studies of EP0 and the stimulation of lytic PRV infections.

CMV-encoded GPCRs in infection, disease, and pathogenesis.

G protein coupled receptors (GPCRs) are seven-transmembrane domain proteins that modulate cellular processes in response to external stimuli. These receptors represent the largest family of membrane proteins, and in mammals, their signaling regulates important physiological functions, such as vision, taste, and olfaction. Many organisms, including yeast, slime molds, and viruses encode GPCRs. Cytomegaloviruses (CMVs) are large, betaherpesviruses, that encode viral GPCRs (vGPCRs). Human CMV (HCMV) encodes four vGPCRs, including UL33, UL78, US27, and US28. Each of these vGPCRs, as well as their rodent and primate orthologues, have been investigated for their contributions to viral infection and disease. Herein, we discuss how the CMV vGPCRs function during lytic and latent infection, as well as our understanding of how they impact viral pathogenesis.

Antagonistic interactions between phage and host factors control arbitrium lysis–lysogeny decision

Phages can use a small-molecule communication arbitrium system to coordinate lysis–lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin–antitoxin system MazE–MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis–lysogeny by domesticating and fine-tuning a phage-defence mechanism.

ВЛИЯНИЕ КРАТКОСРОЧНОЙ ИЗОЛЯЦИИ В ГЕРМООБЪЕКТЕ МАЛОГО ОБЪЕМА НА СУБКЛИНИЧЕСКУЮ РЕАКТИВАЦИЮ ЛАТЕНТНЫХ ПАТОГЕНОВ ЧЕЛОВЕКА ВИРУСНОЙ И БАКТЕРИАЛЬНОЙ ПРИРОДЫ

The paper presents data of the investigations of subclinical reactivation of 7 human herpes viruses and bacteria Mycoplasmahominis, Ureaplasmaurealiticum and Chlamydiatrachomatis from 6 participants in a 14-day isolation study conducted in a small chamber with controlled environment. Purpose of analysis was to establish correlations between DNAs of latent viral and bacterial pathogens in bio-liquids of organism and indices of the natural and adaptive immune system. It was determined that short-term isolation in a small chamber may provoke reactivation of latent pathogens both of the viral and bacterial nature. The events can be triggered by changes in the immune status and appear in consequence of depressed immunological control of latent infections. Dynamics of the latent infections lytic cycle in the course of the study demonstrated a high degree of individual variability.

A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency.

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.

Lambda CI Binding to Related Phage Operator Sequences Validates Alignment Algorithm and Highlights the Importance of Overlooked Bonds.

Bacteriophage λ's CI repressor protein controls a genetic switch between the virus's lysogenic and lytic lifecycles, in part, by selectively binding to six different DNA sequences within the phage genome-collectively referred to as operator sites. However, the minimal level of information needed for CI to recognize and specifically bind these six unique-but-related sequences is unclear. In a previous study, we introduced an algorithm that extracts the minimal direct readout information needed for λ-CI to recognize and bind its six binding sites. We further revealed direct readout information shared among three evolutionarily related lambdoid phages: λ-phage, Enterobacteria phage VT2-Sakai, and Stx2 converting phage I, suggesting that the λ-CI protein could bind to the operator sites of these other phages. In this study, we show that λ-CI can indeed bind the other two phages' cognate binding sites as predicted using our algorithm, validating the hypotheses from that paper. We go on to demonstrate the importance of specific hydrogen bond donors and acceptors that are maintained despite changes to the nucleobase itself, and another that has an important role in recognition and binding. This in vitro validation of our algorithm supports its use as a tool to predict alternative binding sites for DNA-binding proteins.

The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

The mobilome of Lactobacillus crispatus M247 includes two novel genetic elements: Tn7088 coding for a putative bacteriocin and the siphovirus prophage ΦM247.

Lactobacillus crispatus is a member of the vaginal and gastrointestinal human microbiota. Here we determined the complete genome sequence of the probiotic strain M247 combining Nanopore and Illumina technologies. The M247 genome is organized in one circular chromosome of 2 336 109 bp, with a GC content of 37.04 % and 2303 ORFs, of which 1962 could be annotated. Analysis of the M247 mobilome, which accounts for 14 % of the whole genome, revealed the presence of: (i) Tn7088, a novel 14 105 bp long integrative and mobilizable element (IME) containing 16 ORFs; (ii) ΦM247, a novel 42 510 bp long siphovirus prophage containing 52 ORFs; (iii) three clustered regularly interspaced short palindromic repeats (CRISPRs); and (iv) 226 insertion sequences (ISs) belonging to 14 different families. Tn7088 has a modular organization including a mobilization module encoding FtsK homologous proteins and a relaxase, an integration/excision module coding for an integrase and an excisionase, and an adaptation module coding for a class I bacteriocin and homologous to the listeriolysin S (lls) locus of Listeria monocytogenes. Genome-wide homology search analysis showed the presence of Tn7088-like elements in 12 out of 23 L. crispatus complete public genomes. Mobilization and integration/excision modules are essentially conserved, while the adaptation module is variable since it is the target site for the integration of different ISs. Prophage ΦM247 contains genes for phage structural proteins, DNA replication and packaging, lysogenic and lytic cycles. ΦM247-like prophages are present in seven L. crispatus complete genomes, with sequence variability mainly due to the integration of ISs. PCR and sequencing showed that the Tn7088 IME excises from the M247 chromosome producing a circular form at a concentration of 4.32×10-5 copies per chromosome, and reconstitution of the Tn7088 chromosomal target site occurred at 6.65×10-4 copies per chromosome. The ΦM247 prophage produces an excised form and a reconstituted target site at a level of 3.90×10-5 and 2.48×10-5 copies per chromosome, respectively. This study identified two novel genetic elements in L. crispatus. Tn7088 represents the first example of an IME carrying a biosynthetic gene cluster for a class I bacteriocin in L. crispatus.

Identification and characterization of the anti-viral interferon lambda 3 as direct target of the Epstein-Barr virus microRNA-BART7-3p

ABSTRACT The human Epstein–Barr virus (EBV), as a member of the human γ herpes viruses (HHV), is known to be linked with distinct tumor types. It is a double-stranded DNA virus and its genome encodes among others for 48 different microRNAs (miRs). Current research demonstrated a strong involvement of certain EBV-miRs in molecular immune evasion mechanisms of infected cells by, e.g., the disruption of human leukocyte antigen (HLA) class Ia and NKG2D functions. To determine novel targets of EBV-miRs involved in immune surveillance, ebv-miR-BART7-3p, an EBV-encoded miR with high expression levels during the different lytic and latent EBV life cycle phases, was overexpressed in human HEK293T cells. Using a cDNA microarray-based comparative analysis, 234 (229 downregulated and 5 upregulated) deregulated human transcripts were identified in ebv-miR-BART7-3p transfectants, which were mainly involved in cellular processes and molecular binding. A statistically significant downregulation of the anti-proliferative and tumor-suppressive hsa-miR-34A and the anti-viral interferon lambda (IFNL)3 mRNA was found. The ebv-miR-BART7-3p-mediated downregulation of IFNL3 expression was due to a direct interaction with the IFNL3 3’-untranslated region (UTR) as determined by luciferase reporter gene assays including the identification of the accurate ebv-miR-BART7-3p binding site. The effect of ebv-miR-BART7-3p on the IFNL3 expression was validated both in human cell lines in vitro and in human tissue specimen with known EBV status. These results expand the current knowledge of EBV-encoded miRs and their role in immune evasion, pathogenesis and malignant transformation.