Alter Host Cell Research Articles

O.8DUX4 mRNA silencing with CRISPR-Cas13 gene therapy as a prospective treatment for Facioscapulohumeral muscular dystrophy

DUX4 de-repression causes Facioscapulohumeral muscular dystrophy (FSHD), which is associated with progressive muscle wasting and weakness that may lead to wheelchair dependence. There are currently no treatments that alter the course of FSHD and therapy development is an unmet need in the field. DUX4 genome editing using the CRISPR-Cas9 approach is promising but also has challenges, partly because the DUX4 gene is present in potentially hundreds of copies, embedded within identical or nearly identical D4Z4 repeats. The CRISPR-Cas13 system does not cleave DNA, but can be specifically directed to a RNA transcript of interest using a sequence-specific guide RNA (gRNA), thereby reducing the risk of permanent DNA damage. Here, the goal is to develop a new Cas13/CRISPR-mediated DUX4 mRNA silencing method as an effective therapeutic strategy for FSHD, without altering host cell DNA. We designed several Cas13b-gRNAs targeting different parts of DUX4 mRNA and demonstrated their ability to suppress DUX4 and prevent cell death in vitro and in vivo. We detected >90% reduction of DUX4 protein in treated cells, and improved viability in these cells. DUX4 mRNA levels were markedly decreased in treated FSHD myotubes, as determined by RNAscope in situ hybridization. This reduction was accompanied by complete or near complete reduction in levels of DUX4-responsive biomarkers in treated patient myoblasts/myotubes. In our first in vivo studies in tibialis anterior muscles of TIC-DUX4 mouse model, our lead constructs caused nearly 100% reduction of DUX4 expression. As of this writing, additional in vivo studies are underway, including testing the capabilities of our AAV-delivered CRISPR/Cas13 system to suppress DUX4 long-term in our new TIC-DUX4 mouse model, and improve histopathological, functional, and molecular outcomes. This study provides proof of concept for silencing DUX4 expression using the RNA targeting CRISPR-Cas13b system and has implications for future FSHD therapy.

Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex.

Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the β-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophilaIMPORTANCE For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the ∼330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.

Signal Distortion: How Intracellular Pathogens Alter Host Cell Fate by Modulating NF-κB Dynamics.

By uncovering complex dynamics in the expression or localization of transcriptional regulators in single cells that were otherwise hidden at the population level, live cell imaging has transformed our understanding of how cells sense and orchestrate appropriate responses to changes in their internal state or extracellular environment. This has proved particularly true for the nuclear factor-kappaB (NF-κB) family of transcription factors, key regulators of the inflammatory response and innate immune function, which are capable of encoding information about the mode and intensity of stimuli in the dynamics of NF-κB nuclear accumulation and loss. While live cell imaging continues to serve as a useful tool in ongoing efforts to characterize the feedbacks that shape these dynamics and to connect dynamics to downstream gene expression, it is also proving invaluable for recent studies that seek to determine how intracellular pathogens subvert NF-κB signaling to survive and replicate within host cells by providing quantitative information about the pathogen and changes in NF-κB activity during different stages of an infection. Here, we provide a brief overview of NF-κB signaling in innate immune cells and review recent literature that uses live imaging to investigate the mechanisms by which bacterial and yeast pathogens modulate NF-κB in a variety of different host cell types to evade destruction or maintain the viability of an intracellular growth niche.

Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells.

Klebsiella pneumoniae raises significant concerns to the health care industry as these microbes are the source of widespread contamination of medical equipment, cause pneumonia as well as other multiorgan metastatic infections and have gained multidrug resistance. Despite soaring mortality rates, the host cell alterations occurring during these infections remain poorly understood. Here, we show that during in vitro and in vivo K.pneumoniae infections of lung epithelia, microtubules are severed and then eliminated. This destruction does not require direct association of K.pneumoniae with the host cells, as microtubules are disassembled in cells that are distant from the infecting bacteria. This microtubule dismantling is dependent on the K.pneumoniae (Kp) gene ytfL as non-pathogenic Escherichia coli expressing Kp ytfL disassemble microtubules in the absence of K.pneumoniae itself. Our data points to the host katanin catalytic subunit A like 1 protein (KATNAL1) and the katanin regulatory subunit B1 protein (KATNB1) as the gatekeepers to the microtubule severing event as both proteins localise specifically to microtubule cut sites. Infected cells that had either of these proteins knocked out maintained intact microtubules. Taken together, we have identified a novel mechanism that a bacterial pathogen has exploited to cause microtubule destruction within the host epithelia.

O-deGlcNAcylation is required for Entamoeba histolytica-induced HepG2 cell death

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. E. histolytica can induce host-cell apoptosis by initiating various intracellular signaling mechanisms closely associated with tissue pathogenesis and parasitic immune evasion. O-GlcNAcylation, similar to phosphorylation, is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, whether O-GlcNAc alterations in host cells affect E. histolytica-induced cell death and which signal molecules participate in E. histolytica-induced deglycosylation remain unknown. In this study, co-incubation of HepG2 cells with E. histolytica increased DNA fragmentation and LDH release as compared with control cells. Additionally, Gal-lectin-mediated amoebic adherence of live trophozoites to HepG2 cells decreased O-GlcNAcylated protein levels within 5 min. We also observed a rapid decrease in cellular OGT protein level, but not OGA, in HepG2 cells in a contact-dependent manner. Furthermore, HepG2 pretreatment with OGA inhibitors or OGA siRNA prevented E. histolytica-induced O-deGlcNAcylation, DNA fragmentation, and LDH release. Our results suggested that E. histolytica-induced O-deGlcNAcylation in HepG2 cells was an important process required for hepatocyte cell death induced by E. histolytica adherence.

Rhinovirus induces an anabolic reprogramming in host cell metabolism essential for viral replication

Rhinoviruses (RVs) are responsible for the majority of upper airway infections; despite their high prevalence and the resulting economic burden, effective treatment is lacking. We report here that RV induces metabolic alterations in host cells, which offer an efficient target for antiviral intervention. We show that RV-infected cells rapidly up-regulate glucose uptake in a PI3K-dependent manner. In parallel, infected cells enhance the expression of the PI3K-regulated glucose transporter GLUT1. In-depth metabolomic analysis of RV-infected cells revealed a critical role of glucose mobilization from extracellular and intracellular pools via glycogenolysis for viral replication. Infection resulted in a highly anabolic state, including enhanced nucleotide synthesis and lipogenesis. Consistently, we observed that glucose deprivation from medium and via glycolysis inhibition by 2-deoxyglucose (2-DG) potently impairs viral replication. Metabolomic analysis showed that 2-DG specifically reverts the RV-induced anabolic reprogramming. In addition, treatment with 2-DG inhibited RV infection and inflammation in a murine model. Thus, we demonstrate that the specific metabolic fingerprint of RV infection can be used to identify new targets for therapeutic intervention.

CagY-Dependent Regulation of Type IV Secretion in Helicobacter pylori Is Associated with Alterations in Integrin Binding.

ABSTRACTStrains of Helicobacter pylori that cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4+ T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human α5β1 integrin, we tested the hypothesis that recombination in the CagY MRR regulates T4SS function by modulating binding to α5β1 integrin. Using a cell-free microfluidic assay, we found that H. pylori binding to α5β1 integrin under shear flow is dependent on the CagY MRR, but independent of the presence of the T4SS pili, which are only formed when H. pylori is in contact with host cells. Similarly, expression of CagY in the absence of other T4SS genes was necessary and sufficient for whole bacterial cell binding to α5β1 integrin. Bacteria with variant cagY alleles that reduced T4SS function showed comparable reduction in binding to α5β1 integrin, although CagY was still expressed on the bacterial surface. We speculate that cagY-dependent modulation of H. pylori T4SS function is mediated by alterations in binding to α5β1 integrin, which in turn regulates the host inflammatory response so as to maximize persistent infection.

Interplay of Viral Infection, Host Cell Factors and Tumor Microenvironment in the Pathogenesis of Nasopharyngeal Carcinoma.

Undifferentiated nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. In addition, heavy infiltration of leukocytes is a common characteristic of EBV-associated NPC. It has long been suggested that substantial and interactive impacts between cancer and stromal cells create a tumor microenvironment (TME) to promote tumorigenesis. The coexistence of tumor-infiltrating lymphocytes with EBV-infected NPC cells represents a distinct TME which supports immune evasion and cancer development from the early phase of EBV infection. Intracellularly, EBV-encoded viral products alter host cell signaling to facilitate tumor development and progression. Intercellularly, EBV-infected cancer cells communicate with stromal cells through secretion of cytokines and chemokines, or via release of tumor exosomes, to repress immune surveillance and enhance metastasis. Although high expression of miR-BARTs has been detected in NPC patients, contributions of these more recently discovered viral products to the establishment of TME are still vaguely defined. Further investigations are needed to delineate the mechanistic linkage of the interplay between viral and host factors, especially in relation to TME, which can be harnessed in future therapeutic strategies.

Herpesvirus-encoded microRNAs detected in human gingiva alter host cell transcriptome and regulate viral infection

MicroRNAs (miRNAs) are small, non-coding RNAs of ~18–25 nucleotides that have gained extensive attention as critical regulators in complex gene networks including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and function. Many viruses encode miRNAs that directly downregulate the expression of factors of the innate immune system, which includes proteins involved in promoting apoptosis and recruitment. In this study, we examined the expression profiles of three previously identified viral miRNAs (v-miRs) from the human herpesvirus (HHV) family, HSV-1 (miR-H1), KSHV (miR-K12-3-3p), and HCMV (miR-US4) in healthy and diseased periodontal tissues and observed increased levels of v-miRs in diseased tissues. To understand the significance of this increase, we overexpressed v-miRs in human oral keratinocytes (HOK), a common target for various HHV, and analyzed the impact of miR-H1 and miR-K12-3-3p on the host transcriptome. More than 1300 genes were altered in HOK overexpressing miR-H1 and miR-K12-3-3p. Global pathway analysis of deregulated genes identified several key cellular pathways that may favor viral persistence. Using bioinformatic analysis, we predicted hundreds of potential v-miR binding sites on genes downregulated by miR-H1 and miR-K12-3-3p and validated three novel target v-miR sites suggesting widespread direct and indirect modulation of numerous host genes/pathways by a single v-miR. Finally, in vitro HSV-1 infection assays showed that miR-H1 can regulate viral entry and infection in human oral keratinocytes (HOK). Overall, our results demonstrate clinical and functional relevance of pathogenic viral molecules viz., v-miRs that regulate both host and viral functions and may contribute to the pathogenesis of inflammatory oral diseases.

Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses.

Prevalence of the members of herpesvirus family in oral inflammatory diseases is increasingly acknowledged suggesting their likely role as an etiological factor. However, the underlying mechanisms remain obscure. In our recent miRNA profiling of healthy and diseased human tooth pulps, elevated expression of human herpesvirus encoded viral microRNAs (v-miRs) were identified. Based on the fold induction and significance values, we selected three v-miRs namely miR-K12-3-3p [Kaposi sarcoma-associated virus (KSHV)], miR-H1 [herpes simplex virus 1 (HSV1)], and miR-UL-70-3p [human cytomegalovirus (HCMV)] to further examine their impact on host cellular functions. We examined their impact on cellular miRNA profiles of primary human oral keratinocytes (HOK). Our results show differential expression of several host miRNAs in v-miR-transfected HOK. High levels of v-miRs were detected in exosomes derived from v-miR transfected HOK as well as the KSHV-infected cell lines. We show that HOK-derived exosomes release their contents into macrophages (Mφ) and alter expression of endogenous miRNAs. Concurrent expression analysis of precursor (pre)-miRNA and mature miRNA suggest transcriptional or posttranscriptional impact of v-miRs on the cellular miRNAs. Employing bioinformatics, we predicted several pathways targeted by deregulated cellular miRNAs that include cytoskeletal organization, endocytosis, and cellular signaling. We validated three novel targets of miR-K12-3-3p and miR-H1 that are involved in endocytic and intracellular trafficking pathways. To evaluate the functional consequence of this regulation, we performed phagocytic uptake of labeled bacteria and noticed significant attenuation in miR-H1 and miR-K12-3-3p but not miR-UL70-3p transfected primary human Mφ. Multiple cytokine analysis of E. coli challenged Mφ revealed marked reduction of secreted cytokine levels with important roles in innate and adaptive immune responses suggesting a role of v-miRs in immune subversion. Our findings reveal that oral disease associated v-miRs can dysregulate functions of key host cells that shape oral mucosal immunity thus exacerbating disease severity and progression.

Viral manipulation of host mRNA decay.

Viruses alter host-cell gene expression at many biochemical levels, such as transcription, translation, mRNA splicing and mRNA decay in order to create a cellular environment suitable for viral replication. In this review, we discuss mechanisms by which viruses manipulate host-gene expression at the level of mRNA decay in order to enable the virus to evade host antiviral responses to allow viral survival and replication. We discuss different cellular RNA decay pathways, including the deadenylation-dependent mRNA decay pathway, and various strategies that viruses exploit to manipulate these pathways in order to create a virus-friendly cellular environment.

Deletion of the Major Facilitator Superfamily Transporter fptB Alters Host Cell Interactions and Attenuates Virulence of Type A Francisella tularensis.

ABSTRACTFrancisella tularensis is a Gram-negative, facultative, intracellular coccobacillus that can infect a wide variety of hosts. In humans, F. tularensis causes the zoonosis tularemia following insect bites, ingestion, inhalation, and the handling of infected animals. The fact that a very small inoculum delivered by the aerosol route can cause severe disease, coupled with the possibility of its use as an aerosolized bioweapon, has led to the classification of Francisella tularensis as a category A select agent and has renewed interest in the formulation of a vaccine. To this end, we engineered a type A strain SchuS4 derivative containing a targeted deletion of the major facilitator superfamily (MFS) transporter fptB. Based on the attenuating capacity of this deletion in the F. tularensis LVS background, we hypothesized that the deletion of this transporter would alter the intracellular replication and cytokine induction of the type A strain and attenuate virulence in the stringent C57BL/6J mouse model. Here we demonstrate that the deletion of fptB significantly alters the intracellular life cycle of F. tularensis, attenuating intracellular replication in both cell line-derived and primary macrophages and inducing a novel cytosolic escape delay. Additionally, we observed prominent differences in the in vitro cytokine profiles in human macrophage-like cells. The mutant was highly attenuated in the C57BL/6J mouse model and provided partial protection against virulent type A F. tularensis challenge. These results indicate a fundamental necessity for this nutrient transporter in the timely progression of F. tularensis through its replication cycle and in pathogenesis.

Antibiotics directly affect host cell metabolism

Antibiotic treatment alters host cell metabolism in a manner that affects both the efficacy of the drug and the immune response to infection.

Somatic Host Cell Alterations in HPV Carcinogenesis.

High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and phosphatase and tensin homolog (PTEN), human leukocyte antigen A and B (HLA-A and HLA-B)-A/B, and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions.

Dengue Virus Activates the AMP Kinase-mTOR Axis To Stimulate a Proviral Lipophagy.

Robust dengue virus (DENV) replication requires lipophagy, a selective autophagy that targets lipid droplets. The autophagic mobilization of lipids leads to increased β-oxidation in DENV-infected cells. The mechanism by which DENV induces lipophagy is unknown. Here, we show that infection with DENV activates the metabolic regulator 5' adenosine-monophosphate activated kinase (AMPK), and that the silencing or pharmacological inhibition of AMPK activity decreases DENV replication and the induction of lipophagy. The activity of the mechanistic target of rapamycin complex 1 (mTORC1) decreases in DENV-infected cells and is inversely correlated with lipophagy induction. Constitutive activation of mTORC1 by depletion of tuberous sclerosis complex 2 (TSC2) inhibits lipophagy induction in DENV-infected cells and decreases viral replication. While AMPK normally stimulates TSC2-dependent inactivation of mTORC1 signaling, mTORC1 inactivation is independent of AMPK activation during DENV infection. Thus, DENV stimulates and requires AMPK signaling as well as AMPK-independent suppression of mTORC1 activity for proviral lipophagy.IMPORTANCE Dengue virus alters host cell lipid metabolism to promote its infection. One mechanism for altered metabolism is the induction of a selective autophagy that targets lipid droplets, termed lipophagy. Lipophagy mobilizes lipid stores, resulting in enhanced β-oxidation and viral replication. We show here that DENV infection activates and requires the central metabolic regulator AMPK for its replication and the induction of lipophagy. This is required for the induction of lipophagy, but not basal autophagy, in DENV-infected cells.

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8+ T-cells.

The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc.

Current Trends and Alternative Scenarios in EBV Research.

Epstein-Barr virus (EBV) infection is associated with several distinct hematological and epithelial malignancies, e.g., Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and others. The association with several malignant tumors of local and worldwide distribution makes EBV one of the most important tumor viruses. Furthermore, because EBV can cause posttransplant lymphoproliferative disease, transplant medicine has to deal with EBV as a major pathogenic virus second only to cytomegalovirus. In this review, we summarize briefly the natural history of EBV infection and outline some of the recent advances in the pathogenesis of the major EBV-associated neoplasms. We present alternative scenarios and discuss them in the light of most recent experimental data. Emerging research areas including EBV-induced patho-epigenetic alterations in host cells and the putative role of exosome-mediated information transfer in disease development are also within the scope of this review. This book contains an in-depth description of a series of modern methodologies used in EBV research. In this introductory chapter, we thoroughly refer to the applications of these methods and demonstrate how they contributed to the understanding of EBV-host cell interactions. The data gathered using recent technological advancements in molecular biology and immunology as well as the application of sophisticated in vitro and in vivo experimental models certainly provided deep and novel insights into the pathogenetic mechanisms of EBV infection and EBV-associated tumorigenesis. Furthermore, the development of adoptive T cell immunotherapy has provided a novel approach to the therapy of viral disease in transplant medicine and hematology.

Dynamic flux of microvesicles modulate parasite-host cell interaction of Trypanosoma cruzi in eukaryotic cells.

Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell-derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP-1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell-derived trypomastigote (tissue culture-derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP-1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP-1-derived MVs can fuse with parasite-derived MVs. Furthermore, MVs derived from the TCT-THP-1 interaction showed a higher fusogenic capacity than those from META- or EPI-THP-1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite-THP-1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP-1-derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology.

Dynamics of Human and Viral RNA Methylation during Zika Virus Infection.

Infection with the flavivirus Zika (ZIKV) causes neurological, immunological, and developmental defects through incompletely understood mechanisms. We report that ZIKV infection affects viral and human RNAs by altering the topology and function of N6-adenosine methylation (m6A), a modification affecting RNA structure and function. m6A nucleosides are abundant in ZIKV RNA, with twelve m6A peaks identified across full-length ZIKV RNA. m6A in ZIKV RNA is controlled by host methyltransferases METTL3 and METTL14 and demethylases ALKBH5 and FTO, and knockdown of methyltransferases increases, while silencing demethylases decreases, ZIKV production. YTHDF family proteins, which regulate the stability of m6A-modified RNA, bind to ZIKV RNA, and their silencing increases ZIKV replication. Profiling of the m6A methylome of host mRNAs reveals that ZIKV infection alters m6A location in mRNAs, methylation motifs, and target genes modified by methyltransferases. Our results identify a mechanism by which ZIKV interacts with and alters host cell functions.

Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

BackgroundBurkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood.MethodsWe determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS).ResultsWe found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages.ConclusionUnderstanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections.