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Sample records for host cell rna

  1. pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

    PubMed Central

    Sugiyama, Kenji; Kawaguchi, Atsushi; Okuwaki, Mitsuru; Nagata, Kyosuke

    2015-01-01

    Replication of influenza viral genomic RNA (vRNA) is catalyzed by viral RNA-dependent RNA polymerase (vRdRP). Complementary RNA (cRNA) is first copied from vRNA, and progeny vRNAs are then amplified from the cRNA. Although vRdRP and viral RNA are minimal requirements, efficient cell-free replication could not be reproduced using only these viral factors. Using a biochemical complementation assay system, we found a novel activity in the nuclear extracts of uninfected cells, designated IREF-2, that allows robust unprimed vRNA synthesis from a cRNA template. IREF-2 was shown to consist of host-derived proteins, pp32 and APRIL. IREF-2 interacts with a free form of vRdRP and preferentially upregulates vRNA synthesis rather than cRNA synthesis. Knockdown experiments indicated that IREF-2 is involved in in vivo viral replication. On the basis of these results and those of previous studies, a plausible role(s) for IREF-2 during the initiation processes of vRNA replication is discussed. DOI: http://dx.doi.org/10.7554/eLife.08939.001 PMID:26512887

  2. The influence of viral RNA secondary structure on interactions with innate host cell defences

    PubMed Central

    Witteveldt, Jeroen; Blundell, Richard; Maarleveld, Joris J.; McFadden, Nora; Evans, David J.; Simmonds, Peter

    2014-01-01

    RNA viruses infecting vertebrates differ fundamentally in their ability to establish persistent infections with markedly different patterns of transmission, disease mechanisms and evolutionary relationships with their hosts. Although interactions with host innate and adaptive responses are complex and persistence mechanisms likely multi-factorial, we previously observed associations between bioinformatically predicted RNA secondary formation in genomes of positive-stranded RNA viruses with their in vivo fitness and persistence. To analyse this interactions functionally, we transfected fibroblasts with non-replicating, non-translated RNA transcripts from RNA viral genomes with differing degrees of genome-scale ordered RNA structure (GORS). Single-stranded RNA transcripts induced interferon-β mediated though RIG-I and PKR activation, the latter associated with rapid induction of antiviral stress granules. A striking inverse correlation was observed between induction of both cellular responses with transcript RNA structure formation that was independent of both nucleotide composition and sequence length. The consistent inability of cells to recognize RNA transcripts possessing GORS extended to downstream differences from unstructured transcripts in expression of TNF-α, other interferon-stimulated genes and induction of apoptosis. This functional association provides novel insights into interactions between virus and host early after infection and provides evidence for a novel mechanism for evading intrinsic and innate immune responses. PMID:24335283

  3. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

    PubMed Central

    Viktorovskaya, Olga V.; Greco, Todd M.; Cristea, Ileana M.; Thompson, Sunnie R.

    2016-01-01

    Background There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. Methodology/Principal Findings Seventy-nine novel RNA binding proteins for dengue virus (DENV) were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. Conclusions/Significance The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with

  4. MicroRNA profile analysis of host cells before and after wild human rotavirus infection.

    PubMed

    Zhou, Yan; Wu, Jinyuan; Geng, Panpan; Kui, Xiang; Xie, Yuping; Zhang, Lei; Liu, Yaling; Yin, Na; Zhang, Guangming; Yi, Shan; Li, Hongjun; Sun, Maosheng

    2016-09-01

    Rotavirus infection is an important cause of acute gastroenteritis in children, but the interaction between rotavirus and host cells is not completely understood. We isolated a wildtype (wt) rotavirus strain, ZTR-68(P [8] G1), which is derived from an infant with diarrhea in southwest China in 2010. In this study, we investigated host cellular miRNA expression profiles changes in response to ZTR-68 in early stage of infection to investigate the role of miRNAs upon rotavirus infection. Differentially expressed miRNAs were identified by deep sequencing and qRT-PCR and the function of their targets predicted by Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. A total of 36 candidate miRNAs were identified. Comparative analysis indicated that 29 miRNAs were significantly down-regulated and 7 were up-regulated after infection. The data were provided contrasting the types of microRNAs in two different permissive cell lines (HT29 and MA104). The target assays results showed that mml-miR-7 and mml-miR-125a are involved in anti-rotavirus and virus-host interaction in host cells. These results offer clues for identifying potential candidates in vector-based antiviral strategies. J. Med. Virol. 88:1497-1510, 2016. © 2016 Wiley Periodicals, Inc. PMID:26890217

  5. A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Wang, Fei-yu; Zhang, Yu-qing; Wang, Xin-min; Wang, Chan; Wang, Xiao-fang; Wu, Jiang-dong; Wu, Fang; Zhang, Wan-jiang; Zhang, Le

    2016-04-01

    Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy. PMID:27033209

  6. A pathogenic long noncoding RNA redesigns the epigenetic landscape of the infected cells by subverting host Histone Deacetylase 6 activity.

    PubMed

    Castellano, Mayte; Pallas, Vicente; Gomez, Gustavo

    2016-09-01

    Viroids - ancient plant-pathogenic long noncoding RNAs - have developed a singular evolutionary strategy based on reprogramming specific phases of host-metabolism to ensure that their infection cycle can be completed in infected cells. However, the molecular aspects governing this transregulatory phenomenon remain elusive. Here, we use immunoprecipitation assays and bisulfite sequencing of rDNA to shown that, in infected cucumber and Nicotiana benthamina plants, Hop stunt viroid (HSVd) recruits and functionally subverts Histone Deacetylase 6 (HDA6) to promote host-epigenetic alterations that trigger the transcriptional alterations observed during viroid pathogenesis. This notion is supported by the demonstration that, during infection, the HSVd-HDA6 complex occurs in vivo and that endogenous HDA6 expression is increased in HSVd-infected cells. Moreover, transient overexpression of recombinant HDA6 reverts the hypomethylation status of rDNA observed in HSVd-infected plants and reduces viroid accumulation. We hypothesize that the host-transcriptional alterations induced as a consequence of viroid-mediated HDA6 recruitment favor spurious recognition of HSVd-RNA as an RNA Pol II template, thereby improving viroid replication. Our results constitute the first description of a physical and functional interaction between a pathogenic RNA and a component of the host RNA silencing mechanism, providing novel evidence of the potential of these pathogenic lncRNAs to physically redesign the host-cell environment and reprogram their regulatory mechanisms. PMID:27174164

  7. Purification of a soluble template-dependent rhinovirus RNA polymerase and its dependence on a host cell protein for viral RNA synthesis.

    PubMed Central

    Morrow, C D; Lubinski, J; Hocko, J; Gibbons, G F; Dasgupta, A

    1985-01-01

    The soluble phase of the cytoplasm of human rhinovirus type 2-infected cells contains an enzymatic activity able to copy rhinovirion RNA without an added primer. This RNA-dependent RNA polymerase (replicase) makes a specific copy of the added rhinovirion RNA, as shown by hybridization of the product to its template RNA but not to other RNAs. The same replicase preparation also contains a virus-specific polyuridylic acid [poly(U)] polymerase activity which is dependent on added polyadenylic acid-oligouridylic acid template-primer. Both activities purify together until a step at which poly(U) polymerase but no replicase activity is recovered. Addition of a purified HeLa cell protein (host factor) to this poly(U) polymerase completely reconstitutes rhinovirus replicase activity. Host factor activity can be supplied by adding oligouridylic acid, suggesting that the host cell protein acts at the initiation step of rhinovirus RNA replication. A virus-specific 64,000-dalton protein purifies with both poly(U) polymerase and replicase activities. Images PMID:2981346

  8. Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells

    PubMed Central

    Kumari, Bharti; Jain, Pratistha; Das, Shaoli; Ghosal, Suman; Hazra, Bibhabasu; Trivedi, Ashish Chandra; Basu, Anirban; Chakrabarti, Jayprokas; Vrati, Sudhanshu; Banerjee, Arup

    2016-01-01

    Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation. PMID:26838068

  9. A bacterial regulatory RNA attenuates virulence, spread and human host cell phagocytosis

    PubMed Central

    Le Pabic, Hélène; Germain-Amiot, Noëlla; Bordeau, Valérie; Felden, Brice

    2015-01-01

    Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation. PMID:26240382

  10. Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

    SciTech Connect

    Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.; Dasgupta, A.

    1987-11-01

    The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

  11. Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

    PubMed Central

    Baddal, Buket; Muzzi, Alessandro; Censini, Stefano; Calogero, Raffaele A.; Torricelli, Giulia; Guidotti, Silvia; Taddei, Anna R.; Covacci, Antonello; Pizza, Mariagrazia; Rappuoli, Rino; Pezzicoli, Alfredo

    2015-01-01

    ABSTRACT The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies. PMID:26578681

  12. Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition

    SciTech Connect

    Versteeg, Gijs A.; Bredenbeek, Peter J.; Worm, Sjoerd H.E. van den; Spaan, Willy J.M. . E-mail: w.j.m.spaan@lumc.nl

    2007-04-25

    Many viruses encode antagonists to prevent interferon (IFN) induction. Infection of fibroblasts with the murine hepatitis coronavirus (MHV) and SARS-coronavirus (SARS-CoV) did not result in nuclear translocation of interferon-regulatory factor 3 (IRF3), a key transcription factor involved in IFN induction, and induction of IFN mRNA transcription. Furthermore, MHV and SARS-CoV infection could not prevent IFN induction by poly (I:C) or Sendai virus, suggesting that these CoVs do not inactivate IRF3-mediated transcription regulation, but apparently prevent detection of replicative RNA by cellular sensory molecules. Our data indicate that shielding of viral RNA to host cell sensors might be the main general mechanism for coronaviruses to prevent IFN induction.

  13. Solar and temperature treatments affect the ability of human rotavirus wa to bind to host cells and synthesize viral RNA.

    PubMed

    Romero-Maraccini, Ofelia C; Shisler, Joanna L; Nguyen, Thanh H

    2015-06-15

    Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation. PMID:25862222

  14. Solar and Temperature Treatments Affect the Ability of Human Rotavirus Wa To Bind to Host Cells and Synthesize Viral RNA

    PubMed Central

    Shisler, Joanna L.

    2015-01-01

    Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation. PMID:25862222

  15. Requirements for eIF4A and eIF2 during translation of Sindbis virus subgenomic mRNA in vertebrate and invertebrate host cells.

    PubMed

    Garcia-Moreno, Manuel; Sanz, Miguel Angel; Pelletier, Jerry; Carrasco, Luis

    2013-05-01

    We have examined the requirements for the initiation factors (eIFs) eIF4A and eIF2 to translate Sindbis virus (SV) subgenomic mRNA (sgmRNA) in the natural hosts of SV: vertebrate and arthropod cells. Notably, this viral mRNA does not utilize eIF4A in SV-infected mammalian cells. However, eIF4A is required to translate this mRNA in transfected cells. Therefore, SV sgmRNA exhibits a dual mechanism for translation with respect to the use of eIF4A. Interestingly, SV genomic mRNA requires eIF4A for translation during the early phase of infection. In sharp contrast to what is observed in mammalian cells, active eIF2 is necessary to translate SV sgmRNA in mosquito cells. However, eIF4A is not necessary for SV sgmRNA translation in this cell line. In the SV sgmRNA coding region, proximal to the initiation codon is a hairpin structure that confers eIF2 independence only in mammalian cells infected by SV. Strikingly, this structure does not provide independence for eIF4A neither in mammalian nor in mosquito cells. These findings provide the first evidence of different eIF requirements for translation of SV sgmRNA in vertebrate and invertebrate cells. These observations can help to understand the interaction of SV with its host cells. PMID:23189929

  16. Kaposi's sarcoma-associated herpesvirus noncoding polyadenylated nuclear RNA interacts with virus- and host cell-encoded proteins and suppresses expression of genes involved in immune modulation.

    PubMed

    Rossetto, Cyprian C; Pari, Gregory S

    2011-12-01

    During lytic infection, Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a polyadenylated nuclear RNA (PAN RNA). This noncoding RNA (ncRNA) is localized to the nucleus and is the most abundant viral RNA during lytic infection; however, to date, the role of PAN RNA in the virus life cycle is unknown. Many examples exist where ncRNAs have a defined key regulatory function controlling gene expression by various mechanisms. Our goal for this study was to identify putative binding partners for PAN RNA in an effort to elucidate a possible function for the transcript in KSHV infection. We employed an in vitro affinity protocol where PAN RNA was used as bait for factors present in BCBL-1 cell nuclear extract to show that PAN RNA interacts with several virus- and host cell-encoded factors, including histones H1 and H2A, mitochondrial and cellular single-stranded binding proteins (SSBPs), and interferon regulatory factor 4 (IRF4). RNA chromatin immunoprecipitation (ChIP) assays confirmed that PAN RNA interacted with these factors in the infected cell environment. A luciferase reporter assay showed that PAN RNA expression interfered with the ability of IRF4/PU.1 to activate the interleukin-4 (IL-4) promoter, strongly suggesting a role for PAN RNA in immune modulation. Since the proteomic screen and functional data suggested a role in immune responses, we investigated if constitutive PAN RNA expression could affect other genes involved in immune responses. PAN RNA expression decreased expression of gamma interferon, interleukin-18, alpha interferon 16, and RNase L. These data strongly suggest that PAN RNA interacts with viral and cellular proteins and can function as an immune modulator. PMID:21957289

  17. Determination of host RNA helicases activity in viral replication

    PubMed Central

    Sharma, Amit; Boris-Lawrie, Kathleen

    2016-01-01

    RNA helicases are encoded by all eukaryotic and prokaryotic cells and a minority of viruses. Activity of RNA helicases is necessary for all steps in the expression of cells and viruses and the host innate response to virus infection. Their vast functional repertoire is attributable to the core ATPase-dependent helicase domain in conjunction with flanking domains that are interchangeable and engage viral and cellular cofactors. Here, we address the important issue of host RNA helicases that are necessary for replication of a virus. The chapter covers approaches to identification and characterization of candidate helicases and methods to define the biochemical and biophysical parameters of specificity and functional activity of the enzymes. We discuss the context of cellular RNA helicase activity and virion-associated RNA helicases. The methodology and choice of controls fosters the assessment of the virologic scope of RNA helicases across divergent cell lineages and viral replication cycles. PMID:22713331

  18. miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.

    PubMed

    Stiefel, Fabian; Fischer, Simon; Sczyrba, Alexander; Otte, Kerstin; Hesse, Friedemann

    2016-05-10

    Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering. PMID:27002234

  19. Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

    PubMed

    Dhamne, Hemant; Chande, Ajit G; Mukhopadhyaya, Robin

    2014-01-01

    Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance. PMID:24325878

  20. Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection.

    PubMed

    Zhao, Hongxing; Chen, Maoshan; Lind, Sara Bergström; Pettersson, Ulf

    2016-05-01

    The deregulation of cellular long non-coding RNA (lncRNA) expression during a human adenovirus infection was studied by deep sequencing. Expression of lncRNAs increased substantially following the progression of the infection. Among 645 significantly expressed lncRNAs, the expression of 398 was changed more than 2-fold. More than 80% of them were up-regulated and 80% of them were detected during the late phase. Based on the genomic locations of the deregulated lncRNAs in relation to known mRNAs and miRNAs, they were predicted to be involved in growth, structure, apoptosis and wound healing in the early phase, cell proliferation in the intermediate phase and protein synthesis, modification and transport in the late phase. The most significant functions of cellular RNA-binding proteins, previously shown to interact with the deregulated lncRNAs identified here, are involved in RNA splicing, nuclear export and translation events. We hypothesize that adenoviruses exploit the lncRNA network to optimize their reproduction. PMID:27003248

  1. Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs.

    PubMed

    Miesen, Pascal; Ivens, Alasdair; Buck, Amy H; van Rij, Ronald P

    2016-02-01

    In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species. PMID:26914027

  2. Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

    PubMed Central

    Miesen, Pascal; Ivens, Alasdair; Buck, Amy H.; van Rij, Ronald P.

    2016-01-01

    In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species. PMID:26914027

  3. Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

    PubMed Central

    Costales, Jaime A; Daily, Johanna P; Burleigh, Barbara A

    2009-01-01

    Background The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. Results We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change ≥ 2, p-value < 0.01) 24 hours post-invasion. A prominent type I interferon response was observed in each cell type, reflecting a secondary response to secreted cytokine in infected cultures. To identify a core cytokine-independent response in T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. Conclusion Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at

  4. A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion.

    PubMed

    Andritschke, Daniel; Dilling, Sabrina; Emmenlauer, Mario; Welz, Tobias; Schmich, Fabian; Misselwitz, Benjamin; Rämö, Pauli; Rottner, Klemens; Kerkhoff, Eugen; Wada, Teiji; Penninger, Josef M; Beerenwinkel, Niko; Horvath, Peter; Dehio, Christoph; Hardt, Wolf-Dietrich

    2016-01-01

    Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect. PMID:27627128

  5. An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway

    PubMed Central

    Robertson, Kevin A.; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J.; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J.; Enright, Anton J.; Yamamoto, Mami; Pradeepa, Madapura M.; Lennox, Kimberly A.; Behlke, Mark A.; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J.; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

    2016-01-01

    In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway. PMID:26938778

  6. The cag-pathogenicity island encoded CncR1 sRNA oppositely modulates Helicobacter pylori motility and adhesion to host cells.

    PubMed

    Vannini, Andrea; Roncarati, Davide; Danielli, Alberto

    2016-08-01

    Small regulatory RNAs (sRNAs) are emerging as key post-transcriptional regulators in many bacteria. In the human pathobiont Helicobacter pylori a plethora of trans- and cis-encoded sRNAs have been pinpointed by a global transcriptome study. However, only two have been studied in depth at the functional level. Here we report the characterization of CncR1, an abundant and conserved sRNA encoded by the virulence-associated cag pathogenicity island (cag-PAI) of H. pylori. Growth-phase dependent transcription of CncR1 is directed by the PcagP promoter, which resulted to be a target of the essential transcriptional regulator HsrA (HP1043). We demonstrate that the 213 nt transcript arising from this promoter ends at an intrinsic terminator, few bases upstream of the annotated cagP open reading frame, establishing CncR1 as the predominant gene product encoded by the cagP (cag15) locus. Interestingly, the deletion of the locus resulted in the deregulation en masse of σ(54)-dependent genes, linking CncR1 to flagellar functions. Accordingly, the enhanced motility recorded for cncR1 deletion mutants was complemented by ectopic reintroduction of the allele in trans. In silico prediction identified fliK, encoding a flagellar checkpoint protein, as likely regulatory target of CncR1. The interaction of CncR1 with the fliK mRNA was thus further investigated in vitro, demonstrating the formation of strand-specific interactions between the two RNA molecules. Accordingly, the full-length translational fusions of fliK with a lux reporter gene were induced in a cncR1 deletion mutant in vivo. These data suggest the involvement of CncR1 in the post-transcriptional modulation of H. pylori motility functions through down-regulation of a critical flagellar checkpoint factor. Concurrently, the cncR1 mutant revealed a decrease of transcript levels for several H. pylori adhesins, resulting in a phenotypically significant impairment of bacterial adhesion to a host gastric cell line. The data

  7. Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

    PubMed Central

    Krumm, Stefanie A.; Ndungu, J. Maina; Yoon, Jeong-Joong; Dochow, Melanie; Sun, Aiming; Natchus, Michael; Snyder, James P.; Plemper, Richard K.

    2011-01-01

    Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen

  8. Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions.

    PubMed

    Westermann, Alexander J; Förstner, Konrad U; Amman, Fabian; Barquist, Lars; Chao, Yanjie; Schulte, Leon N; Müller, Lydia; Reinhardt, Richard; Stadler, Peter F; Vogel, Jörg

    2016-01-28

    Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes. PMID:26789254

  9. Circulating miRNA panel for prediction of acute graft-versus-host disease in lymphoma patients undergoing matched unrelated hematopoietic stem cell transplantation.

    PubMed

    Gimondi, Silvia; Dugo, Matteo; Vendramin, Antonio; Bermema, Anisa; Biancon, Giulia; Cavané, Alessandra; Corradini, Paolo; Carniti, Cristiana

    2016-07-01

    Acute graft-versus-host disease (aGVHD) results in significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Noninvasive diagnostic and prognostic tests for aGVHD are currently lacking, but would be beneficial in predicting aGVHD and improving the safety of allo-HSCT. Circulating microRNAs exhibit marked stability and may serve as biomarkers in several clinical settings. Here, we evaluated the use of circulating microRNAs as predictive biomarkers of aGVHD in lymphoma patients after allo-HSCT from matched unrelated donors (MUDs). After receiving informed consent, we prospectively collected plasma samples from 24 lymphoma patients before and after unmanipulated MUD allo-HSCT; microRNAs were then isolated. Fourteen patients developed aGVHD symptoms at a median of 48 days (range: 32-90) post-transplantation. Two patients developed intestinal GVHD, eight cutaneous GVHD, and four multiorgan GVHD. The microRNA expression profile was examined using quantitative real-time polymerase chain reaction (qRT-PCR). MicroRNAs 194 and 518f were significantly upregulated in aGVHD samples compared with samples taken from non-aGVHD patients. Remarkably, these upregulated microRNAs could be detected before the onset of aGVHD. Pathway prediction analysis indicated that these microRNAs may regulate critical pathways involved in aGVHD pathogenesis. Considering the noninvasive characteristics of plasma sampling and the feasibility of detecting miRNAs after allo-HSCT using real-time polymerase chain reaction, our results indicate that circulating microRNAs have the potential to enable an earlier aGVHD diagnosis and might assist in individualizing therapeutic strategies after MUD allo-HSCT. Nevertheless, standardization of blood sampling and analysis protocols is mandatory for the introduction of miRNA profiling into routine clinical use. PMID:27013207

  10. Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein

    PubMed Central

    Larkins-Ford, Jonah; McCormick, Craig; Gaglia, Marta M.

    2016-01-01

    Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5’->3’-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3’ end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus. PMID:26849127

  11. Host RNA Packaging by Retroviruses: A Newly Synthesized Story

    PubMed Central

    Eckwahl, Matthew J.; Telesnitsky, Alice

    2016-01-01

    ABSTRACT A fascinating aspect of retroviruses is their tendency to nonrandomly incorporate host cell RNAs into virions. In addition to the specific tRNAs that prime reverse transcription, all examined retroviruses selectively package multiple host cell noncoding RNAs (ncRNAs). Many of these ncRNAs appear to be encapsidated shortly after synthesis, before assembling with their normal protein partners. Remarkably, although some packaged ncRNAs, such as pre-tRNAs and the spliceosomal U6 small nuclear RNA (snRNA), were believed to reside exclusively within mammalian nuclei, it was demonstrated recently that the model retrovirus murine leukemia virus (MLV) packages these ncRNAs from a novel pathway in which unneeded nascent ncRNAs are exported to the cytoplasm for degradation. The finding that retroviruses package forms of ncRNAs that are rare in cells suggests several hypotheses for how these RNAs could assist retrovirus assembly and infectivity. Moreover, recent experiments in several laboratories have identified additional ways in which cellular ncRNAs may contribute to the retrovirus life cycle. This review focuses on the ncRNAs that are packaged by retroviruses and the ways in which both encapsidated ncRNAs and other cellular ncRNAs may contribute to retrovirus replication. PMID:26861021

  12. Down-regulation of a host microRNA by a viral noncoding RNA.

    PubMed

    Cazalla, D; Steitz, J A

    2010-01-01

    Primate herpesviruses express more noncoding RNAs (ncRNAs) than any other class of mammalian viruses during either latency or the lytic phase of the viral life cycle. T cells transformed by the monkey virus Herpesvirus saimiri (HVS) express seven viral U-rich ncRNAs called HSURs. Conserved sequences in HSURs1 and 2 exhibit complementarity to three host-cell microRNAs (miRNAs). The predicted interactions of HSURs1 and 2 with these miRNAs were confirmed by coimmuno-precipitation experiments performed on extracts of marmoset T cells transformed by a wild-type or a mutant HVS lacking these two HSURs. Mutational analyses demonstrated that the binding of miR-27 to HSUR1 and that of miR-16 to HSUR2 involves base pairing. One of these miRNAs, miR-27, is dramatically lowered in abundance in HVS-transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR1 demonstrated that degradation of mature miR-27 occurs in a sequence-specific and binding-dependent manner but does not occur by AU-rich element (ARE)-mediated decay, which controls the intracellular level of HSUR1 itself. This viral strategy exemplifies the use of an ncRNA to control host-cell gene expression via the miRNA pathway and has potential applications both experimentally and therapeutically. PMID:21139068

  13. Diverse roles of host RNA binding proteins in RNA virus replication.

    PubMed

    Li, Zhenghe; Nagy, Peter D

    2011-01-01

    Plus-strand +RNA viruses co-opt host RNA-binding proteins (RBPs) to perform many functions during viral replication. A few host RBPs have been identified that affect the recruitment of viral +RNAs for replication. Other subverted host RBPs help the assembly of the membrane-bound replicase complexes, regulate the activity of the replicase and control minus- or plus-strand RNA synthesis. The host RBPs also affect the stability of viral RNAs, which have to escape cellular RNA degradation pathways. While many host RBPs seem to have specialized functions, others participate in multiple events during infection. Several conserved RBPs, such as eEF1A, hnRNP proteins and Lsm 1-7 complex, are co-opted by evolutionarily diverse +RNA viruses, underscoring some common themes in virus-host interactions. On the other hand, viruses also hijack unique RBPs, suggesting that +RNA viruses could utilize different RBPs to perform similar functions. Moreover, different +RNA viruses have adapted unique strategies for co-opting unique RBPs. Altogether, a deeper understanding of the functions of the host RBPs subverted for viral replication will help development of novel antiviral strategies and give new insights into host RNA biology. PMID:21505273

  14. The Virus-Host Interplay: Biogenesis of +RNA Replication Complexes

    PubMed Central

    Reid, Colleen R.; Airo, Adriana M.; Hobman, Tom C.

    2015-01-01

    Positive-strand RNA (+RNA) viruses are an important group of human and animal pathogens that have significant global health and economic impacts. Notable members include West Nile virus, Dengue virus, Chikungunya, Severe acute respiratory syndrome (SARS) Coronavirus and enteroviruses of the Picornaviridae family.Unfortunately, prophylactic and therapeutic treatments against these pathogens are limited. +RNA viruses have limited coding capacity and thus rely extensively on host factors for successful infection and propagation. A common feature among these viruses is their ability to dramatically modify cellular membranes to serve as platforms for genome replication and assembly of new virions. These viral replication complexes (VRCs) serve two main functions: To increase replication efficiency by concentrating critical factors and to protect the viral genome from host anti-viral systems. This review summarizes current knowledge of critical host factors recruited to or demonstrated to be involved in the biogenesis and stabilization of +RNA virus VRCs. PMID:26287230

  15. Identification of linc-NeD125, a novel long non coding RNA that hosts miR-125b-1 and negatively controls proliferation of human neuroblastoma cells.

    PubMed

    Bevilacqua, Valeria; Gioia, Ubaldo; Di Carlo, Valerio; Tortorelli, Anna F; Colombo, Teresa; Bozzoni, Irene; Laneve, Pietro; Caffarelli, Elisa

    2015-01-01

    The human genome contains some thousands of long non coding RNAs (lncRNAs). Many of these transcripts are presently considered crucial regulators of gene expression and functionally implicated in developmental processes in Eukaryotes. Notably, despite a huge number of lncRNAs are expressed in the Central Nervous System (CNS), only a few of them have been characterized in terms of molecular structure, gene expression regulation and function. In the present study, we identify linc-NeD125 as a novel cytoplasmic, neuronal-induced long intergenic non coding RNA (lincRNA). Linc-NeD125 represents the host gene for miR-125b-1, a microRNA with an established role as negative regulator of human neuroblastoma cell proliferation. Here, we demonstrate that these two overlapping non coding RNAs are coordinately induced during in vitro neuronal differentiation, and that their expression is regulated by different mechanisms. While the production of miR-125b-1 relies on transcriptional regulation, linc-NeD125 is controlled at the post-transcriptional level, through modulation of its stability. We also demonstrate that linc-NeD125 functions independently of the hosted microRNA, by reducing cell proliferation and activating the antiapoptotic factor BCL-2. PMID:26480000

  16. Host cells and cell banking.

    PubMed

    Stacey, Glyn N; Merten, Otto-Wilhelm

    2011-01-01

    Gene therapy based on the use of viral vectors is entirely dependent on the use of animal cell lines, mainly of mammalian origin, but also of insect origin. As for any biotechnology product for clinical use, viral -vectors have to be produced with cells derived from an extensively characterized cell bank to maintain the appropriate standard for assuring the lowest risk for the patients to be treated. Although many different cell types and lines have been used for the production of viral vectors, HEK293 cells or their derivatives have been extensively used for production of different vector types: adenovirus, oncorectrovirus, lentivirus, and AAV vectors, because of their easy handling and the possibility to grow them adherently in serum-containing medium as well as in suspension in serum-free culture medium. Despite this, these cells are not necessarily the best for the production of a given viral vector, and there are many other cell lines with significant advantages including superior growth and/or production characteristics, which have been tested and also used for the production of clinical vector batches. This chapter presents basic -considerations concerning the characterization of cell banks, in the first part, and, in the second part, practically all cell lines (at least when public information was available) established and developed for the production of the most important viral vectors (adenoviral, oncoretroviral, lentiviral, AAV, baculovirus). PMID:21590393

  17. Host Cell Factors Involved in the Life Cycle of FMDV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-Mouth Disease Virus (FMDV), like other RNA viruses, recruits various host cell factors to assist in translation and replication of the virus genome. While FMDV translation has been extensively investigated, much remains unknown regarding replication of the positive-sense RNA genome. In thi...

  18. siRNA Screening Identifies the Host Hexokinase 2 (HK2) Gene as an Important Hypoxia-Inducible Transcription Factor 1 (HIF-1) Target Gene in Toxoplasma gondii-Infected Cells

    PubMed Central

    Menendez, Matthew T.; Teygong, Crystal; Wade, Kristin; Florimond, Celia

    2015-01-01

    ABSTRACT Although it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth. Toxoplasma increases host HK2 transcript and protein levels in a HIF-1-dependent manner. In addition, parasite growth at 3% oxygen is restored in HIF-1-deficient cells transfected with HK2 expression plasmids. Both HIF-1 activation and HK2 expression were accompanied by increases in host glycolytic flux, suggesting that enhanced HK2 expression in parasite-infected cells is functionally significant. Parasite dependence on host HK2 and HIF-1 expression is not restricted to transformed cell lines, as both are required for parasite growth in nontransformed C2C12 myoblasts and HK2 is upregulated in vivo following infection. While HK2 is normally associated with the cytoplasmic face of the outer mitochondrial membrane at physiological O2 levels, HK2 relocalizes to the host cytoplasm following infection, a process that is required for parasite growth at 3% oxygen. Taken together, our findings show that HIF-1-dependent expression and relocalization of HK2 represent a novel mechanism by which Toxoplasma establishes its replicative niche at tissue oxygen tensions. PMID:26106078

  19. Viral Evasion and Manipulation of Host RNA Quality Control Pathways.

    PubMed

    Hogg, J Robert

    2016-08-15

    Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. PMID:27226372

  20. Repression of host RNA polymerase II transcription by herpes simplex virus type 1.

    PubMed Central

    Spencer, C A; Dahmus, M E; Rice, S A

    1997-01-01

    Lytic infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in rapid repression of host gene expression and selective activation of the viral genome. This transformation in gene expression is thought to involve repression of host transcription and diversion of the host RNA polymerase (RNAP II) transcription machinery to the viral genome. However, the extent of virus-induced host transcription repression and the mechanisms responsible for these major shifts in transcription specificities have not been examined. To determine how HSV-1 accomplishes repression of host RNAP II transcription, we assayed transcription patterns on several cellular genes in cells infected with mutant and wild-type HSV-1. Our results suggest that HSV-1 represses RNAP II transcription on most cellular genes. However, each cellular gene we examined responds differently to the transcription repressive effects of virus infection, both quantitatively and with respect to the involvement of viral gene products. Virus-induced shutoff of host RNAP II transcription requires expression of multiple immediate-early genes. In contrast, expression of delayed-early and late genes and viral DNA replication appear to contribute little to repression of host cell RNAP II transcription. Modification of RNAP II to the intermediately phosphorylated (II(I)) form appears unlinked to virus-induced repression of host cell transcription. However, full repression of host transcription is correlated with depletion of the hyperphosphorylated (IIO) form of RNAP II. PMID:9032335

  1. VIGS, HIGS and FIGS: small RNA silencing in the interactions of viruses or filamentous organisms with their plant hosts.

    PubMed

    Baulcombe, David C

    2015-08-01

    Recent evidence indicates two-way traffic of silencing RNA between filamentous organisms and their plant hosts. There are also indications that suppressors of RNA silencing are transferred from filamentous organisms into host plant cells where they influence the innate immune system. Here I use virus disease as a template for interpretation of RNA silencing in connection with filamentous organisms and infected plant cells. I propose that host plant interactions of these organisms are influenced by RNA silencing networks in which there are: small interfering RNAs from the host that are transported into the filamentous organism and vice versa; silencing suppressors from the organism that are transported into the host; endogenous small interfering RNAs and micro RNAs that target components of the innate immune system or endogenous suppressors of the innate immune system. PMID:26247121

  2. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways.

    PubMed

    Weiberg, Arne; Wang, Ming; Lin, Feng-Mao; Zhao, Hongwei; Zhang, Zhihong; Kaloshian, Isgouhi; Huang, Hsien-Da; Jin, Hailing

    2013-10-01

    Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers "virulent" sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism. PMID:24092744

  3. A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

    PubMed Central

    Koeppen, Katja; Hampton, Thomas H.; Jarek, Michael; Scharfe, Maren; Gerber, Scott A.; Mielcarz, Daniel W.; Demers, Elora G.; Dolben, Emily L.; Hammond, John H.; Hogan, Deborah A.; Stanton, Bruce A.

    2016-01-01

    Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response. PMID:27295279

  4. MicroRNA-17-92 controls T-cell responses in graft-versus-host disease and leukemia relapse in mice.

    PubMed

    Wu, Yongxia; Heinrichs, Jessica; Bastian, David; Fu, Jianing; Nguyen, Hung; Schutt, Steven; Liu, Yuejun; Jin, Junfei; Liu, Chen; Li, Qi-Jing; Xia, Changqing; Yu, Xue-Zhong

    2015-09-10

    MicroRNAs (miRs) play important roles in orchestrating many aspects of the immune response. The miR-17-92 cluster, which encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1, and 92-1, is among the best characterized of these miRs. The miR-17-92 cluster has been shown to regulate a variety of immune responses including infection, tumor, and autoimmunity, but the role of this cluster in T-cell response to alloantigens has not been previously explored. By using major histocompatibility complex (MHC)-matched, -mismatched, and haploidentical murine models of allogeneic bone marrow transplantation (allo-BMT), we demonstrate that the expression of miR-17-92 on donor T cells is essential for the induction of graft-versus-host disease (GVHD), but dispensable for the graft-versus-leukemia (GVL) effect. The miR-17-92 plays a major role in promoting CD4 T-cell activation, proliferation, survival, and Th1 differentiation, while inhibiting Th2 and iTreg differentiation. Alternatively, miR-17-92 may promote migration of CD8 T cells to GVHD target organs, but has minimal impact on CD8 T-cell proliferation, survival, or cytolytic function, which could contribute to the preserved GVL effect mediated by T cells deficient for miR-17-92. Furthermore, we evaluated a translational approach and found that systemic administration of antagomir to block miR-17 or miR-19b in this cluster significantly inhibited alloreactive T-cell expansion and interferon-γ (IFNγ) production, and prolonged the survival in recipients afflicted with GVHD while preserving the GVL effect. Taken together, the current work provides a strong rationale and demonstrates the feasibility to target miR-17-92 for the control of GVHD while preserving GVL activity after allo-BMT. PMID:26138686

  5. Host miRNA degradation by Herpesvirus saimiri small nuclear RNA requires an unstructured interacting region.

    PubMed

    Pawlica, Paulina; Moss, Walter N; Steitz, Joan A

    2016-08-01

    Herpesvirus saimiri, an oncogenic herpesvirus, during latency produces seven small nuclear RNAs, called the Herpesvirus saimiri U RNAs (HSUR1-7). HSUR1 mediates degradation of the host microRNA, miR-27, via a process that requires imperfect base-pairing. The decreased levels of miR-27 lead to prolonged T-cell activation and likely contribute to oncogenesis. To gain insight into HSUR1-mediated degradation of miR-27, we probed the in vivo secondary structure of HSUR1 and coupled this with bioinformatic structural analyses. The results suggest that HSUR1 adopts a conformation different than previously believed and that the region complementary to miR-27 lacks stable structure. To determine whether HSUR1 structural flexibility is important for its ability to mediate miR-27 degradation, we performed structurally informative mutagenic analyses of HSUR1. HSUR1 mutants in which the miR-27 binding site sequence is preserved, but sequestered in predicted helices, lose their ability to decrease miR-27 levels. These results indicate that the HSUR1 miR27-binding region must be available in a conformationally flexible segment for noncoding RNA function. PMID:27335146

  6. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens

    PubMed Central

    Meliopoulos, Victoria A.; Andersen, Lauren E.; Birrer, Katherine F.; Simpson, Kaylene J.; Lowenthal, John W.; Bean, Andrew G. D.; Stambas, John; Stewart, Cameron R.; Tompkins, S. Mark; van Beusechem, Victor W.; Fraser, Iain; Mhlanga, Musa; Barichievy, Samantha; Smith, Queta; Leake, Devin; Karpilow, Jon; Buck, Amy; Jona, Ghil; Tripp, Ralph A.

    2012-01-01

    Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.—Meliopoulos, V. A., Andersen, L. E., Birrer, K. F., Simpson, K. J., Lowenthal, J. W., Bean, A. G. D., Stambas, J., Stewart, C. R., Tompkins, S. M., van Beusechem, V. W., Fraser, I., Mhlanga, M., Barichievy, S., Smith, Q., Leake, D., Karpilow, J., Buck, A., Jona, G., Tripp, R. A. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens. PMID:22247330

  7. Virus-host interactions: new insights from the small RNA world

    PubMed Central

    Browne, Edward P; Li, Junjie; Chong, Mark; Littman, Dan R

    2005-01-01

    RNA silencing has a known role in the antiviral responses of plants and insects. Recent evidence, including the finding that the Tat protein of human immunodeficiency virus (HIV) can suppress the host's RNA-silencing pathway and may thus counteract host antiviral RNAs, suggests that RNA-silencing pathways could also have key roles in mammalian virus-host interactions. PMID:16277756

  8. Identification of RNA Helicase A (RHA) as a New Host Factor in the Replication Cycle of FMDV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV) as with other RNA viruses, recruits various host cell factors to assist in the translation and replication of the virus genome. In this study we investigated the role of RNA helicase A (RHA) in the life cycle of FMDV. Immunofluorescent microscopy combined with b...

  9. Host-virus interaction: the antiviral defense function of small interfering RNAs can be enhanced by host microRNA-7 in vitro

    PubMed Central

    Zhang, Xiaoying; Liu, Dongyun; Zhang, Sheng; Wei, Xiujuan; Song, Jie; Zhang, Yupei; Jin, Min; Shen, Zhiqiang; Wang, Xinwei; Feng, Zhichun; Li, Junwen

    2015-01-01

    Small interfering RNAs (siRNAs) directed against poliovirus (PV) and other viruses effectively inhibit viral replication and have been developed as antiviral agents. Here, we demonstrate that a specific siRNA targeting the region between nucleotides 100–125 (siRNA-100) from the 5′-untranslated region (5′-UTR) of PV plays a critical role in inhibiting PV replication. Our data demonstrate that siRNA-100 treatment can greatly reduce PV titers, resulting in up-regulation of host microRNA-7 (miR-7), which in turn, leads to enhance inhibition of PV infection further. Moreover, our results suggest that siRNA-100 can also impair the spread of PV to uninfected cells by increasing host resistance to PV, resulting in decreasing necrosis and cytopathic effects (CPE) levels, as well as prolonging the survival of infected cells. Indeed, the active antiviral effect of siRNA-100 was potentially supplemented by the activity of miR-7, and both of them can serve as stabilizing factors for maintenance of cellular homeostasis. Results of this study identify a molecular mechanism of RNAi for antiviral defense, and extend our knowledge of the complex interplay between host and PV, which will provide a basis for the development of effective RNAi-based therapies designed to inhibit PV replication and protect host cells. PMID:26067353

  10. Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7

    PubMed Central

    Takata, Akemi; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yoshikawa, Takeshi; Koike, Kazuhiko

    2016-01-01

    The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection. PMID:26979389

  11. Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

    PubMed

    Takata, Akemi; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yoshikawa, Takeshi; Koike, Kazuhiko

    2016-01-01

    The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection. PMID:26979389

  12. Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells.

    PubMed

    Schnettler, Esther; Sterken, Mark G; Leung, Jason Y; Metz, Stefan W; Geertsema, Corinne; Goldbach, Rob W; Vlak, Just M; Kohl, Alain; Khromykh, Alexander A; Pijlman, Gorben P

    2012-12-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  13. Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

    PubMed Central

    Schnettler, Esther; Sterken, Mark G.; Leung, Jason Y.; Metz, Stefan W.; Geertsema, Corinne; Goldbach, Rob W.; Vlak, Just M.; Kohl, Alain

    2012-01-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  14. A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes.

    PubMed

    Avraham, Roi; Haseley, Nathan; Fan, Amy; Bloom-Ackermann, Zohar; Livny, Jonathan; Hung, Deborah T

    2016-08-01

    The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host-pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host-pathogen interactions in infection models. PMID:27442864

  15. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites.

    PubMed

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A; Li, Jianhui; Randall, Glenn; Belov, George A; Wang, Xiaofeng

    2016-02-23

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

  16. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

    PubMed Central

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A.; Li, Jianhui; Randall, Glenn; Belov, George A.; Wang, Xiaofeng

    2016-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

  17. Dual Analysis of the Murine Cytomegalovirus and Host Cell Transcriptomes Reveal New Aspects of the Virus-Host Cell Interface

    PubMed Central

    Juranic Lisnic, Vanda; Babic Cac, Marina; Lisnic, Berislav; Trsan, Tihana; Mefferd, Adam; Das Mukhopadhyay, Chitrangada; Cook, Charles H.; Jonjic, Stipan; Trgovcich, Joanne

    2013-01-01

    Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV) and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq). We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases. PMID:24086132

  18. Penetration of Bdellovibrio bacteriovorus into Host Cells

    PubMed Central

    Abram, Dinah; e Melo, J. Castro; Chou, D.

    1974-01-01

    Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite. Images PMID:4208138

  19. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

    PubMed Central

    Phillips, Stacia L.; Soderblom, Erik J.

    2016-01-01

    ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. PMID:26733069

  20. Differential regulation of host mRNA translation during obligate pathogen-plant interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus infection reprograms the plant messenger RNA (mRNA) transcriptome by activating or interfering with a variety of signaling pathways, but the effects on host mRNA translation have not been explored on a genome-wide scale. To address this issue, Arabidopsis thaliana mRNA transcripts were quantif...

  1. A Host Small GTP-binding Protein ARL8 Plays Crucial Roles in Tobamovirus RNA Replication

    PubMed Central

    Nishikiori, Masaki; Mori, Masashi; Dohi, Koji; Okamura, Hideyasu; Katoh, Etsuko; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-01-01

    Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5′-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions. PMID:22174675

  2. A host small GTP-binding protein ARL8 plays crucial roles in tobamovirus RNA replication.

    PubMed

    Nishikiori, Masaki; Mori, Masashi; Dohi, Koji; Okamura, Hideyasu; Katoh, Etsuko; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-12-01

    Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5'-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions. PMID:22174675

  3. Extreme resistance as a host counter-counter defense against viral suppression of RNA silencing.

    PubMed

    Sansregret, Raphaël; Dufour, Vanessa; Langlois, Mathieu; Daayf, Fouad; Dunoyer, Patrice; Voinnet, Olivier; Bouarab, Kamal

    2013-01-01

    RNA silencing mediated by small RNAs (sRNAs) is a conserved regulatory process with key antiviral and antimicrobial roles in eukaryotes. A widespread counter-defensive strategy of viruses against RNA silencing is to deploy viral suppressors of RNA silencing (VSRs), epitomized by the P19 protein of tombusviruses, which sequesters sRNAs and compromises their downstream action. Here, we provide evidence that specific Nicotiana species are able to sense and, in turn, antagonize the effects of P19 by activating a highly potent immune response that protects tissues against Tomato bushy stunt virus infection. This immunity is salicylate- and ethylene-dependent, and occurs without microscopic cell death, providing an example of "extreme resistance" (ER). We show that the capacity of P19 to bind sRNA, which is mandatory for its VSR function, is also necessary to induce ER, and that effects downstream of P19-sRNA complex formation are the likely determinants of the induced resistance. Accordingly, VSRs unrelated to P19 that also bind sRNA compromise the onset of P19-elicited defense, but do not alter a resistance phenotype conferred by a viral protein without VSR activity. These results show that plants have evolved specific responses against the damages incurred by VSRs to the cellular silencing machinery, a likely necessary step in the never-ending molecular arms race opposing pathogens to their hosts. PMID:23785291

  4. Diagnosis of Childhood Tuberculosis and Host RNA Expression in Africa

    PubMed Central

    Banwell, Claire M.; Chagaluka, George; Crampin, Amelia C.; Dockrell, Hazel M.; French, Neil; Hamilton, Melissa S.; Hibberd, Martin L.; Kern, Florian; Langford, Paul R.; Ling, Ling; Mlotha, Rachel; Ottenhoff, Tom H.M.; Pienaar, Sandy; Pillay, Vashini; Scott, J. Anthony G.; Twahir, Hemed; Wilkinson, Robert J.

    2014-01-01

    BACKGROUND Improved diagnostic tests for tuberculosis in children are needed. We hypothesized that transcriptional signatures of host blood could be used to distinguish tuberculosis from other diseases in African children who either were or were not infected with the human immunodeficiency virus (HIV). METHODS The study population comprised prospective cohorts of children who were undergoing evaluation for suspected tuberculosis in South Africa (655 children), Malawi (701 children), and Kenya (1599 children). Patients were assigned to groups according to whether the diagnosis was culture-confirmed tuberculosis, culture-negative tuberculosis, diseases other than tuberculosis, or latent tuberculosis infection. Diagnostic signatures distinguishing tuberculosis from other diseases and from latent tuberculosis infection were identified from genomewide analysis of RNA expression in host blood. RESULTS We identified a 51-transcript signature distinguishing tuberculosis from other diseases in the South African and Malawian children (the discovery cohort). In the Kenyan children (the validation cohort), a risk score based on the signature for tuberculosis and for diseases other than tuberculosis showed a sensitivity of 82.9% (95% confidence interval [CI], 68.6 to 94.3) and a specificity of 83.6% (95% CI, 74.6 to 92.7) for the diagnosis of culture-confirmed tuberculosis. Among patients with cultures negative for Mycobacterium tuberculosis who were treated for tuberculosis (those with highly probable, probable, or possible cases of tuberculosis), the estimated sensitivity was 62.5 to 82.3%, 42.1 to 80.8%, and 35.3 to 79.6%, respectively, for different estimates of actual tuberculosis in the groups. In comparison, the sensitivity of the Xpert MTB/RIF assay for molecular detection of M. tuberculosis DNA in cases of culture-confirmed tuberculosis was 54.3% (95% CI, 37.1 to 68.6), and the sensitivity in highly probable, probable, or possible cases was an estimated 25.0 to 35

  5. The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends

    PubMed Central

    Xie, Mingyi; Zhang, Wei; Shu, Mei-Di; Xu, Acer; Lenis, Diana A.; DiMaio, Daniel; Steitz, Joan A.

    2015-01-01

    Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3′ end processing signal (3′ box), generates the 5′ ends of HVS pre-miRNA hairpins. Here, we identify a novel 3′ box-like sequence (miRNA 3′ box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3′ end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3′ box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA–protein interactions inside cells. Surprisingly, in contrast to snRNA 3′ end processing, HVS pre-miRNA 3′ end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology. PMID:26220997

  6. Allogeneic T cell responses are regulated by a specific miRNA-mRNA network

    PubMed Central

    Sun, Yaping; Tawara, Isao; Zhao, Meng; Qin, Zhaohui S.; Toubai, Tomomi; Mathewson, Nathan; Tamaki, Hiroya; Nieves, Evelyn; Chinnaiyan, Arul M.; Reddy, Pavan

    2013-01-01

    Donor T cells that respond to host alloantigens following allogeneic bone marrow transplantation (BMT) induce graft-versus-host (GVH) responses, but their molecular landscape is not well understood. MicroRNAs (miRNAs) regulate gene (mRNA) expression and fine-tune the molecular responses of T cells. We stimulated naive T cells with either allogeneic or nonspecific stimuli and used argonaute cross-linked immunoprecipitation (CLIP) with subsequent ChIP microarray analyses to profile miR responses and their direct mRNA targets. We identified a unique expression pattern of miRs and mRNAs following the allostimulation of T cells and a high correlation between the expression of the identified miRs and a reduction of their mRNA targets. miRs and mRNAs that were predicted to be differentially regulated in allogeneic T cells compared with nonspecifically stimulated T cells were validated in vitro. These analyses identified wings apart-like homolog (Wapal) and synaptojanin 1 (Synj1) as potential regulators of allogeneic T cell responses. The expression of these molecular targets in vivo was confirmed in MHC-mismatched experimental BMT. Targeted silencing of either Wapal or Synj1 prevented the development of GVH response, confirming a role for these regulators in allogeneic T cell responses. Thus, this genome-wide analysis of miRNA-mRNA interactions identifies previously unrecognized molecular regulators of T cell responses. PMID:24216511

  7. Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection.

    PubMed

    Flores, R; Minoia, S; Carbonell, A; Gisel, A; Delgado, S; López-Carrasco, A; Navarro, B; Di Serio, F

    2015-11-01

    The discovery of viroids about 45 years ago heralded a revolution in Biology: small RNAs comprising around 350 nt were found to be able to replicate autonomously-and to incite diseases in certain plants-without encoding proteins, fundamental properties discriminating these infectious agents from viruses. The initial focus on the pathological effects usually accompanying infection by viroids soon shifted to their molecular features-they are circular molecules that fold upon themselves adopting compact secondary conformations-and then to how they manipulate their hosts to be propagated. Replication of viroids-in the nucleus or chloroplasts through a rolling-circle mechanism involving polymerization, cleavage and circularization of RNA strands-dealt three surprises: (i) certain RNA polymerases are redirected to accept RNA instead of their DNA templates, (ii) cleavage in chloroplastic viroids is not mediated by host enzymes but by hammerhead ribozymes, and (iii) circularization in nuclear viroids is catalyzed by a DNA ligase redirected to act upon RNA substrates. These enzymes (and ribozymes) are most probably assisted by host proteins, including transcription factors and RNA chaperones. Movement of viroids, first intracellularly and then to adjacent cells and distal plant parts, has turned out to be a tightly regulated process in which specific RNA structural motifs play a crucial role. More recently, the advent of RNA silencing has brought new views on how viroids may cause disease and on how their hosts react to contain the infection; additionally, viroid infection may be restricted by other mechanisms. Representing the lowest step on the biological size scale, viroids have also attracted considerable interest to get a tentative picture of the essential characteristics of the primitive replicons that populated the postulated RNA world. PMID:25738582

  8. Contrasting Lifestyles Within the Host Cell.

    PubMed

    Di Russo Case, Elizabeth; Samuel, James E

    2016-02-01

    Intracellular bacterial pathogens have evolved to exploit the protected niche provided within the boundaries of a eukaryotic host cell. Upon entering a host cell, some bacteria can evade the adaptive immune response of its host and replicate in a relatively nutrient-rich environment devoid of competition from other host flora. Growth within a host cell is not without their hazards, however. Many pathogens enter their hosts through receptor-mediated endocytosis or phagocytosis, two intracellular trafficking pathways that terminate in a highly degradative organelle, the phagolysosome. This usually deadly compartment is maintained at a low pH and contains degradative enzymes and reactive oxygen species, resulting in an environment to which few bacterial species are adapted. Some intracellular pathogens, such as Shigella, Listeria, Francisella, and Rickettsia, escape the phagosome to replicate within the cytosol of the host cell. Bacteria that remain within a vacuole either alter the trafficking of their initial phagosomal compartment or adapt to survive within the harsh environment it will soon become. In this chapter, we focus on the mechanisms by which different vacuolar pathogens either evade lysosomal fusion, as in the case of Mycobacterium and Chlamydia, or allow interaction with lysosomes to varying degrees, such as Brucella and Coxiella, and their specific adaptations to inhabit a replicative niche. PMID:26999394

  9. Contrasting Lifestyles Within the Host Cell

    PubMed Central

    Case, Elizabeth Di Russo; Samuel, James E.

    2015-01-01

    CHAPTER SUMMARY Intracellular bacterial pathogens have evolved to exploit the protected niche provided within the boundaries of a eukaryotic host cell. Upon entering a host cell, some bacteria can evade the adaptive immune response of its host, and replicate in a relatively nutrient-rich environment devoid of competition from other host flora. Growth within a host cell is not without its hazards, however. Many pathogens enter their hosts through receptor-mediated endocytosis or phagocytosis, two intracellular trafficking pathways that terminate in a highly degradative organelle, the phagolysosome. This usually deadly compartment is maintained at a low pH, and contains degradative enzymes, and reactive oxygen species resulting in an environment to which few bacterial species are adapted. Some intracellular pathogens, like Shigella, Listeria, Francisella, and Rickettsia escape the phagosome to replicate within the cytosol of the host cell. Bacteria that remain within a vacuole either alter the trafficking of their initial phagosomal compartment or adapt to survive within the harsh environment it will soon become. In this chapter, we focus on the mechanisms by which different vacuolar pathogens either evade lysosomal fusion, as in the case of Mycobacterium and Chlamydia, or allow interaction with lysosomes to varying degrees, such as Brucella and Coxiella, and their specific adaptations to inhabit a replicative niche. PMID:26999394

  10. Cooperative Treatment of Metastatic Breast Cancer Using Host-Guest Nanoplatform Coloaded with Docetaxel and siRNA.

    PubMed

    Wang, Dangge; Wang, Tingting; Xu, Zhiai; Yu, Haijun; Feng, Bing; Zhang, Junying; Guo, Chengyue; Yin, Qi; Zhang, Zhiwen; Li, Yaping

    2016-01-27

    Conventional chemotherapy shows moderate efficiency against metastatic cancer since it targets only part of the mechanisms regulating tumor growth and metastasis. Here, gold nanorod (GNR)-based host-guest nanoplatforms loaded with docetaxel (DTX) and small interfering RNA (siRNA)-p65 (referred to as DTX-loaded GNR (GDTX)/p65) for chemo-, RNA interference (RNAi), and photothermal ablation (PTA) cooperative treatment of metastatic breast cancer are reported. To prepare the nanoplatform, GNRs are first coated with cyclodextrin (CD)-grafted polyethylenimine (PEI) and then loaded with DTX and siRNA through host-guest interaction with CD and electrostatic interaction with PEI, respectively. Upon near-infrared laser irradiation, GNRs generate a significant hyperthermia effect to trigger siRNA and DTX release. DTX reduces tumor growth by inhibiting mitosis of cancer cells. Meanwhile, siRNA-p65 suppresses lung metastasis and proliferation of cancer cells by blocking the nuclear factor kappa B (NF-κB) pathway and downregulating the downstream genes matrix metalloproteinase-9 (MMP-9) and B cell lymphoma-2 (Bcl-2). It is demonstrated that GDTX/p65 in combination with laser irradiation significantly inhibits the growth and lung metastasis of 4T1 breast tumors. The antitumor results suggest promising potential of the host-guest nanoplatform for combinational treatment of metastatic cancer by using RNAi, chemotherapy, and PTA. PMID:26662850

  11. Cell-host, LINE and environment

    PubMed Central

    Del Re, Brunella; Giorgi, Gianfranco

    2013-01-01

    Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship. PMID:23734298

  12. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response

    PubMed Central

    Hempstead, Andrew D.; Isberg, Ralph R.

    2015-01-01

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR. PMID:26598709

  13. Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions.

    PubMed

    Boudreault, Simon; Martenon-Brodeur, Camille; Caron, Marie; Garant, Jean-Michel; Tremblay, Marie-Pier; Armero, Victoria E S; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Lemay, Guy; Bisaillon, Martin

    2016-01-01

    Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection. PMID:27598998

  14. Host Control of Insect Endogenous Retroviruses: Small RNA Silencing and Immune Response

    PubMed Central

    Fablet, Marie

    2014-01-01

    Endogenous retroviruses are relics of ancient infections from retroviruses that managed to integrate into the genome of germline cells and remained vertically transmitted from parent to progeny. Subsequent to the endogenization process, these sequences can move and multiply in the host genome, which can have deleterious consequences and disturb genomic stability. Natural selection favored the establishment of silencing pathways that protect host genomes from the activity of endogenous retroviruses. RNA silencing mechanisms are involved, which utilize piRNAs. The response to exogenous viral infections uses siRNAs, a class of small RNAs that are generated via a distinct biogenesis pathway from piRNAs. However, interplay between both pathways has been identified, and interactions with anti-bacterial and anti-fungal immune responses are also suspected. This review focuses on Diptera (Arthropods) and intends to compile pieces of evidence showing that the RNA silencing pathway of endogenous retrovirus regulation is not independent from immunity and the response to infections. This review will consider the mechanisms that allow the lasting coexistence of viral sequences and host genomes from an evolutionary perspective. PMID:25412365

  15. Viral suppressors of RNA-based viral immunity: Host targets

    PubMed Central

    Wu, Qingfa; Wang, Xianbing

    2010-01-01

    Discovery of diverse plant and animal viral proteins as suppressors of RNA silencing has provided strong support for an RNA-based viral immunity (RVI), which is now known to specifically destroy viral RNAs by RNA interference in fungi, plants and invertebrates. Here we review several recent studies that have revealed new mechanistic insights into plant and insect viral suppressors of RVI or suggested a role for RNA silencing suppression during mammalian viral infection. PMID:20638637

  16. Complexity of Host Micro-RNA Response to Cytomegalovirus Reactivation After Organ Transplantation.

    PubMed

    Egli, A; Lisboa, L F; O'Shea, D; Asberg, A; Mueller, T; Emery, V; Kumar, D; Humar, A

    2016-02-01

    Human (Homo sapiens) micro-RNAs (hsa-miRNAs) regulate virus and host-gene translation, but the biological impact in patients with human cytomegalovirus (hCMV) infection is not well defined in a clinically relevant model. First, we compared hsa-miRNA expression profiles in peripheral blood mononuclear cells from 35 transplant recipients with and without CMV viremia by using a microarray chip covering 847 hsa-miRNAs. This approach demonstrated a set of 142 differentially expressed hsa-miRNAs. Next, we examined the effect of each of these miRNAs on viral growth by using human fibroblasts (human foreskin fibroblast-1) infected with the hCMV Towne strain, identifying a subset of proviral and antiviral hsa-miRNAs. miRNA-target prediction software indicated potential binding sites within the hCMV genome (e.g., hCMV-UL52 and -UL100 [UL = unique long]) and host-genes (e.g., interleukin-1 receptor, IRF1). Luciferase-expressing plasmid constructs and immunoblotting confirmed several predicted miRNA targets. Finally, we determined the expression of selected proviral and antiviral hsa-miRNAs in 242 transplant recipients with hCMV-viremia. We measured hsa-miRNAs before and after antiviral therapy and correlated hsa-miRNA expression levels to hCMV-replication dynamics. One of six antiviral hsa-miRNAs showed a significant increase during treatment, concurrent with viral decline. In contrast, six of eight proviral hsa-miRNAs showed a decrease during viral decline. Our results indicate that a complex and multitargeted hsa-miRNA response occurs during CMV replication in immunosuppressed patients. This study provides mechanistic insight and potential novel biomarkers for CMV replication. PMID:26460801

  17. Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems.

    PubMed

    Parameswaran, Poornima; Sklan, Ella; Wilkins, Courtney; Burgon, Trever; Samuel, Melanie A; Lu, Rui; Ansel, K Mark; Heissmeyer, Vigo; Einav, Shirit; Jackson, William; Doukas, Tammy; Paranjape, Suman; Polacek, Charlotta; dos Santos, Flavia Barreto; Jalili, Roxana; Babrzadeh, Farbod; Gharizadeh, Baback; Grimm, Dirk; Kay, Mark; Koike, Satoshi; Sarnow, Peter; Ronaghi, Mostafa; Ding, Shou-Wei; Harris, Eva; Chow, Marie; Diamond, Michael S; Kirkegaard, Karla; Glenn, Jeffrey S; Fire, Andrew Z

    2010-02-01

    We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts. PMID:20169186

  18. Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

    PubMed Central

    Parameswaran, Poornima; Sklan, Ella; Wilkins, Courtney; Burgon, Trever; Samuel, Melanie A.; Lu, Rui; Ansel, K. Mark; Heissmeyer, Vigo; Einav, Shirit; Jackson, William; Doukas, Tammy; Paranjape, Suman; Polacek, Charlotta; dos Santos, Flavia Barreto; Jalili, Roxana; Babrzadeh, Farbod; Gharizadeh, Baback; Grimm, Dirk; Kay, Mark; Koike, Satoshi; Sarnow, Peter; Ronaghi, Mostafa; Ding, Shou-Wei; Harris, Eva; Chow, Marie; Diamond, Michael S.; Kirkegaard, Karla; Glenn, Jeffrey S.; Fire, Andrew Z.

    2010-01-01

    We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts. PMID:20169186

  19. A short treatment of cells with the lanthanide ions La3+, Ce3+, Pr3+ or Nd3+ changes the cellular chemistry into a state in which RNA replication of flaviviruses is specifically blocked without interference with host-cell multiplication

    PubMed Central

    Wengler, Gerd; Wengler, Gisela; Koschinski, Andreas

    2007-01-01

    Alpha- and flaviviruses contain class II fusion proteins, which form ion-permeable pores in the target membrane during virus entry. The pores generated during entry of the alphavirus Semliki Forest virus have been shown previously to be blocked by lanthanide ions. Here, analyses of the influence of rare earth ions on the entry of the flaviviruses West Nile virus and Uganda S virus revealed an unexpected effect of lanthanide ions. The results showed that a 30 s treatment of cells with an appropriate lanthanide ion changed the cellular chemistry into a state in which the cells no longer supported the multiplication of flaviviruses. This change occurred in cells treated before, during or after infection, did not inhibit multiplication of Semliki Forest virus and did not interfere with host-cell multiplication. The change was generated in vertebrate and insect cells, and was elicited in the presence of actinomycin D. In vertebrate cells, the change was elicited specifically by La3+, Ce3+, Pr3+ and Nd3+. In insect cells, additional lanthanide ions had this activity. Further analyses showed that lanthanide ion treatment blocked the ability of the host cell to support the replication of flavivirus RNA. These results open two areas of research: the study of molecular alterations induced by lanthanide ion treatment in uninfected cells and the analysis of the resulting modifications of the flavivirus RNA replicase complex. The findings possibly open the way for the development of a general chemotherapy against flavivirus diseases such as Dengue fever, Japanese encephalitis, West Nile fever and yellow fever. PMID:17947525

  20. Noncoding RNAs of Plant Viruses and Viroids: Sponges of Host Translation and RNA Interference Machinery.

    PubMed

    Miller, W Allen; Shen, Ruizhong; Staplin, William; Kanodia, Pulkit

    2016-03-01

    Noncoding sequences in plant viral genomes are well-known to control viral replication and gene expression in cis. However, plant viral and viroid noncoding (nc)RNA sequences can also regulate gene expression acting in trans, often acting like 'sponges' that bind and sequester host cellular machinery to favor viral infection. Noncoding sequences of small subgenomic (sg)RNAs of Barley yellow dwarf virus (BYDV) and Red clover necrotic mosaic virus (RCNMV) contain a cap-independent translation element that binds translation initiation factor eIF4G. We provide new evidence that a sgRNA of BYDV can globally attenuate host translation, probably by sponging eIF4G. Subgenomic ncRNA of RCNMV is generated via 5' to 3' degradation by a host exonuclease. The similar noncoding subgenomic flavivirus (sf)RNA, inhibits the innate immune response, enhancing viral pathogenesis. Cauliflower mosaic virus transcribes massive amounts of a 600-nt ncRNA, which is processed into small RNAs that overwhelm the host's RNA interference (RNAi) system. Viroids use the host RNAi machinery to generate viroid-derived ncRNAs that inhibit expression of host defense genes by mimicking a microRNA. More examples of plant viral and viroid ncRNAs are likely to be discovered, revealing fascinating new weaponry in the host-virus arms race. PMID:26900786

  1. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis

    PubMed Central

    Millet, Jean Kaoru; Whittaker, Gary R.

    2015-01-01

    Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity. PMID:25445340

  2. RNA-Free and Ribonucleoprotein-Associated Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal Domain of Host RNA Polymerase II

    PubMed Central

    Martínez-Alonso, Mónica; Hengrung, Narin

    2016-01-01

    ABSTRACT Influenza viruses subvert the transcriptional machinery of their hosts to synthesize their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap snatching, the virus steals short 5′ capped RNA fragments from host capped RNAs and uses them to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that the influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5-phosphorylated form of the Pol II CTD, both in the presence and in the absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation. IMPORTANCE Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health, as well as represent a large economic burden to health care systems globally. Existing vaccines are not always effective, as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed, it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for

  3. Construction of a host-independent T7 expression system with small RNA regulation.

    PubMed

    Wang, Gang; Li, Qiang; Xu, Dikai; Cui, Mingxin; Sun, Xiao; Xu, Yanyan; Wang, Wenya

    2014-11-10

    It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572. PMID:25193711

  4. Interaction of Mycobacterium tuberculosis with Host Cell Death Pathways

    PubMed Central

    Srinivasan, Lalitha; Ahlbrand, Sarah; Briken, Volker

    2014-01-01

    Mycobacterium tuberculosis (Mtb) has coevolved with humans for tens of thousands of years. It is thus highly adapted to its human host and has evolved multiple mechanisms to manipulate host immune responses to its advantage. One central host pathogen interaction modality is host cell death pathways. Host cell apoptosis is associated with a protective response to Mtb infection, whereas a necrotic response favors the pathogen. Consistently, Mtb inhibits host cell apoptosis signaling but promotes induction of programmed necrosis. The molecular mechanisms involved in Mtb-mediated host cell death manipulation, the consequences for host immunity, and the potential for therapeutic and preventive approaches will be discussed. PMID:24968864

  5. Picornavirus IRES elements: RNA structure and host protein interactions.

    PubMed

    Martínez-Salas, Encarnación; Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Lozano, Gloria; Diaz-Toledano, Rosa

    2015-08-01

    Internal ribosome entry site (IRES) elements were discovered in picornaviruses. These elements are cis-acting RNA sequences that adopt diverse three-dimensional structures and recruit the translation machinery using a 5' end-independent mechanism assisted by a subset of translation initiation factors and various RNA binding proteins termed IRES transacting factors (ITAFs). Many of these factors suffer important modifications during infection including cleavage by picornavirus proteases, changes in the phosphorylation level and/or redistribution of the protein from the nuclear to the cytoplasm compartment. Picornavirus IRES are amongst the most potent elements described so far. However, given their large diversity and complexity, the mechanistic basis of its mode of action is not yet fully understood. This review is focused to describe recent advances on the studies of RNA structure and RNA-protein interactions modulating picornavirus IRES activity. PMID:25617758

  6. HIV-Induced Epigenetic Alterations in Host Cells.

    PubMed

    Abdel-Hameed, Enass A; Ji, Hong; Shata, Mohamed Tarek

    2016-01-01

    Human immunodeficiency virus (HIV), a member of the Retroviridae family, is a positive-sense, enveloped RNA virus. HIV, the causative agent of acquired immunodeficiency syndrome (AIDS) has two major types, HIV-1 and HIV-2 In HIV-infected cells the single stranded viral RNA genome is reverse transcribed and the double-stranded viral DNA integrates into the cellular DNA, forming a provirus. The proviral HIV genome is controlled by the host epigenetic regulatory machinery. Cellular epigenetic regulators control HIV latency and reactivation by affecting the chromatin state in the vicinity of the viral promoter located to the 5' long terminal repeat (LTR) sequence. In turn, distinct HIV proteins affect the epigenotype and gene expression pattern of the host cells. HIV-1 infection of CD4(+) T cells in vitro upregulated DNMT activity and induced hypermethylation of distinct cellular promoters. In contrast, in the colon mucosa and peripheral blood mononuclear cells from HIV-infected patients demethylation of the FOXP3 promoter was observed, possibly due to the downregulation of DNA methyltransferase 1. For a curative therapy of HIV infected individuals and AIDS patients, a combination of antiretroviral drugs with epigenetic modifying compounds have been suggested for the reactivation of latent HIV-1 genomes. These epigenetic drugs include histone deacetylase inhibitors (HDACI), histone methyltransferase inhibitors (HMTI), histone demethylase inhibitors, and DNA methyltransferase inhibitors (DNMTI). PMID:26659262

  7. Movement of regulatory RNA between animal cells

    PubMed Central

    Jose, Antony M.

    2015-01-01

    Summary Recent studies suggest that RNA can move from one cell to another and regulate genes through specific base-pairing. Mechanisms that modify or select RNA for secretion from a cell are unclear. Secreted RNA can be stable enough to be detected in the extracellular environment and can enter the cytosol of distant cells to regulate genes. Mechanisms that import RNA into the cytosol of an animal cell can enable uptake of RNA from many sources including other organisms. This role of RNA is akin to that of steroid hormones, which cross cell membranes to regulate genes. The potential diagnostic use of RNA in human extracellular fluids has ignited interest in understanding mechanisms that enable the movement of RNA between animal cells. Genetic model systems will be essential to gain more confidence in proposed mechanisms of RNA transport and to connect an extracellular RNA with a specific biological function. Studies in the worm C. elegans and in other animals have begun to reveal parts of this novel mechanism of cell-to-cell communication. Here, I summarize the current state of this nascent field, highlight the many unknowns, and suggest future directions. PMID:26138457

  8. Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase

    PubMed Central

    Gowher, Ali; Smirnov, Alexandre; Tarassov, Ivan; Entelis, Nina

    2013-01-01

    In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNALysCUU into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNALysCUU and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms. PMID:23799079

  9. A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants.

    PubMed

    Martinez, German; Castellano, Mayte; Tortosa, Maria; Pallas, Vicente; Gomez, Gustavo

    2014-02-01

    Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the 'Hop stunt viroid' accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle. PMID:24178032

  10. Regulation of Cell Death by Transfer RNA

    PubMed Central

    2013-01-01

    Abstract Significance: Both transfer RNA (tRNA) and cytochrome c are essential molecules for the survival of cells. tRNA decodes mRNA codons into amino-acid-building blocks in protein in all organisms, whereas cytochrome c functions in the electron transport chain that powers ATP synthesis in mitochondrion-containing eukaryotes. Additionally, in vertebrates, cytochrome c that is released from mitochondria is a potent inducer of apoptosis, activating apoptotic proteins (caspases) in the cytoplasm to dismantle cells. A better understanding of both tRNA and cytochrome c is essential for an insight into the regulation of cell life and death. Recent Advances: A recent study showed that the mitochondrion-released cytochrome c can be removed from the cell-death pathway by tRNA molecules. The direct binding of cytochrome c by tRNA provides a mechanism for tRNA to regulate cell death, beyond its role in gene expression. Critical Issues: The nature of the tRNA–cytochrome c binding interaction remains unknown. The questions of how this interaction affects tRNA function, cellular metabolism, and apoptotic sensitivity are unanswered. Future Directions: Investigations into the critical issues raised above will improve the understanding of tRNA in the fundamental processes of cell death and metabolism. Such knowledge will inform therapies in cell death-related diseases. Antioxid. Redox Signal. 19, 583–594. PMID:23350625

  11. Influenza virus mRNA trafficking through host nuclear speckles.

    PubMed

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W; García-Sastre, Adolfo; Fontoura, Beatriz M A

    2016-01-01

    Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression. PMID:27572970

  12. Current techniques for visualizing RNA in cells

    PubMed Central

    Mannack, Lilith V.J.C.; Eising, Sebastian; Rentmeister, Andrea

    2016-01-01

    Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations. PMID:27158473

  13. Parasite calcineurin regulates host cell recognition and attachment by apicomplexans

    PubMed Central

    Paul, Aditya S.; Saha, Sudeshna; Engelberg, Klemens; Jiang, Rays H.Y.; Coleman, Bradley I.; Kosber, Aziz L.; Chen, Chun-Ti; Ganter, Markus; Espy, Nicole; Gilberger, Tim W.; Gubbels, Marc-Jan; Duraisingh, Manoj T.

    2015-01-01

    SUMMARY Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans. PMID:26118996

  14. Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

    PubMed Central

    Helentjaris, T; Ehrenfeld, E

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell. Images PMID:189067

  15. Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

    PubMed Central

    Routh, Andrew; Domitrovic, Tatiana; Johnson, John E.

    2012-01-01

    Next-generation sequencing is a valuable tool in our growing understanding of the genetic diversity of viral populations. Using this technology, we have investigated the RNA content of a purified nonenveloped single-stranded RNA virus, flock house virus (FHV). We have also investigated the RNA content of virus-like particles (VLPs) of FHV and the related Nudaurelia capensis omega virus. VLPs predominantly package ribosomal RNA and transcripts of their baculoviral expression vectors. In addition, we find that 5.3% of the packaged RNAs are transposable elements derived from the Sf21 genome. This observation may be important when considering the therapeutic use of VLPs. We find that authentic FHV virions also package a variety of host RNAs, accounting for 1% of the packaged nucleic acid. Significant quantities of host messenger RNAs, ribosomal RNA, noncoding RNAs, and transposable elements are readily detected. The packaging of these host RNAs elicits the possibility of horizontal gene transfer between eukaryotic hosts that share a viral pathogen. We conclude that the genetic content of nonenveloped RNA viruses is variable, not just by genome mutation, but also in the diversity of RNA transcripts that are packaged. PMID:22308402

  16. Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.

    PubMed Central

    Krueger, D M; Cavanaugh, C M

    1997-01-01

    The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins. PMID:8979342

  17. Silencing suppressors: viral weapons for countering host cell defenses.

    PubMed

    Song, Liping; Gao, Shijuan; Jiang, Wei; Chen, Shuai; Liu, Yanjun; Zhou, Ling; Huang, Wenlin

    2011-04-01

    RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21-23 nt RNA molecules. During infection, RNAi can act as an innate immune system to defend against viruses. As a counter-defensive strategy, silencing suppressors are encoded by viruses to inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and act via different mechanisms. In this review, we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses. We also review the modes of action proposed for some silencing suppressors. PMID:21528352

  18. Natural Killer Cells and Antifungal Host Response

    PubMed Central

    Schmidt, Stanislaw; Zimmermann, Stefanie-Yvonne; Tramsen, Lars; Koehl, Ulrike

    2013-01-01

    As a result of improved experimental methodologies and a better understanding of the immune system, there is increasing insight into the antifungal activity of natural killer (NK) cells. Murine and human NK cells are able to damage fungi of different genera and species in vitro, and they exert both direct and indirect antifungal activity through cytotoxic molecules such as perforin and through cytokines and interferons, respectively. On the other hand, recent data suggest that fungi exhibit immunosuppressive effects on NK cells. Whereas clear in vivo data are lacking in humans, the importance of NK cells in the host response against fungi has been demonstrated in animal models. Further knowledge of the interaction of NK cells with fungi might help to better understand the pathogenesis of invasive fungal infections and to improve treatment strategies. PMID:23365210

  19. Interaction between viral RNA silencing suppressors and host factors in plant immunity.

    PubMed

    Nakahara, Kenji S; Masuta, Chikara

    2014-08-01

    To elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity, a zigzag model is useful for uncovering aspects common to different host-pathogen interactions. By analogy of the steps in virus-host interactions with the steps in the standard zigzag model outlined in recent papers, we may regard RNA silencing as pattern-triggered immunity (PTI) against viruses, RNA silencing suppressors (RSSs) as effectors to overcome host RNA silencing and resistance gene (R-gene)-mediated defense as effector-triggered immunity (ETI) recognizing RSSs as avirulence proteins. However, because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus, we here defined a model especially designed for viruses. Although we simplified the phenomena involved in the virus-host interactions in the model, certain specific interactive steps can be explained by integrating additional host factors into the model. These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus-plant interactions. For example, we propose candidates that may interact with viral RSSs to induce the resistance response. PMID:24875766

  20. MicroRNA target prediction by expression analysis of host genes.

    PubMed

    Gennarino, Vincenzo Alessandro; Sardiello, Marco; Avellino, Raffaella; Meola, Nicola; Maselli, Vincenza; Anand, Santosh; Cutillo, Luisa; Ballabio, Andrea; Banfi, Sandro

    2009-03-01

    MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression by inducing RNA cleavage or translational inhibition. Most human miRNAs are intragenic and are transcribed as part of their hosting transcription units. We hypothesized that the expression profiles of miRNA host genes and of their targets are inversely correlated and devised a novel procedure, HOCTAR (host gene oppositely correlated targets), which ranks predicted miRNA target genes based on their anti-correlated expression behavior relative to their respective miRNA host genes. HOCTAR is the first tool for systematic miRNA target prediction that utilizes the same set of microarray experiments to monitor the expression of both miRNAs (through their host genes) and candidate targets. We applied the procedure to 178 human intragenic miRNAs and found that it performs better than currently available prediction softwares in pinpointing previously validated miRNA targets. The high-scoring HOCTAR predicted targets were enriched in Gene Ontology categories, which were consistent with previously published data, as in the case of miR-106b and miR-93. By means of overexpression and loss-of-function assays, we also demonstrated that HOCTAR is efficient in predicting novel miRNA targets and we identified, by microarray and qRT-PCR procedures, 34 and 28 novel targets for miR-26b and miR-98, respectively. Overall, we believe that the use of HOCTAR significantly reduces the number of candidate miRNA targets to be tested compared to the procedures based solely on target sequence recognition. Finally, our data further confirm that miRNAs have a significant impact on the mRNA levels of most of their targets. PMID:19088304

  1. New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: Conservation, folding, and host adaptation

    PubMed Central

    Kieft, Jeffrey S; Rabe, Jennifer L; Chapman, Erich G

    2015-01-01

    Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3′UTR that interact with Xrn1 to halt processive 5′ to 3′ decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3′ UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3′ UTR architecture, sfRNA production, and host adaptation. PMID:26399159

  2. New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: Conservation, folding, and host adaptation.

    PubMed

    Kieft, Jeffrey S; Rabe, Jennifer L; Chapman, Erich G

    2015-01-01

    Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3'UTR that interact with Xrn1 to halt processive 5' to 3' decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3' UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3' UTR architecture, sfRNA production, and host adaptation. PMID:26399159

  3. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

    PubMed

    Rainey, Stephanie M; Martinez, Julien; McFarlane, Melanie; Juneja, Punita; Sarkies, Peter; Lulla, Aleksei; Schnettler, Esther; Varjak, Margus; Merits, Andres; Miska, Eric A; Jiggins, Francis M; Kohl, Alain

    2016-04-01

    The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced

  4. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways

    PubMed Central

    McFarlane, Melanie; Juneja, Punita; Sarkies, Peter; Lulla, Aleksei; Schnettler, Esther; Varjak, Margus; Merits, Andres; Miska, Eric A.; Jiggins, Francis M.; Kohl, Alain

    2016-01-01

    The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3´ open reading frame than the 5´ non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia’s antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced

  5. Concepts of papillomavirus entry into host cells.

    PubMed

    Day, Patricia M; Schelhaas, Mario

    2014-02-01

    Papillomaviruses enter basal cells of stratified epithelia. Assembly of new virions occurs in infected cells during terminal differentiation. This unique biology is reflected in the mechanism of entry. Extracellularly, the interaction of nonenveloped capsids with several host cell proteins, after binding, results in discrete conformational changes. Asynchronous internalization occurs over several hours by an endocytic mechanism related to, but distinct from macropinocytosis. Intracellular trafficking leads virions through the endosomal system, and from late endosomes to the trans-Golgi-network, before nuclear delivery. Here, we discuss the existing data with the aim to synthesize an integrated model of the stepwise process of entry, thereby highlighting key open questions. Additionally, we relate data from experiments with cultured cells to in vivo results. PMID:24525291

  6. Nuclear Localization of Flavivirus RNA Synthesis in Infected Cells

    PubMed Central

    Uchil, Pradeep Devappa; Kumar, Anil V. A.; Satchidanandam, Vijaya

    2006-01-01

    Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex. PMID:16699025

  7. A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell.

    PubMed

    Herbert, Kristina M; Nag, Anita

    2016-01-01

    Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage. PMID:27271653

  8. A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell

    PubMed Central

    Herbert, Kristina M.; Nag, Anita

    2016-01-01

    Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage. PMID:27271653

  9. Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses

    PubMed Central

    Tsetsarkin, Konstantin A.; Liu, Guangping; Kenney, Heather; Bustos-Arriaga, Jose; Hanson, Christopher T.; Whitehead, Stephen S.; Pletnev, Alexander G.

    2015-01-01

    Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics. PMID:25906260

  10. A capsidless ssRNA virus hosted by an unrelated dsRNA virus.

    PubMed

    Zhang, Rui; Hisano, Sakae; Tani, Akio; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2016-01-01

    Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication(1). Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix(2). YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses(3), while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses(4). Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus. PMID:27571749

  11. hnRNPL and nucleolin bind LINE-1 RNA and function as host factors to modulate retrotransposition

    PubMed Central

    Peddigari, Suresh; Li, Patrick Wai-Lun; Rabe, Jennifer L.; Martin, Sandra L.

    2013-01-01

    Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition. PMID:23161687

  12. hnRNPL and nucleolin bind LINE-1 RNA and function as host factors to modulate retrotransposition.

    PubMed

    Peddigari, Suresh; Li, Patrick Wai-Lun; Rabe, Jennifer L; Martin, Sandra L

    2013-01-01

    Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition. PMID:23161687

  13. Counting Legionella cells within single amoeba host cells

    EPA Science Inventory

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  14. Global Analysis of Mouse Polyomavirus Infection Reveals Dynamic Regulation of Viral and Host Gene Expression and Promiscuous Viral RNA Editing

    PubMed Central

    Garren, Seth B.; Kondaveeti, Yuvabharath; Duff, Michael O.; Carmichael, Gordon G.

    2015-01-01

    Mouse polyomavirus (MPyV) lytically infects mouse cells, transforms rat cells in culture, and is highly oncogenic in rodents. We have used deep sequencing to follow MPyV infection of mouse NIH3T6 cells at various times after infection and analyzed both the viral and cellular transcriptomes. Alignment of sequencing reads to the viral genome illustrated the transcriptional profile of the early-to-late switch with both early-strand and late-strand RNAs being transcribed at all time points. A number of novel insights into viral gene expression emerged from these studies, including the demonstration of widespread RNA editing of viral transcripts at late times in infection. By late times in infection, 359 host genes were seen to be significantly upregulated and 857 were downregulated. Gene ontology analysis indicated transcripts involved in translation, metabolism, RNA processing, DNA methylation, and protein turnover were upregulated while transcripts involved in extracellular adhesion, cytoskeleton, zinc finger binding, SH3 domain, and GTPase activation were downregulated. The levels of a number of long noncoding RNAs were also altered. The long noncoding RNA MALAT1, which is involved in splicing speckles and used as a marker in many late-stage cancers, was noticeably downregulated, while several other abundant noncoding RNAs were strongly upregulated. We discuss these results in light of what is currently known about the MPyV life cycle and its effects on host cell growth and metabolism. PMID:26407100

  15. An Integrative Analysis of microRNA and mRNA Profiling in CML Stem Cells.

    PubMed

    Nassar, Farah J; El Eit, Rabab; Nasr, Rihab

    2016-01-01

    Integrative analysis of microRNA (miRNA) and messenger RNA (mRNA) in Chronic Myeloid leukemia (CML) stem cells is an important technique to study the involvement of miRNA and their targets in CML stem cells self-renewal, maintenance, and therapeutic resistance. Here, we describe a simplified integrative analysis using Ingenuity Pathway Analysis software after performing proper RNA extraction, miRNA and mRNA microarray and data analysis. PMID:27581151

  16. RNA Hairpin Folding in the Crowded Cell.

    PubMed

    Gao, Mimi; Gnutt, David; Orban, Axel; Appel, Bettina; Righetti, Francesco; Winter, Roland; Narberhaus, Franz; Müller, Sabine; Ebbinghaus, Simon

    2016-02-24

    Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin-structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high-molecular-weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer. PMID:26833452

  17. RNA Hairpin Folding in the Crowded Cell

    PubMed Central

    Gao, Mimi; Gnutt, David; Orban, Axel; Appel, Bettina; Righetti, Francesco; Winter, Roland; Narberhaus, Franz; Müller, Sabine

    2016-01-01

    Abstract Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer. PMID:26833452

  18. Genome rearrangement affects RNA virus adaptability on prostate cancer cells.

    PubMed

    Pesko, Kendra; Voigt, Emily A; Swick, Adam; Morley, Valerie J; Timm, Collin; Yin, John; Turner, Paul E

    2015-01-01

    Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene

  19. Early Bunyavirus-Host Cell Interactions

    PubMed Central

    Albornoz, Amelina; Hoffmann, Anja B.; Lozach, Pierre-Yves; Tischler, Nicole D.

    2016-01-01

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion. PMID:27213430

  20. Early Bunyavirus-Host Cell Interactions.

    PubMed

    Albornoz, Amelina; Hoffmann, Anja B; Lozach, Pierre-Yves; Tischler, Nicole D

    2016-01-01

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion. PMID:27213430

  1. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  2. Helicobacter pylori interferes with an embryonic stem cell micro RNA cluster to block cell cycle progression

    PubMed Central

    2011-01-01

    Background MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. Results Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. Conclusions These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections. PMID:22027184

  3. Cellular resistance to HIV-1 infection in target cells coincides with a rapid induction of X-DING-CD4 mRNA: indication of the unique host innate response to virus regulated through function of the X-DING-CD4 gene.

    PubMed

    Shilpi, Rasheda Y; Sachdeva, Rakhee; Simm, Malgorzata

    2012-08-01

    Clinical reports indicate that some infected individuals control HIV-1 replication through undefined mechanisms. Our group reported that a human protein named X-DING-CD4 holds a potent antiviral activity, blocking transcription of HIV-1 LTR through the inhibition of NF-κB/DNA binding. Based on observations that transformed HIV-1 resistant CD4(+) T cells produce higher levels of soluble X-DING-CD4 protein upon their exposure to virus, we hypothesized that resistance to HIV-1 in these cells may be regulated through function of the X-DING-CD4 gene. Real-time PCR evaluations of X-DING-CD4 mRNA expression confirmed our hypothesis; HIV-1 exposure caused rapid up-regulation of X-DING-CD4 mRNA in resistant, but not susceptible, cells; and the burst of X-DING-CD4 mRNA expression correlated with restriction of HIV-1 transcription. Subsequently, we examined the activity of the X-DING-CD4 gene in monocytes and macrophages from (n = 13) HIV-negative donors. The assessment of HIV-1 gag mRNA showed that the majority of cells were permissive to virus replication; however, macrophages from four donors were refractory to HIV-1 infection. In response to virus, these cells up-regulated X-DING-CD4 gene expression by 2- to 1000-fold. These data provide evidence that the X-DING-CD4 gene contributes to early cellular protection from HIV infection in some individuals and this protection depends solely on the unique genetic regulation of the host. PMID:22042911

  4. Visual detection of Flavivirus RNA in living cells.

    PubMed

    Miorin, Lisa; Maiuri, Paolo; Marcello, Alessandro

    2016-04-01

    Flaviviruses include a wide range of important human pathogens delivered by insects or ticks. These viruses have a positive-stranded RNA genome that is replicated in the cytoplasm of the infected cell. The viral RNA genome is the template for transcription by the virally encoded RNA polymerase and for translation of the viral proteins. Furthermore, the double-stranded RNA intermediates of viral replication are believed to trigger the innate immune response through interaction with cytoplasmic cellular sensors. Therefore, understanding the subcellular distribution and dynamics of Flavivirus RNAs is of paramount importance to understand the interaction of the virus with its cellular host, which could be of insect, tick or mammalian, including human, origin. Recent advances on the visualization of Flavivirus RNA in living cells together with the development of methods to measure the dynamic properties of viral RNA are reviewed and discussed in this essay. In particular the application of bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are analysed in the context of tick-borne encephalitis virus replication. Conclusions driven by this approached are discussed in the wider context Flavivirus infection. PMID:26542763

  5. RNA regulators of host immunity and pathogen adaptive responses in the oral cavity

    PubMed Central

    Kreth, Jens; Liu, Nan; Chen, Zhiyun; Merritt, Justin

    2015-01-01

    The recent explosion of RNA-seq studies has resulted in a newfound appreciation for the importance of riboregulatory RNAs in the posttranscriptional control of eukaryotic and prokaryotic genetic networks. The current review will explore the role of trans-riboregulatory RNAs in various adaptive responses of host and pathogen in the oral cavity. PMID:25790757

  6. RNA CoMPASS: a dual approach for pathogen and host transcriptome analysis of RNA-seq datasets.

    PubMed

    Xu, Guorong; Strong, Michael J; Lacey, Michelle R; Baribault, Carl; Flemington, Erik K; Taylor, Christopher M

    2014-01-01

    High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism's transcriptome. Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at

  7. RNA CoMPASS: A Dual Approach for Pathogen and Host Transcriptome Analysis of RNA-Seq Datasets

    PubMed Central

    Lacey, Michelle R.; Baribault, Carl; Flemington, Erik K.; Taylor, Christopher M.

    2014-01-01

    High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism's transcriptome. Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at

  8. Probing the Virus Host Interaction in High Containment: An Approach Using Pooled Short Hairpin RNA

    PubMed Central

    Filone, Claire Marie; Dower, Ken; Cowley, Glenn S.; Hensley, Lisa E.

    2015-01-01

    Abstract The study of viruses in high containment offers unique challenges for technology-intense approaches. These approaches include high-throughput screening for small-molecule antivirals and genetic perturbation-based screens for host factors required for viral replication. Here, we describe the use of whole-genome scale pooled short hairpin RNA (shRNA) libraries to screen for host factors necessary for viral infection at BSL2, and the transition of this technique into the BSL4 environment. Pooled screening provides a unique way to circumvent many of the technological challenges associated with other high-throughput screening approaches in high containment. Our pooled screening approach identified host factors involved in the replication of orthopoxviruses (Vaccinia and Monkeypox) and filoviruses (Ebola and Marburg) under conditions that enable straightforward screen-to-follow-up approaches. PMID:25646658

  9. Analysis of Host Gene Expression Profile in HIV-1 and HIV-2 Infected T-Cells

    PubMed Central

    Devadas, Krishnakumar; Biswas, Santanu; Haleyurgirisetty, Mohan; Wood, Owen; Ragupathy, Viswanath; Lee, Sherwin; Hewlett, Indira

    2016-01-01

    HIV replication is closely regulated by a complex pathway of host factors, many of them being determinants of cell tropism and host susceptibility to HIV infection. These host factors are known to exert a positive or negative influence on the replication of the two major types of HIV, HIV-1 and HIV-2, thereby modulating virus infectivity, host response to infection and ultimately disease progression profiles characteristic of these two types. Understanding the differential regulation of host cellular factors in response to HIV-1 and HIV-2 infections will help us to understand the apparent differences in rates of disease progression and pathogenesis. This knowledge would aid in the discovery of new biomarkers that may serve as novel targets for therapy and diagnosis. The objective of this study was to determine the differential expression of host genes in response to HIV-1/HIV-2 infection. To achieve this, we analyzed the effects of HIV-1 (MN) and HIV-2 (ROD) infection on the expression of host factors in PBMC at the RNA level using the Agilent Whole Human Genome Oligo Microarray. Differentially expressed genes were identified and their biological functions determined. Host gene expression profiles were significantly changed. Gene expression profiling analysis identified a subset of differentially expressed genes in HIV-1 and HIV-2 infected cells. Genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected cells. Relatively few genes were differentially expressed in cells infected with HIV-2. PMID:26821323

  10. Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus

    PubMed Central

    Zhang, Jiantao; Diaz, Arturo; Mao, Lan; Ahlquist, Paul

    2012-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly. PMID:22345450

  11. Host ESCRT Proteins Are Required for Bromovirus RNA Replication Compartment Assembly and Function

    PubMed Central

    Diaz, Arturo; Zhang, Jiantao; Ollwerther, Abigail; Wang, Xiaofeng; Ahlquist, Paul

    2015-01-01

    Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance. PMID:25748299

  12. RNA transport during TMV cell-to-cell movement

    PubMed Central

    Peña, Eduardo J.; Heinlein, Manfred

    2012-01-01

    Studies during the last 25 years have provided increasing evidence for the ability of plants to support the cell-to-cell and systemic transport of RNA molecules and that this process plays a role in plant development and in the systemic orchestration of cellular responses against pathogens and other environmental challenges. Since RNA viruses exploit the cellular RNA transport machineries for spreading their genomes between cells they represent convenient models to investigate the underlying mechanisms. In this regard, the intercellular spread of Tobacco mosaic virus (TMV) has been studied for many years. The RNA of TMV moves cell-to-cell in a non-encapsidated form in a process depending on virus-encoded movement protein (MP). Here, we discuss the current state of the art in studies using TMV and its MP as a model for RNA transport. While the ability of plants to transport viral and cellular RNA molecules is consistent with RNA transport phenomena in other systems, further studies are needed to increase our ability to visualize viral RNA (vRNA) in vivo and to distinguish RNA-transport related processes from those involved in antiviral defense. PMID:22973280

  13. Investigating host dependence of xylose utilization in recombinant Saccharomyces cerevisiae strains using RNA-seq analysis

    PubMed Central

    2013-01-01

    Background Xylose-based ethanol production by recombinant S. cerevisiae is of great interest to basic and applied bioenergy research. By expressing three different fungal pathways in two S. cerevisiae hosts respectively, we found that the xylose utilization efficiency by recombinant S. cerevisiae depends not only on the choice of xylose pathway but also on the choice of host, exhibiting an obvious host or context dependence. To investigate molecular mechanisms of this context dependence, we applied RNA-seq analysis in this study for a systematic characterization of the xylose utilization via different pathways in different S. cerevisiae hosts. Results Based on the RNA-seq analysis, the transcripts that were regulated during xylose utilization have been identified. Three transcription factors involved in regulation of amino acid metabolism, responses to oxidative stresses, and degradation of aggregated proteins, respectively, were found to participate in xylose metabolism regulation regardless of which pathway was expressed and which host the xylose pathway was expressed in. Nine transcription factors, involved in homeostasis, regulation of amino acid metabolism, and stress responses, were identified as the key modules responsible for the host-specific responses to the same xylose pathway. In addition, the transcriptional regulations of xylose utilization in different yeast hosts were compared to two reference regulation patterns, which indicated that diverse regulation strategies were adopted by different hosts for improved xylose utilization. Conclusions This study provides the first transcriptomic study of the host dependence of xylose utilization in S. cerevisiae. Both the conserved regulatory modules for xylose metabolism and the key modules responsible for host dependence were identified. As indicated by the functions of the conserved transcription factors involved in xylose metabolism regulation, the xylose utilization in recombinant S. cerevisiae may be

  14. The Type III Secretion Translocation Pore Senses Host Cell Contact

    PubMed Central

    Armentrout, Erin I.; Rietsch, Arne

    2016-01-01

    Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip. PMID:27022930

  15. Proteome-Wide Overexpression of Host Proteins for Identification of Factors Affecting Tombusvirus RNA Replication: an Inhibitory Role of Protein Kinase C

    PubMed Central

    Shah Nawaz-ul-Rehman, Muhammad; Martinez-Ochoa, Natalia; Pascal, Helene; Sasvari, Zsuzsanna; Herbst, Christin; Xu, Kai; Baker, Jannine; Sharma, Monika; Herbst, Alan

    2012-01-01

    To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts. PMID:22718827

  16. Replication of Mengovirus I. Effect on Synthesis of Macromolecules by Host Cell

    PubMed Central

    Plagemann, Peter G. W.; Swim, H. Earle

    1966-01-01

    Plagemann, Peter G. W. (Western Reserve University, Cleveland, Ohio), and H. Earle Swim. Replication of mengovirus. I. Effect on synthesis of macromolecules by host cell. J. Bacteriol. 91:2317–2326. 1966.—The replication of mengovirus was studied in two strains of Novikoff (rat) hepatoma cells propagated in vitro. The replicative cycle in both strains required 6.5 to 7 hr. Infection resulted in a marked depression of ribonucleic acid (RNA) and protein synthesis by strain N1S1-63. Inhibition of RNA synthesis was reflected by a decrease in the deoxyribonucleic acid (DNA)-dependent RNA polymerase activity of isolated nuclei. Mengovirus had no effect on either protein or RNA synthesis or on the DNA-dependent RNA polymerase activity of a second strain, N1S1-67. The time course of viral-induced synthesis of RNA by cells was studied in cells treated with actinomycin D. It was first detectable between 2.5 and 3 hr after infection and continued until 6.5 to 7 hr. The formation of mature virus was estimated biochemically by measuring the amount of RNA synthesized as a result of viral infection which was resistant to degradation by ribonuclease in the presence of deoxycholate. Approximately 70% of the deoxycholate-ribonuclease-resistant RNA was located in mature virus, and the remainder was double-stranded. The formation of mature virus began about 45 min after viral-directed (actinomycin-resistant) synthesis of RNA was detectable in the cell, and only about 18 to 20% of the total RNA synthesized was incorporated into virus. Release of virus from cells began about 1 hr after maturation was first detectable. Release of virus from cells was accompanied by a loss of a large proportion of their cytoplasmic RNA and protein. PMID:4287585

  17. Salmonella – At Home in the Host Cell

    PubMed Central

    Malik-Kale, Preeti; Jolly, Carrie E.; Lathrop, Stephanie; Winfree, Seth; Luterbach, Courtney; Steele-Mortimer, Olivia

    2011-01-01

    The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic “trigger”-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on cohorts of effector proteins translocated into host cells by two type III secretion systems (T3SS), although T3SS-independent mechanisms of entry may be important for invasion of certain host cell types. Recent studies into the intracellular lifestyle of Salmonella have provided new insights into the mechanisms used by this pathogen to modulate its intracellular environment. Here we discuss current knowledge of Salmonella-host interactions including invasion and establishment of an intracellular niche within the host. PMID:21687432

  18. miRNA and Vascular Cell Movement

    PubMed Central

    Yue, Junming

    2011-01-01

    miRNAs are a new class of endogenous small RNAs that negatively regulate gene expression at the posttranscriptional level. Accumulating experimental evidence shows that miRNAs regulate cellular apoptosis, proliferation, differentiation, and migration. Dysregulation of miRNA expression leads to various human diseases including cancer and cardiovascular disease. miRNA maturation is regulated at multiple steps by different mechanisms, including miRNA editing, hairpin loop binding, self-regulation, and cross-talk with other signaling pathways. Vascular cell movement plays a pivotal role in the development of various cancers and cardiovascular diseases. miRNAs have been found to regulate vascular cell movement. Presently the chemically synthesized antagomir, miRNA mimics have been widely used in investigating the biological functions of miRNA genes. The viral vectors, including adenoviral, lentiviral, and adeno-associated viral vectors, have been used to efficiently overexpress or knockdown miRNAs in vitro and in vivo. Therefore, targeting vascular cell movement using miRNA-based drug or gene therapy would provide a novel therapeutic approach in the treatment of cancers and vascular diseases. PMID:21241758

  19. RNA Granules in Germ Cells

    PubMed Central

    Voronina, Ekaterina; Seydoux, Geraldine; Sassone-Corsi, Paolo; Nagamori, Ippei

    2011-01-01

    “Germ granules” are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program. PMID:21768607

  20. Detection of Persistent West Nile Virus RNA in Experimentally and Naturally Infected Avian Hosts

    PubMed Central

    Wheeler, Sarah S.; Langevin, Stanley A.; Brault, Aaron C.; Woods, Leslie; Carroll, Brian D.; Reisen, William K.

    2012-01-01

    To determine whether West Nile virus (WNV) persistent infection in avian hosts may potentially serve as an overwintering mechanism, House Sparrows and House Finches, experimentally and naturally infected with several strains of WNV, and two naturally infected Western Scrub-Jays were held in mosquito-proof outdoor aviaries from 2007–March 2008. Overall, 94% (n = 36) of House Sparrows, 100% (n = 14) of House Finches and 2 Western Scrub-Jays remained WNV antibody positive. When combined by species, 37% of the House Sparrows, 50% of the House Finches, and 2 Western Scrub-Jays were WNV RNA positive at necropsy, up to 36 weeks post-infection. Infectious WNV was not detected. Our study supports the hypothesis that some avian hosts support the long-term persistence of WNV RNA, but it remains unresolved whether these infections relapse to restart an avian-arthropod transmission cycle and thereby serve as an overwintering mechanism for WNV. PMID:22826479

  1. Mechanisms of host cell invasion by Trypanosoma cruzi.

    PubMed

    Caradonna, Kacey L; Burleigh, Barbara A

    2011-01-01

    One of the more accepted concepts in our understanding of the biology of early Trypanosoma cruzi-host cell interactions is that the mammalian-infective trypomastigote forms of the parasite must transit the host cell lysosomal compartment in order to establish a productive intracellular infection. The acidic environment of the lysosome provides the appropriate conditions for parasite-mediated disruption of the parasitophorous vacuole and release of T. cruzi into the host cell cytosol, where replication of intracellular amastigotes occurs. Recent findings indicate a level of redundancy in the lysosome-targeting process where T. cruzi trypomastigotes exploit different cellular pathways to access host cell lysosomes in non-professional phagocytic cells. In addition, the reversible nature of the host cell penetration process was recently demonstrated when conditions for fusion of the nascent parasite vacuole with the host endosomal-lysosomal system were not met. Thus, the concept of parasite retention as a critical component of the T. cruzi invasion process was introduced. Although it is clear that host cell recognition, attachment and signalling are required to initiate invasion, integration of this knowledge with our understanding of the different routes of parasite entry is largely lacking. In this chapter, we focus on current knowledge of the cellular pathways exploited by T. cruzi trypomastigotes to invade non-professional phagocytic cells and to gain access to the host cell lysosome compartment. PMID:21884886

  2. Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting.

    PubMed

    Maynard, Nathaniel D; Macklin, Derek N; Kirkegaard, Karla; Covert, Markus W

    2012-01-01

    Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA(Lys) uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron-sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans. PMID:22294093

  3. Interaction of chlamydiae and host cells in vitro.

    PubMed Central

    Moulder, J W

    1991-01-01

    The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models. PMID:2030670

  4. Plant subviral RNAs as a long noncoding RNA (lncRNA): Analogy with animal lncRNAs in host-virus interactions.

    PubMed

    Shimura, Hanako; Masuta, Chikara

    2016-01-01

    Satellite RNAs (satRNAs) and viroids belong to the group called subviral agents and are the smallest pathogens of plants. In general, small satRNAs and viroids are 300-400 nt in size and do not encode any functional proteins; they are thus regarded as so-called long noncoding RNAs (lncRNAs). These lncRNAs are receiving great attention as a new RNA class involved in gene regulation to control important biological processes such as gene transcription and epigenetic regulation. A substantial number of lncRNAs in animal cells have been found to play important roles in the interactions between a virus and its host. We here discuss the pathogenicity of subviral RNAs (especially satRNAs) in plant cells and their functions as lncRNAs associated with viral diseases, using animal lncRNAs as an analogy. Because, unlike animal lncRNAs, plant subviral RNAs can replicate and accumulate at very high levels in infected cells, we here considered the unique possibility that the RNA silencing machinery of plants, an important defense mechanism against virus infection, may have brought about the replication ability of subviral molecules. In addition, we also discuss the possibility that satRNAs may have arisen from plant-virus interactions in virus-infected cells. Understanding the molecular functions of these unique lncRNAs in plants will enable us to reveal the most plausible origins of these subviral RNAs. PMID:26116900

  5. Host cells and methods for production of isobutanol

    DOEpatents

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2016-08-23

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  6. Methods for production of proteins in host cells

    DOEpatents

    Donnelly, Mark; Joachimiak, Andrzej

    2004-01-13

    The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

  7. Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

    PubMed Central

    Robert, Catherine; Pascalis, Hervé; Michelle, Caroline; Jardot, Priscilla; Charrel, Rémi; Raoult, Didier; Desnues, Christelle

    2015-01-01

    Background Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles. Principal Findings We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method. Conclusion The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses. PMID:26431175

  8. Cold-Sensitive Pseudomonas RNA Polymerase I. Characterization of the Host Dependent Cold-Sensitive Restriction of Phage CB3

    PubMed Central

    Sobieski, Rodney J.; Olsen, Ronald H.

    1973-01-01

    Analysis of bacteriophage CB3 infection of Pseudomonas aeruginosa strain PAT2 establishes that phage induced changes in net macromolecular synthesis are absent at nonpermissive phage growth temperatures (32 C). Alterations which are evident in the PAT2 strain at 37 C or in the fully permissive strain, PAO1C, at either warm or cold temperatures do not occur in PAT2 at low temperatures. CB3 DNA synthesis and the degradation of host DNA to approximately 78S components occur at 37 C, but are absent in PAT2 at 20 C. Nevertheless, attachment of phage DNA to host cytoplasmic material occurs under permissive and nonpermissive conditions. This binding of phage DNA at 20 C is identical in nature to phage DNA bound at 37 C. Thus, the conditional cold-sensitive PAT2 host function in the growth of CB3 is expressed subsequent to membrane binding of the infecting genomes but prior to the onset of the initiation of CB3 DNA synthesis, the inhibition of host DNA synthesis, and the transient depression in RNA synthesis which occurs in permissive cells. PMID:4202617

  9. Ehrlichia chaffeensis TRP32 Interacts with Host Cell Targets That Influence Intracellular Survival

    PubMed Central

    Luo, Tian

    2012-01-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions of E. chaffeensis tandem repeat proteins 47 and 120 (TRP47 and -120) and the eukaryotic host cell have been described. In this investigation, yeast two-hybrid analysis demonstrated that an E. chaffeensis type 1 secretion system substrate, TRP32, interacts with a diverse group of human proteins associated with major biological processes of the host cell, including protein synthesis, trafficking, degradation, immune signaling, cell signaling, iron metabolism, and apoptosis. Eight target proteins, including translation elongation factor 1 alpha 1 (EF1A1), deleted in azoospermia (DAZ)-associated protein 2 (DAZAP2), ferritin light polypeptide (FTL), CD63, CD14, proteasome subunit beta type 1 (PSMB1), ring finger and CCCH-type domain 1 (RC3H1), and tumor protein p53-inducible protein 11 (TP53I11) interacted with TRP32 as determined by coimmunoprecipitation assays, colocalization with TRP32 in HeLa and THP-1 cells, and/or RNA interference. Interactions between TRP32 and host targets localized to the E. chaffeensis morulae or in the host cell cytoplasm adjacent to morulae. Common or closely related interacting partners of E. chaffeensis TRP32, TRP47, and TRP120 demonstrate a molecular convergence on common cellular processes and molecular cross talk between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that promote intracellular survival. PMID:22547548

  10. Knockdown of specific host factors protects against influenza virus-induced cell death

    PubMed Central

    Tran, A T; Rahim, M N; Ranadheera, C; Kroeker, A; Cortens, J P; Opanubi, K J; Wilkins, J A; Coombs, K M

    2013-01-01

    Cell death is a characteristic consequence of cellular infection by influenza virus. Mounting evidence indicates the critical involvement of host-mediated cellular death pathways in promoting efficient influenza virus replication. Furthermore, it appears that many signaling pathways, such as NF-κB, formerly suspected to solely promote cell survival, can also be manipulated to induce cell death. Current understanding of the cell death pathways involved in influenza virus-mediated cytopathology and in virus replication is limited. This study was designed to identify host genes that are required for influenza-induced cell death. The approach was to perform genome-wide lentiviral-mediated human gene silencing in A549 cells and determine which genes could be silenced to provide resistance to influenza-induced cell death. The assay proved to be highly reproducible with 138 genes being identified in independent screens. The results were independently validated using siRNA to each of these candidates. Graded protection was observed in this screen with the silencing of any of 19 genes, each providing >85% protection. Three gene products, TNFSF13 (APRIL), TNFSF12-TNFSF13 (TWE-PRIL) and USP47, were selected because of the high levels of protection conferred by their silencing. Protein and mRNA silencing and protection from influenza-induced cell death was confirmed using multiple shRNA clones and siRNA, indicating the specificity of the effects. USP47 knockdown prevented proper viral entry into the host cell, whereas TNFSF12-13/TNFSF13 knockdown blocked a late stage in viral replication. This screening approach offers the means to identify a large number of potential candidates for the analysis of viral-induced cell death. These results may also have much broader applicability in defining regulatory mechanisms involved in cell survival. PMID:23949218

  11. Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

    PubMed

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-07-01

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

  12. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    PubMed

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. PMID:25329362

  13. Genome-wide whole blood microRNAome and transcriptome analyses reveal miRNA-mRNA regulated host response to foodborne pathogen Salmonella infection in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNAs (miRNAs) are important regulators of gene expression and play key roles in several biological processes. However, little is known about the role of miRNAs in regulating genes involved in host response to bacterial infection. Here, we present a systematic study of miRNA and mRNA profiles fr...

  14. RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model

    PubMed Central

    Brady, Rebecca A.; Bruno, Vincent M.; Burns, Drusilla L.

    2015-01-01

    Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTI), which are primarily self-limiting. We conducted a comprehensive analysis of the host transcriptome during a S. aureus SSTI to provide insight on the protective mechanisms that thwart these infections. We utilized a murine SSTI model in which one ear is epicutaneously challenged while the other is not. We then harvested these infected and uninfected ears, as well as ears from naïve mice, at one, four, and seven days post-challenge, and performed RNA sequencing (RNA-seq) using the Illumina platform. RNA-seq data demonstrated a robust response at the site of infection. Comparison of gene expression profiles between infected ears and the non-infected ears of challenged mice defined the local response to infection, while comparisons of expression profiles of non-infected ears from challenged mice to ears of naïve mice revealed changes in gene expression levels away from the site indicative of a systemic response. Over 1000 genes exhibited increased expression locally at all tested time points. The local response was more robust than the systemic response. Through evaluation of the RNA-seq data using the Upstream Regulator Analytic as part of the Ingenuity Pathway Analysis software package, we found that changes in the activation and inhibition of regulatory pathways happen first locally, and lag behind systemically. The activated pathways are highly similar at all three time points during SSTI, suggesting a stable global response over time. Transcript increases and pathway activation involve pro- and anti-inflammatory mediators, chemotaxis, cell signaling, keratins, and TH1/TH17 cytokines. Transcript decreases and pathway inhibition demonstrate that metabolic genes and anti-inflammatory pathways are repressed. These data provide insight on the host responses that may aid in resolution of this self-limited S. aureus infection, and may shed light on potential immune correlates of

  15. RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model.

    PubMed

    Brady, Rebecca A; Bruno, Vincent M; Burns, Drusilla L

    2015-01-01

    Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTI), which are primarily self-limiting. We conducted a comprehensive analysis of the host transcriptome during a S. aureus SSTI to provide insight on the protective mechanisms that thwart these infections. We utilized a murine SSTI model in which one ear is epicutaneously challenged while the other is not. We then harvested these infected and uninfected ears, as well as ears from naïve mice, at one, four, and seven days post-challenge, and performed RNA sequencing (RNA-seq) using the Illumina platform. RNA-seq data demonstrated a robust response at the site of infection. Comparison of gene expression profiles between infected ears and the non-infected ears of challenged mice defined the local response to infection, while comparisons of expression profiles of non-infected ears from challenged mice to ears of naïve mice revealed changes in gene expression levels away from the site indicative of a systemic response. Over 1000 genes exhibited increased expression locally at all tested time points. The local response was more robust than the systemic response. Through evaluation of the RNA-seq data using the Upstream Regulator Analytic as part of the Ingenuity Pathway Analysis software package, we found that changes in the activation and inhibition of regulatory pathways happen first locally, and lag behind systemically. The activated pathways are highly similar at all three time points during SSTI, suggesting a stable global response over time. Transcript increases and pathway activation involve pro- and anti-inflammatory mediators, chemotaxis, cell signaling, keratins, and TH1/TH17 cytokines. Transcript decreases and pathway inhibition demonstrate that metabolic genes and anti-inflammatory pathways are repressed. These data provide insight on the host responses that may aid in resolution of this self-limited S. aureus infection, and may shed light on potential immune correlates of

  16. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  17. New Hosts of Simplicimonas similis and Trichomitus batrachorum Identified by 18S Ribosomal RNA Gene Sequences

    PubMed Central

    Dimasuay, Kris Genelyn B.; Lavilla, Orlie John Y.; Rivera, Windell L.

    2013-01-01

    Trichomonads are obligate anaerobes generally found in the digestive and genitourinary tract of domestic animals. In this study, four trichomonad isolates were obtained from carabao, dog, and pig hosts using rectal swab. Genomic DNA was extracted using Chelex method and the 18S rRNA gene was successfully amplified through novel sets of primers and undergone DNA sequencing. Aligned isolate sequences together with retrieved 18S rRNA gene sequences of known trichomonads were utilized to generate phylogenetic trees using maximum likelihood and neighbor-joining analyses. Two isolates from carabao were identified as Simplicimonas similis while each isolate from dog and pig was identified as Pentatrichomonas hominis and Trichomitus batrachorum, respectively. This is the first report of S. similis in carabao and the identification of T. batrachorum in pig using 18S rRNA gene sequence analysis. The generated phylogenetic tree yielded three distinct groups mostly with relatively moderate to high bootstrap support and in agreement with the most recent classification. Pathogenic potential of the trichomonads in these hosts still needs further investigation. PMID:23936631

  18. A Neuron-Specific Host MicroRNA Targets Herpes Simplex Virus-1 ICP0 Expression and Promotes Latency

    PubMed Central

    Pan, Dongli; Flores, Omar; Umbach, Jennifer L.; Pesola, Jean M.; Bentley, Peris; Rosato, Pamela C.; Leib, David A.; Cullen, Bryan R.; Coen, Donald M.

    2014-01-01

    Summary After infecting peripheral sites, herpes simplex virus (HSV) invades the nervous system and initiates latent infection in sensory neurons. Establishment and maintenance of HSV latency requires host survival, and entails repression of productive cycle (“lytic”) viral gene expression. We find that a neuron-specific microRNA, miR-138, represses expression of ICP0, a viral transactivator of lytic gene expression. A mutant HSV-1 (M138) with disrupted miR-138 target sites in ICP0 mRNA exhibits enhanced expression of ICP0 and other lytic proteins in infected neuronal cells in culture. Following corneal inoculation, M138-infected mice have higher levels of ICP0 and lytic transcripts in trigeminal ganglia during establishment of latency, and exhibit increased mortality and encephalitis symptoms. After full establishment of latency, the fraction of trigeminal ganglia harboring detectable lytic transcripts is greater in M138-infected mice. Thus, miR-138 is a neuronal factor that represses HSV-1 lytic gene expression, promoting host survival and viral latency. PMID:24721573

  19. Host manipulation by cancer cells: Expectations, facts, and therapeutic implications.

    PubMed

    Tissot, Tazzio; Arnal, Audrey; Jacqueline, Camille; Poulin, Robert; Lefèvre, Thierry; Mery, Frédéric; Renaud, François; Roche, Benjamin; Massol, François; Salzet, Michel; Ewald, Paul; Tasiemski, Aurélie; Ujvari, Beata; Thomas, Frédéric

    2016-03-01

    Similar to parasites, cancer cells depend on their hosts for sustenance, proliferation and reproduction, exploiting the hosts for energy and resources, and thereby impairing their health and fitness. Because of this lifestyle similarity, it is predicted that cancer cells could, like numerous parasitic organisms, evolve the capacity to manipulate the phenotype of their hosts to increase their own fitness. We claim that the extent of this phenomenon and its therapeutic implications are, however, underappreciated. Here, we review and discuss what can be regarded as cases of host manipulation in the context of cancer development and progression. We elaborate on how acknowledging the applicability of these principles can offer novel therapeutic and preventive strategies. The manipulation of host phenotype by cancer cells is one more reason to adopt a Darwinian approach in cancer research. PMID:26849295

  20. An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

    PubMed Central

    Schmid, Maximilian; Dufner, Bianca; Dürk, Julius; Bedal, Konstanze; Stricker, Kristina; Prokoph, Lukas Ali; Koch, Christoph; Wege, Anja K.; Zirpel, Henner; van Zandbergen, Ger; Ecker, Rupert; Boghiu, Bogdan; Ritter, Uwe

    2015-01-01

    Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load. PMID:26488169

  1. Control of Host Cell Phosphorylation by Legionella Pneumophila

    PubMed Central

    Haenssler, Eva; Isberg, Ralph R.

    2011-01-01

    Phosphorylation is one of the most frequent modifications in intracellular signaling and is implicated in many processes ranging from transcriptional control to signal transduction in innate immunity. Many pathogens modulate host cell phosphorylation pathways to promote growth and establish an infectious disease. The intracellular pathogen Legionella pneumophila targets and exploits the host phosphorylation system throughout the infection cycle as part of its strategy to establish an environment beneficial for replication. Key to this manipulation is the L. pneumophila Icm/Dot type IV secretion system, which translocates bacterial proteins into the host cytosol that can act directly on phosphorylation cascades. This review will focus on the different stages of L. pneumophila infection, in which host kinases and phosphatases contribute to infection of the host cell and promote intracellular survival of the pathogen. This includes the involvement of phosphatidylinositol 3-kinases during phagocytosis as well as the role of phosphoinositide metabolism during the establishment of the replication vacuole. Furthermore, L. pneumophila infection modulates the NF-κB and mitogen-activated protein kinase pathways, two signaling pathways that are central to the host innate immune response and involved in regulation of host cell survival. Therefore, L. pneumophila infection manipulates host cell signal transduction by phosphorylation at multiple levels. PMID:21747787

  2. Epistasis between mutations is host-dependent for an RNA virus

    PubMed Central

    Lalić, Jasna; Elena, Santiago F.

    2013-01-01

    How, and to what extent, does the environment influence the way mutations interact? Do environmental changes affect both the sign and the magnitude of epistasis? Are there any correlations between environments in the variability, sign or magnitude of epistasis? Very few studies have tackled these questions. Here, we addressed them in the context of viral emergence. Most emerging viruses are RNA viruses with small genomes, overlapping reading frames and multifunctional proteins for which epistasis is abundant. Understanding the effect of host species in the sign and magnitude of epistasis will provide insights into the evolutionary ecology of infectious diseases and the predictability of viral emergence. PMID:22809724

  3. Diversity of host species and strains of Pneumocystis carinii is based on rRNA sequences.

    PubMed Central

    Shah, J S; Pieciak, W; Liu, J; Buharin, A; Lane, D J

    1996-01-01

    We have amplified by PCR Pneumocystis carinii cytoplasmic small-subunit rRNA (variously referred to as 16S-like or 18S-like rRNA) genes from DNA extracted from bronchoalveolar lavage and induced sputum specimens from patients positive for P. carinii and from infected ferret lung tissue. The amplification products were cloned into pUC18, and individual clones were sequenced. Comparison of the determined sequences with each other and with published rat and partial human P.carinii small-subunit rRNA gene sequences reveals that, although all P. carinii small-subunit rRNAs are closely related (approximately 96% identity), small-subunit rRNA genes isolated from different host species (human, rat, and ferret) exhibit distinctive patterns of sequence variation. Two types of sequences were isolated from the infected ferret lung tissue, one as a predominant species and the other as a minor species. There was 96% identity between the two types. In situ hybridization of the infected ferret lung tissue with oligonucleotide probes specific for each type revealed that there were two distinct strains of P. carinii present in the ferret lung tissue. Unlike the ferret P. carinii isolates, the small-subunit rRNA gene sequences from different human P. carinii isolates have greater than 99% identity and are distinct from all rat and ferret sequences so far inspected or reported in the literature. Southern blot hybridization analysis of PCR amplification products from several additional bronchoalveolar lavage or induced sputum specimens from P. carinii-infected patients, using a 32P-labeled oligonucleotide probe specific for human P. carinii, also suggests that all of the human P. carinii isolates are identical. These findings indicate that human P. carinii isolates may represent a distinct species of P. carinii distinguishable from rat and ferret P. carinii on the basis of characterization of small-subunit rRNA gene sequences. PMID:8770515

  4. Characterization of the mutant spectra of a fish RNA virus within individual hosts during natural infections

    USGS Publications Warehouse

    Emmenegger, Eveline J.; Troyer, Ryan M.; Kurath, Gael

    2003-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an RNA virus that causes significant mortalities of salmonids in the Pacific Northwest of North America. RNA virus populations typically contain genetic variants that form a heterogeneous virus pool, referred to as a quasispecies or mutant spectrum. This study characterized the mutant spectra of IHNV populations within individual fish reared in different environmental settings by RT–PCR of genomic viral RNA and determination of partial glycoprotein gene sequences of molecular clones. The diversity of the mutant spectra from ten in vivo populations was low and the average mutation frequencies of duplicate populations did not significantly exceed the background mutation level expected from the methodology. In contrast, two in vitro populations contained variants with an identical mutational hot spot. These results indicated that the mutant spectra of natural IHNV populations is very homogeneous, and does not explain the different magnitudes of genetic diversity observed between the different IHNV genogroups. Overall the mutant frequency of IHNV within its host is one of the lowest reported for RNA viruses.

  5. Process of Bipolaris sorghicola invasion of host cells.

    PubMed

    Peng, C; Ge, T T; He, X L; Huang, Y H; Xu, Z L; Zhang, D Y; Shao, H B; Guo, S W

    2016-01-01

    Target leaf spot is a sorghum leaf disease caused by Bipolaris sorghicola, a species of fungus with a global distribution. In this study, we investigated the process by which B. sorghicola invades cells of barley, onion, Arabidopsis thaliana species, and sorghum. The results showed that within 8 h of coming into contact with host cells, the hyphal ends of B. sorghicola expand and form a uniform infective penetration pegbolt-like structure; a primary infection mycelium can be formed inside host cells within 24 h after contact, which can infect closed cells after 48 h. A mycelium can grow within the gap between cells and form infective hyphae. The pathogen infection process was the same in different host cells. B. sorghicola can affect root cells through soil infection, indicating that it may also have characteristics of soil-borne pathogens. PMID:26985945

  6. Malaria Sporozoites Traverse Host Cells within Transient Vacuoles.

    PubMed

    Risco-Castillo, Veronica; Topçu, Selma; Marinach, Carine; Manzoni, Giulia; Bigorgne, Amélie E; Briquet, Sylvie; Baudin, Xavier; Lebrun, Maryse; Dubremetz, Jean-François; Silvie, Olivier

    2015-11-11

    Plasmodium sporozoites are deposited in the host skin by Anopheles mosquitoes. The parasites migrate from the dermis to the liver, where they invade hepatocytes through a moving junction (MJ) to form a replicative parasitophorous vacuole (PV). Malaria sporozoites need to traverse cells during progression through host tissues, a process requiring parasite perforin-like protein 1 (PLP1). We find that sporozoites traverse cells inside transient vacuoles that precede PV formation. Sporozoites initially invade cells inside transient vacuoles by an active MJ-independent process that does not require vacuole membrane remodeling or release of parasite secretory organelles typically involved in invasion. Sporozoites use pH sensing and PLP1 to exit these vacuoles and avoid degradation by host lysosomes. Next, parasites enter the MJ-dependent PV, which has a different membrane composition, precluding lysosome fusion. The malaria parasite has thus evolved different strategies to evade host cell defense and establish an intracellular niche for replication. PMID:26607162

  7. Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.

    PubMed

    Wei, Zhiyun; Batagov, Arsen O; Carter, David R F; Krichevsky, Anna M

    2016-01-01

    Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA. PMID:27503761

  8. Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA

    PubMed Central

    Wei, Zhiyun; Batagov, Arsen O.; Carter, David R. F.; Krichevsky, Anna M.

    2016-01-01

    Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA. PMID:27503761

  9. Host cells and methods for producing isoprenyl alkanoates

    SciTech Connect

    Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-12-01

    The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

  10. Coxiella burnetii Nine Mile II proteins modulate gene expression of monocytic host cells during infection

    PubMed Central

    2010-01-01

    Background Coxiella burnetii is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV) of eukaryotic cells, the biogenesis and maintenance of which is dependent on C. burnetii protein synthesis. These observations suggest that C. burnetii actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by C. burnetii proteins during infection. Results We have identified 36 host cell genes that are specifically regulated when de novo C. burnetii protein synthesis occurs during infection using comparative microarray analysis. Two parallel sets of infected and uninfected THP-1 cells were grown for 48 h followed by the addition of chloramphenicol (CAM) to 10 μg/ml in one set. Total RNA was harvested at 72 hpi from all conditions, and microarrays performed using Phalanx Human OneArray™ slides. A total of 784 (mock treated) and 901 (CAM treated) THP-1 genes were up or down regulated ≥2 fold in the C. burnetii infected vs. uninfected cell sets, respectively. Comparisons between the complementary data sets (using >0 fold), eliminated the common gene expression changes. A stringent comparison (≥2 fold) between the separate microarrays revealed 36 host cell genes modulated by C. burnetii protein synthesis. Ontological analysis of these genes identified the innate immune response, cell death and proliferation, vesicle trafficking and development, lipid homeostasis, and cytoskeletal organization as predominant cellular functions modulated by C. burnetii protein synthesis. Conclusions Collectively, these data indicate that C. burnetii proteins actively regulate the expression of specific host cell genes and pathways. This is in addition to host cell genes that respond to the presence of the pathogen whether or not

  11. Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells.

    PubMed Central

    Anderson, K P; Klessig, D F

    1982-01-01

    Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection. Images PMID:6283181

  12. Recruitment of Host Progenitor Cells in Rat Liver Transplants

    PubMed Central

    Sun, Zhaoli; Zhang, Xiuying; Locke, Jayme E.; Zheng, Qizhi; Tachibana, Shingo; Diehl, Anna Mae; Williams, George Melville

    2015-01-01

    Despite MHC incompatibility, Lewis to DA rat liver transplants survive indefinitely without immunosuppression, and the studies we report sought the mechanism(s) responsible for this. At one year most of the liver reacted positively to host anti-DA antibody. When small (50%) grafts were transplanted, recruitment was more rapid as most of the organ assumed the host phenotype at 3 months. After transplantation the Y-chromosome was detected in the hepatocytes of XX to XY grafts by both in-situ hybridization and PCR. Further, livers from transgenic Lewis rats carrying strong GFP markers lost the marker with time after transplantation to DA, GFP− hosts. Few liver cells contained the Y chromosome in syngeneic XX to XY liver grafts or when the hosts of Lewis XX to DA XY allografts were treated with cyclosporine A (CsA) 10mgs/kg/day. This dosage also impeded enlargement of the liver at ten days. Using GFP+ XX Lewis donors transplanted to GFP− XY DA hosts, we found little Y DNA in GFP+ cells at 10 days. Host derived OV-6 and c-kit positive, albumen positive cells were present at 3-10 days, but cells with the CD34 marker were less common and some clearly still had the donor phenotype at ten days. CXCR-4 positive cells increased with time and were abundant at 1 month after transplantation. We conclude: 1. extra-hepatic cells can differentiate into liver tissues; 2. regenerative stimuli accelerate stem cell recruitment; 3. both regeneration and recruitment are impeded by CsA immunosuppression, and 4. donor GFP positive cells contained little host Y-chromosome after transplantation suggesting that cell fusion was uncommon and, therefore, unlikely to be the mechanism leading to the changes in genotype and phenotype we observed. PMID:18972402

  13. Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study the Innate Immune System.

    PubMed

    Gardner, Christina L; Trobaugh, Derek W; Ryman, Kate D; Klimstra, William B

    2016-01-01

    The ability to transfect synthetic mRNAs into cells to measure processes such as translation efficiency or mRNA decay has been an invaluable tool in cell biology. The use of electroporation over other methods of transfection is an easy, inexpensive, highly efficient, and scalable method to introduce synthetic mRNA into a wide range of cell types. More recently, coupling of noncoding RNA sequences or protein coding regions from viral pathogens to fluorescent or bioluminescence proteins in RNA "reporters" has permitted study of host-pathogen interactions. These can range from virus infection of cells to translation of the viral genome, replication and stability of viral RNAs, or the efficacy of host antiviral responses. In this chapter, we describe a method for electroporating viral RNA reporters into both fibroblastic and myeloid cells that encode firefly or Renilla luciferase, whose reaction with specific substrates and light emitting activity is a measure of viral RNA translation efficiency. We have used this method to examine host interferon-dependent responses that inhibit viral translation along with identifying secondary structures in the 5' nontranslated region (NTR) and microRNA binding sites in the 3' NTR that are responsible for antagonizing the host innate immune responses and restricting viral cell tropism. PMID:27236796

  14. Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis.

    PubMed

    Nans, Andrea; Ford, Charlotte; Hayward, Richard D

    2015-01-01

    Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses. PMID:26320027

  15. Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis

    PubMed Central

    Nans, Andrea; Ford, Charlotte; Hayward, Richard D.

    2015-01-01

    Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses. PMID:26320027

  16. Cryptosporidia: epicellular parasites embraced by the host cell membrane.

    PubMed

    Valigurová, Andrea; Jirků, Miloslav; Koudela, Bretislav; Gelnar, Milan; Modrý, David; Slapeta, Jan

    2008-07-01

    The ultrastructure of two gastric cryptosporidia, Cryptosporidium muris from experimentally infected rodents (Mastomys natalensis) and Cryptosporidium sp. 'toad' from naturally infected toads (Duttaphrynus melanostictus), was studied using electron microscopy. Observations presented herein allowed us to map ultrastructural aspects of the cryptosporidian invasion process and the origin of a parasitophorous sac. Invading parasites attach to the host cell, followed by gradual envelopment, with the host's cell membrane folds, eventually forming the parasitophorous sac. Cryptosporidian developmental stages remain epicellular during the entire life cycle. The parasite development is illustrated in detail using high resolution field emission scanning electron microscopy. This provides a new insight into the ultrastructural detail of host-parasite interactions and species-specific differences manifested in frequency of detachment of the parasitophorous sac, radial folds of the parasitophorous sac and stem-formation of the parasitised host cell. PMID:18158154

  17. Recruitment of BAD by the Chlamydia trachomatis Vacuole Correlates with Host-Cell Survival

    PubMed Central

    Verbeke, Philippe; Welter-Stahl, Lynn; Ying, Songmin; Hansen, Jon; Häcker, Georg; Darville, Toni; Ojcius, David M

    2006-01-01

    Chlamydiae replicate intracellularly in a vacuole called an inclusion. Chlamydial-infected host cells are protected from mitochondrion-dependent apoptosis, partly due to degradation of BH3-only proteins. The host-cell adapter protein 14-3-3β can interact with host-cell apoptotic signaling pathways in a phosphorylation-dependent manner. In Chlamydia trachomatis-infected cells, 14-3-3β co-localizes to the inclusion via direct interaction with a C. trachomatis-encoded inclusion membrane protein. We therefore explored the possibility that the phosphatidylinositol-3 kinase (PI3K) pathway may contribute to resistance of infected cells to apoptosis. We found that inhibition of PI3K renders C. trachomatis-infected cells sensitive to staurosporine-induced apoptosis, which is accompanied by mitochondrial cytochrome c release. 14-3-3β does not associate with the Chlamydia pneumoniae inclusion, and inhibition of PI3K does not affect protection against apoptosis of C. pneumoniae-infected cells. In C. trachomatis-infected cells, the PI3K pathway activates AKT/protein kinase B, which leads to maintenance of the pro-apoptotic protein BAD in a phosphorylated state. Phosphorylated BAD is sequestered via 14-3-3β to the inclusion, but it is released when PI3K is inhibited. Depletion of AKT through short-interfering RNA reverses the resistance to apoptosis of C. trachomatis-infected cells. BAD phosphorylation is not maintained and it is not recruited to the inclusion of Chlamydia muridarum, which protects poorly against apoptosis. Thus, sequestration of BAD away from mitochondria provides C. trachomatis with a mechanism to protect the host cell from apoptosis via the interaction of a C. trachomatis-encoded inclusion protein with a host-cell phosphoserine-binding protein. PMID:16710454

  18. Host Cell Factors in Filovirus Entry: Novel Players, New Insights

    PubMed Central

    Hofmann-Winkler, Heike; Kaup, Franziska; Pöhlmann, Stefan

    2012-01-01

    Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1) might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism. PMID:23342362

  19. Fidelity of a viral RNA polymerase in an intact host: template structure and host factors affect rates of deletion mutations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant RNA viruses often have very diverse populations. One of the commonly proposed mechanisms for generating these diverse populations is the error prone RNA-dependent RNA polymerase, based on examples from a number of human and bacterial viruses. This paper describes work to determine the insertio...

  20. Crosstalk between Mycobacterium tuberculosis and the host cell

    PubMed Central

    Dey, Bappaditya; Bishai, William R.

    2014-01-01

    The successful establishment and maintenance of a bacterial infection depends on the pathogen’s ability to subvert the host cell’s defense response and successfully survive, proliferate, or persist within the infected cell. To circumvent host defense systems, bacterial pathogens produce a variety of virulence factors that potentiate bacterial adherence and invasion and usurp host cell signaling cascades that regulate intracellular microbial survival and trafficking. Mycobacterium tuberculosis, probably one of the most successful pathogens on earth, has coexisted with humanity for centuries, and this intimate and persistent connection between these two organisms suggests that the pathogen has evolved extensive mechanisms to evade the human immune system at multiple levels. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are hijacked to prevent the generation of an effective immune response thus benefiting the survival of M. tuberculosis within the host cell. Here, we describe several of these mechanisms, with an emphasis on the cyclic nucleotide signaling and subversion of host responses that occur at the intracellular level when tubercle bacilli encounter macrophages, a cell that becomes a safe-house for M. tuberculosis although it is specialized to kill most microbes. PMID:25303934

  1. Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression.

    PubMed

    Ambite, Ines; Lutay, Nataliya; Stork, Christoph; Dobrindt, Ulrich; Wullt, Björn; Svanborg, Catharina

    2016-01-01

    Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease-associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that

  2. The Plant Host Can Affect the Encapsidation of Brome Mosaic Virus (BMV) RNA; BMV Virions Are Surprisingly Heterogeneous

    PubMed Central

    Ni, Peng; Vaughan, Robert C.; Tragesser, Brady; Hoover, Haley; Kao, C. Cheng

    2013-01-01

    Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3′ untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses. PMID:24036424

  3. [How does the apicomplexan parasite Theileria control host cell identity?].

    PubMed

    Marsolier, Justine; Weitzman, Jonathan B

    2014-01-01

    Infectious agents, like bacteria or virus, are responsible for a large number of pathologies in mammals. Microbes have developed mechanisms for interacting with host cell pathways and hijacking cellular machinery to change the phenotypic state. In this review, we focus on an interesting apicomplexan parasite called Theileria. Infection by the tick-transmitted T. annulata parasite causes Tropical Theileriosis in North Africa and Asia, and the related T. parva parasite causes East Coast Fever in Sub-Saharan Africa. This parasite is the only eukaryote known to induce the transformation of its mammalian host cells. Indeed, T. annulata and T. parva infect bovine leukocytes leading to transforming phenotypes, which partially mirror human lymphoma pathologies. Theileria infection causes hyperproliferation, invasiveness and escape from apoptosis, presumably through the manipulation of host cellular pathways. Several host-signaling mechanisms have been implicated. Here we describe the mechanisms involved in parasite-induced transformation phenotypes. PMID:25840458

  4. Ehrlichia's molecular tricks to manipulate their host cells.

    PubMed

    Moumène, Amal; Meyer, Damien F

    2016-03-01

    Ehrlichia is a large genus of obligate intracellular Gram-negative bacteria transmitted by ticks that cause several emerging infectious diseases in humans and are pathogenic on rodents, ruminants, and dogs. Ehrlichia spp. invade and replicate either in endothelial cells, white blood cells, or within midgut cells and salivary glands of their vector ticks. In this review, we discuss the insights that functional studies are providing on how this group of bacteria exploits their host by subverting host innate immunity and hijacking cellular processes. PMID:26617397

  5. Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor.

    PubMed

    Mata, Miguel A; Satterly, Neal; Versteeg, Gijs A; Frantz, Doug; Wei, Shuguang; Williams, Noelle; Schmolke, Mirco; Peña-Llopis, Samuel; Brugarolas, James; Forst, Christian V; White, Michael A; García-Sastre, Adolfo; Roth, Michael G; Fontoura, Beatriz M A

    2011-10-01

    A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy. PMID:21909097

  6. Variations in hypovirus interactions with the fungal-host RNA-silencing antiviral-defense response.

    PubMed

    Zhang, Xuemin; Shi, Diane; Nuss, Donald L

    2012-12-01

    Hypoviruses Cryphonectria hypovirus 1 (CHV-1)/EP713, CHV-1/Euro7, and CHV-1/EP721, which infect the chestnut blight fungus Cryphonectria parasitica, differ in their degrees of virulence attenuation (hypovirulence), symptom expression, and viral RNA accumulation, even though they share between 90% and 99% amino acid sequence identity. In this report we examine whether this variability is influenced by interactions with the C. parasitica Dicer gene dcl2-dependent RNA-silencing antiviral defense response. The mild symptoms exhibited by strains infected with CHV-1/Euro7 and CHV-1/EP721 relative to those with severe hypovirus CHV-1/EP713 did not correlate with a higher induction of the RNA-silencing pathway. Rather, dcl2 transcripts accumulated to a higher level (∼8-fold) following infection by CHV-1/EP713 than following infection by CHV-1/Euro7 (1.2-fold) or CHV-1/EP721 (1.4-fold). The differences in dcl2 transcript accumulation in response to CHV-1/EP713 and CHV-1/EP721 were unrelated to the suppressor of RNA silencing, p29, encoded by the two viruses. Moreover, the coding strand viral RNA levels increased by 33-, 32-, and 16-fold for CHV-1/EP713, CHV-1/Euro7, and CHV-1/EP721, respectively, in Δdcl2 mutant strains. This indicates that a very robust antiviral RNA-silencing response was induced against all three viruses, even though significant differences in the levels of dcl2 transcript accumulation were observed. Unexpectedly, the severe debilitation previously reported for CHV-1/EP713-infected Δdcl2 mutant strains, and observed here for the CHV-1/Euro7-infected Δdcl2 mutant strains, was not observed with infection by CHV-1/EP721. By constructing chimeric viruses containing portions of CHV-1/EP713 and CHV-1/EP721, it was possible to map the region that is associated with the severe debilitation of the Δdcl2 mutant hosts to a 4.1-kb coding domain located in the central part of the CHV-1/EP713 genome. PMID:22993160

  7. Interactions of Histophilus somni with Host Cells.

    PubMed

    Behling-Kelly, Erica; Rivera-Rivas, Jose; Czuprynski, Charles J

    2016-01-01

    Histophilus somni resides as part of the normal microflora in the upper respiratory tract of healthy cattle. From this site, the organism can make its way into the lower respiratory tract, where it is one of the important bacterial agents of the respiratory disease complex. If H. somni cells disseminate to the bloodstream, they frequently result in thrombus formation. A series of in vitro investigations have examined potential mechanisms that might contribute to such thrombus formation. Earlier work showed that H. somni can stimulate some bovine endothelial cells to undergo apoptosis. More recent studies indicate that H. somni stimulates endothelial cell tissue factor activity and disrupts intercellular junctions. The net effect is to enhance procoagulant activity on the endothelium surface and to make the endothelial monolayer more permeable to molecules, leukocytes, and perhaps H. somni cells. H. somni also activates bovine platelets, which also can enhance tissue factor activity on the endothelium surface. When exposed to H. somni, bovine neutrophils and mononuclear phagocytes form extracellular traps in vitro. Ongoing research is investigating how the interplay among endothelial cells, platelets, and leukocytes might contribute to the thrombus formation seen in infected cattle. PMID:26728064

  8. Battle against RNA oxidation: molecular mechanisms for reducing oxidized RNA to protect cells.

    PubMed

    Li, Zhongwei; Malla, Sulochan; Shin, Brian; Li, James M

    2014-01-01

    Oxidation is probably the most common type of damage that occurs in cellular RNA. Oxidized RNA may be dysfunctional and is implicated in the pathogenesis of age-related human diseases. Cellular mechanisms controlling oxidized RNA have begun to be revealed. Currently, a number of ribonucleases and RNA-binding proteins have been shown to reduce oxidized RNA and to protect cells under oxidative stress. Although information about how these factors work is still very limited, we suggest several mechanisms that can be used to minimize oxidized RNA in various organisms. PMID:24375979

  9. BATTLE AGAINST RNA OXIDATION: Molecular mechanisms for reducing oxidized RNA to protect cells

    PubMed Central

    Li, Zhongwei; Malla, Sulochan; Shin, Brian; Li, James M.

    2014-01-01

    Oxidation is probably the most common type of damage that occurs in cellular RNA. Oxidized RNA may be dysfunctional and is implicated in the pathogenesis of age-related human diseases. Cellular mechanisms controlling oxidized RNA have begun to be revealed. Currently, a number of ribonucleases and RNA binding proteins have been shown to reduce oxidized RNA and to protect cells under oxidative stress. Although information about how these factors work is still very limited, we suggest several mechanisms that can be used to minimize oxidized RNA in various organisms. PMID:24375979

  10. Malaria: targeting parasite and host cell kinomes.

    PubMed

    Doerig, Christian; Abdi, Abdirahman; Bland, Nicholas; Eschenlauer, Sylvain; Dorin-Semblat, Dominique; Fennell, Clare; Halbert, Jean; Holland, Zoe; Nivez, Marie-Paule; Semblat, Jean-Philippe; Sicard, Audrey; Reininger, Luc

    2010-03-01

    Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases. PMID:19840874

  11. Different serotypes of dengue viruses differently regulate the expression of the host cell antigen processing machinery.

    PubMed

    Gan, Chye Sheng; Yusof, Rohana; Othman, Shatrah

    2015-09-01

    Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile. PMID:25981524

  12. Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens.

    PubMed

    Warnock, Neil D; Wilson, Leonie; Canet-Perez, Juan V; Fleming, Thomas; Fleming, Colin C; Maule, Aaron G; Dalzell, Johnathan J

    2016-07-01

    Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants. PMID:27033013

  13. A Potential Regulatory Role for Intronic microRNA-338-3p for Its Host Gene Encoding Apoptosis-Associated Tyrosine Kinase

    PubMed Central

    Kos, Aron; Olde Loohuis, Nikkie F. M.; Wieczorek, Martha L.; Glennon, Jeffrey C.; Martens, Gerard J. M.; Kolk, Sharon M.; Aschrafi, Armaz

    2012-01-01

    MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3′ untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration. PMID:22363537

  14. Identification of host genes leading to West Nile virus encephalitis in mice brain using RNA-seq analysis

    PubMed Central

    Kumar, Mukesh; Belcaid, Mahdi; Nerurkar, Vivek R.

    2016-01-01

    Differential host responses may be critical determinants of distinct pathologies of West Nile virus (WNV) NY99 (pathogenic) and WNV Eg101 (non-pathogenic) strains. We employed RNA-seq technology to analyze global differential gene expression in WNV-infected mice brain and to identify the host cellular factors leading to lethal encephalitis. We identified 1,400 and 278 transcripts, which were differentially expressed after WNV NY99 and WNV Eg101 infections, respectively, and 147 genes were common to infection with both the viruses. Genes that were up-regulated in infection with both the viruses were mainly associated with interferon signaling. Genes associated with inflammation and cell death/apoptosis were only expressed after WNV NY99 infection. We demonstrate that differences in the activation of key pattern recognition receptors resulted in the induction of unique innate immune profiles, which corresponded with the induction of interferon and inflammatory responses. Pathway analysis of differentially expressed genes indicated that after WNV NY99 infection, TREM-1 mediated activation of toll-like receptors leads to the high inflammatory response. In conclusion, we have identified both common and specific responses to WNV NY99 and WNV Eg101 infections as well as genes linked to potential resistance to infection that may be targets for therapeutics. PMID:27211830

  15. Identification of host genes leading to West Nile virus encephalitis in mice brain using RNA-seq analysis.

    PubMed

    Kumar, Mukesh; Belcaid, Mahdi; Nerurkar, Vivek R

    2016-01-01

    Differential host responses may be critical determinants of distinct pathologies of West Nile virus (WNV) NY99 (pathogenic) and WNV Eg101 (non-pathogenic) strains. We employed RNA-seq technology to analyze global differential gene expression in WNV-infected mice brain and to identify the host cellular factors leading to lethal encephalitis. We identified 1,400 and 278 transcripts, which were differentially expressed after WNV NY99 and WNV Eg101 infections, respectively, and 147 genes were common to infection with both the viruses. Genes that were up-regulated in infection with both the viruses were mainly associated with interferon signaling. Genes associated with inflammation and cell death/apoptosis were only expressed after WNV NY99 infection. We demonstrate that differences in the activation of key pattern recognition receptors resulted in the induction of unique innate immune profiles, which corresponded with the induction of interferon and inflammatory responses. Pathway analysis of differentially expressed genes indicated that after WNV NY99 infection, TREM-1 mediated activation of toll-like receptors leads to the high inflammatory response. In conclusion, we have identified both common and specific responses to WNV NY99 and WNV Eg101 infections as well as genes linked to potential resistance to infection that may be targets for therapeutics. PMID:27211830

  16. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment. PMID:26810036

  17. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    PubMed

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants. PMID:24476152

  18. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  19. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

    PubMed Central

    Lampe, Elisabeth O.; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C.

    2015-01-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  20. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

    PubMed

    Lampe, Elisabeth O; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C; Hagedorn, Monica

    2016-03-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  1. Autogenous Translational Regulation of the Borna Disease Virus Negative Control Factor X from Polycistronic mRNA Using Host RNA Helicases

    PubMed Central

    Watanabe, Yohei; Ohtaki, Naohiro; Hayashi, Yohei; Ikuta, Kazuyoshi; Tomonaga, Keizo

    2009-01-01

    Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs): upstream ORF (uORF), X, and P in the 5′ to 3′ direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5′ untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases. PMID:19893625

  2. Host cell infiltration into PDT-treated tumor

    NASA Astrophysics Data System (ADS)

    Korbelik, Mladen; Krosl, Gorazd; Dougherty, Graeme J.; Chaplin, David J.

    1992-06-01

    C3H mice bearing SCCVII squamous cell carcinoma were treated with photodynamic therapy (PDT) 24 hours after receiving Photofrin (25 mg/kg, i.v.). Single cell suspensions obtained by the enzymatic digestion of tumors excised either 30 minutes or 4 hours after PDT were analyzed for the content of host immune cells and colony forming ability of malignant cells. The results were compared to the data obtained with non-treated tumors. It is shown that there is a marked increase in the content of cells expressing Mac-1 (monocytes/macrophages or granulocytes) in the tumor 30 minutes post PDT, while a high level of other leucocytes are found within the tumors by 4 hours after PDT. As elaborated in Discussion, the infiltration rate of host immune cells, dying of malignant tumor cells, and yet unknown death rate of host cells originally present in PDT treated tumor occurring concomitantly during this time period complicates this analysis. The results of this study suggest a massive infiltration of macrophages and other leucocytes in PDT treated SCCVII tumor, supporting the suggestion that a potent immune reaction is one of the main characteristics of PDT action in solid tumors. It remains to be determined to what extent is the activity of tumor infiltrating immune cells responsible for its eradication by PDT.

  3. Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein

    PubMed Central

    Tang, Wen-Fang; Huang, Ru-Ting; Chien, Kun-Yi; Huang, Jo-Yun; Lau, Kean-Seng; Jheng, Jia-Rong; Chiu, Cheng-Hsun; Wu, Tzong-Yuan; Chen, Chung-Yung

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3′ untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. IMPORTANCE Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the

  4. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  5. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells

    PubMed Central

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K.; Das, Mahua R.; Sen, Shamik; Sarkar, Debi P.; Jana, Siddhartha S.

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (−) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  6. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

    PubMed

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K; Das, Mahua R; Sen, Shamik; Sarkar, Debi P; Jana, Siddhartha S

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  7. Lipid Exchange between Borrelia burgdorferi and Host Cells

    PubMed Central

    Crowley, Jameson T.; Toledo, Alvaro M.; LaRocca, Timothy J.; Coleman, James L.; London, Erwin; Benach, Jorge L.

    2013-01-01

    Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or 3H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease. PMID:23326230

  8. Toxoplasma Co-opts Host Cells It Does Not Invade

    PubMed Central

    Koshy, Anita A.; Dietrich, Hans K.; Christian, David A.; Melehani, Jason H.; Shastri, Anjali J.; Hunter, Christopher A.; Boothroyd, John C.

    2012-01-01

    Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large. PMID:22910631

  9. Toxoplasma co-opts host cells it does not invade.

    PubMed

    Koshy, Anita A; Dietrich, Hans K; Christian, David A; Melehani, Jason H; Shastri, Anjali J; Hunter, Christopher A; Boothroyd, John C

    2012-01-01

    Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large. PMID:22910631

  10. Viral Genome Tethering to Host Cell Chromatin: Cause and Consequences.

    PubMed

    Aydin, Inci; Schelhaas, Mario

    2016-04-01

    Viruses are small infectious agents that replicate in cells of a host organism and that evolved to use cellular machineries for all stages of the viral life cycle. Here, we critically assess current knowledge on a particular mechanism of persisting viruses, namely, how they tether their genomes to host chromatin, and what consequences arise from this process. A group of persisting DNA viruses, i.e. gamma-herpesviruses and papillomaviruses (PV), uses this tethering strategy to maintain their genomes in the nuclei during cell division. Thus, these viruses face the challenge of viral genome loss during mitosis, as they are transported with the host chromosomes to the nascent daughter nuclei. Incidentally, another group of viruses, certain retroviruses and PV, have adopted this tethering strategy to deliver their genomes into the nuclei of dividing cells during cell entry. By exploiting a phase in the cell cycle when the nuclear envelope is disassembled, viruses bypass the need to engage with the nuclear import machinery. Recent reports suggest that tethering may induce severe cellular consequences that involve activation of mitotic checkpoints, causing missegregation of host chromosomes and genomic instability, which may contribute to cancer. PMID:26787361

  11. Maternal mRNA knockdown studies: antisense experiments using the host-transfer technique in X. laevis and X. tropicalis

    PubMed Central

    Olson, David J.; Hulstrand, Alissa M.; Houston, Douglas W.

    2014-01-01

    SUMMARY The ability to inhibit the activity of maternally stored gene products in Xenopus has led to numerous insights into early developmental mechanisms. Oocytes can be cultured and manipulated in vitro and then implanted into the body cavity of a host female to make them competent for fertilization. Here, we summarize the methods for obtaining, culturing and fertilizing Xenopus oocytes, with the goal of inhibiting maternal gene function through antisense oligonucleotide-mediated mRNA knockdown. We describe a simplified technique for implanting donor oocytes into host females using intraperitoneal injection. Also, we present optimized methods for performing the host-transfer procedure with X. tropicalis oocytes. PMID:22956088

  12. Plasmodium species: master renovators of their host cells.

    PubMed

    de Koning-Ward, Tania F; Dixon, Matthew W A; Tilley, Leann; Gilson, Paul R

    2016-08-01

    Plasmodium parasites, the causative agents of malaria, have developed elaborate strategies that they use to survive and thrive within different intracellular environments. During the blood stage of infection, the parasite is a master renovator of its erythrocyte host cell, and the changes in cell morphology and function that are induced by the parasite promote survival and contribute to the pathogenesis of severe malaria. In this Review, we discuss how Plasmodium parasites use the protein trafficking motif Plasmodium export element (PEXEL), protease-mediated polypeptide processing, a novel translocon termed the Plasmodium translocon of exported proteins (PTEX) and exomembranous structures to export hundreds of proteins to discrete subcellular locations in the host erythrocytes, which enables the parasite to gain access to vital nutrients and to evade the immune defence mechanisms of the host. PMID:27374802

  13. Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

    PubMed Central

    Meier, Roger; Franceschini, Andrea; Horvath, Peter; Tetard, Marilou; Mancini, Roberta; von Mering, Christian; Helenius, Ari

    2014-01-01

    ABSTRACT The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3+ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3+ virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCE Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we

  14. Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization

    PubMed Central

    Capel, Elena; Zomer, Aldert L.; Nussbaumer, Thomas; Bole, Christine; Izac, Brigitte; Frapy, Eric; Meyer, Julie; Bouzinba-Ségard, Haniaa; Bille, Emmanuelle; Jamet, Anne; Cavau, Anne; Letourneur, Franck; Bourdoulous, Sandrine; Rattei, Thomas; Coureuil, Mathieu

    2016-01-01

    ABSTRACT Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. PMID:27486197

  15. RNA-Sequencing Reveals the Progression of Phage-Host Interactions between φR1-37 and Yersinia enterocolitica

    PubMed Central

    Leskinen, Katarzyna; Blasdel, Bob G.; Lavigne, Rob; Skurnik, Mikael

    2016-01-01

    Despite the expanding interest in bacterial viruses (bacteriophages), insights into the intracellular development of bacteriophage and its impact on bacterial physiology are still scarce. Here we investigate during lytic infection the whole-genome transcription of the giant phage vB_YecM_φR1-37 (φR1-37) and its host, the gastroenteritis causing bacterium Yersinia enterocolitica. RNA sequencing reveals that the gene expression of φR1-37 does not follow a pattern typical observed in other lytic bacteriophages, as only selected genes could be classified as typically early, middle or late genes. The majority of the genes appear to be expressed constitutively throughout infection. Additionally, our study demonstrates that transcription occurs mainly from the positive strand, while the negative strand encodes only genes with low to medium expression levels. Interestingly, we also detected the presence of antisense RNA species, as well as one non-coding intragenic RNA species. Gene expression in the phage-infected cell is characterized by the broad replacement of host transcripts with phage transcripts. However, the host response in the late phase of infection was also characterized by up-regulation of several specific bacterial gene products known to be involved in stress response and membrane stability, including the Cpx pathway regulators, ATP-binding cassette (ABC) transporters, phage- and cold-shock proteins. PMID:27110815

  16. RNA-Sequencing Reveals the Progression of Phage-Host Interactions between φR1-37 and Yersinia enterocolitica.

    PubMed

    Leskinen, Katarzyna; Blasdel, Bob G; Lavigne, Rob; Skurnik, Mikael

    2016-04-01

    Despite the expanding interest in bacterial viruses (bacteriophages), insights into the intracellular development of bacteriophage and its impact on bacterial physiology are still scarce. Here we investigate during lytic infection the whole-genome transcription of the giant phage vB_YecM_φR1-37 (φR1-37) and its host, the gastroenteritis causing bacterium Yersinia enterocolitica. RNA sequencing reveals that the gene expression of φR1-37 does not follow a pattern typical observed in other lytic bacteriophages, as only selected genes could be classified as typically early, middle or late genes. The majority of the genes appear to be expressed constitutively throughout infection. Additionally, our study demonstrates that transcription occurs mainly from the positive strand, while the negative strand encodes only genes with low to medium expression levels. Interestingly, we also detected the presence of antisense RNA species, as well as one non-coding intragenic RNA species. Gene expression in the phage-infected cell is characterized by the broad replacement of host transcripts with phage transcripts. However, the host response in the late phase of infection was also characterized by up-regulation of several specific bacterial gene products known to be involved in stress response and membrane stability, including the Cpx pathway regulators, ATP-binding cassette (ABC) transporters, phage- and cold-shock proteins. PMID:27110815

  17. Metabolism and expression of RNA polymerase II transcripts in Influenza virus-infected cells

    SciTech Connect

    Katze, M.G.; Krug, R.M.

    1984-10-01

    Influenza virus infection has adverse effects on the metabolism of two representative RNA polymerase II transcripts in chicken embryo fibroblasts, those coding for BETA-actin and for avian leukosis virus (ALV) proteins. Proviral ALV DNA was integrated into host cell DNA by prior infection with ALV. By S1 endonuclease assay, it was confirmed that nuclear ALV transcripts disappeared very early after infection, already decreasing ca. 80% by 1 h postinfection. A plausible explanation for this nuclear degradation is that the viral cap-dependent endonuclease in the nucleas cleaves the 5' ends of new polymerase II transcripts, rendering the resulting decapped RNAs susceptible to hydrolysis by cellular nucleases. Similar stability of cytoplasmic host cell mRNAs was observed in infected HeLa cells, in which the levels of actin mRNA and two HeLa cell mRNAs (pHe 7 and pHe 28) remained at undiminished levels for 3 h of infection and decreased only slightly by 4.5 h postinfection. The cytoplamic actin and pHe 7 mRNAs isolated from infected HeLa cells were shown to be translated in reticulocyte extracts in biro, indicating that host mRNAs were not inactivated by a virus-induced modification. Despite the continued presence of high levels of functional host cell mRNAs, host cell protein synthesis was effectively shut off by about 3 h postinfection in both chicken embryo fibroblasts and HeLa cells. These results are consistent with the establishment of an influenza virus-specific translational system that selectively translates viral and not host mRNAs.

  18. Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis

    PubMed Central

    Durkin, Charlotte H.; Helaine, Sophie; Boucrot, Emmanuel

    2016-01-01

    Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S. Typhimurium delays epithelial cell turnover in the intestine. PMID:27185791

  19. Next-generation sequencing identifies the natural killer cell microRNA transcriptome

    PubMed Central

    Fehniger, Todd A.; Wylie, Todd; Germino, Elizabeth; Leong, Jeffrey W.; Magrini, Vincent J.; Koul, Sunita; Keppel, Catherine R.; Schneider, Stephanie E.; Koboldt, Daniel C.; Sullivan, Ryan P.; Heinz, Michael E.; Crosby, Seth D.; Nagarajan, Rakesh; Ramsingh, Giridharan; Link, Daniel C.; Ley, Timothy J.; Mardis, Elaine R.

    2010-01-01

    Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3′ untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology. PMID:20935160

  20. Analysis of the miRNA-mRNA-lncRNA networks in ER+ and ER- breast cancer cell lines.

    PubMed

    Wu, Qian; Guo, Li; Jiang, Fei; Li, Lei; Li, Zhong; Chen, Feng

    2015-12-01

    Recently, rapid advances in bioinformatics analysis have expanded our understanding of the transcriptome to a genome-wide level. miRNA-mRNA-lncRNA interactions have been shown to play critical regulatory role in cancer biology. In this study, we discussed the use of an integrated systematic approach to explore new facets of the oestrogen receptor (ER)-regulated transcriptome. The identification of RNAs that are related to the expression status of the ER may be useful in clinical therapy and prognosis. We used a network modelling strategy. First, microarray expression profiling of mRNA, lncRNA and miRNA was performed in MCF-7 (ER-positive) and MDA-MB-231 cells (ER- negative). A co-expression network was then built using co-expression relationships of the differentially expressed mRNAs and lncRNAs. Finally, the selected miRNA-mRNA network was added to the network. The key miRNA-mRNA-lncRNA interaction can be inferred from the network. The mRNA and non-coding RNA expression profiles of the cells with different ER phenotypes were distinct. Among the aberrantly expressed miRNAs, the expression levels of miR-19a-3p, miR-19b-3p and miR-130a-3p were much lower in the MCF-7 cells, whereas that of miR-148b-3p was much higher. In a cluster of miR-17-92, the expression levels of six of seven miRNAs were lower in the MCF-7 cells, in addition to miR-20b in the miR-106a-363 cluster. However, the levels of all the miRNAs in the miR-106a-25 cluster were higher in the MCF-7 cells. In the co-expression networking, CD74 and FMNL2 gene which is involved in the immune response and metastasis, respectively, had a stronger correlation with ER. Among the aberrantly expressed lncRNAs, lncRNA-DLEU1 was highly expressed in the MCF-7 cells. A statistical analysis revealed that there was a co-expression relationship between ESR1 and lncRNA-DLEU1. In addition, miR-19a and lncRNA-DLEU1 are both located on the human chromosome 13q. We speculate that miR-19a might be co-expressed with lncRNA-DLEU1

  1. Single mRNA Tracking in Live Cells

    PubMed Central

    Park, Hye Yoon; Buxbaum, Adina R.; Singer, Robert H.

    2011-01-01

    Asymmetric distribution of mRNA is a prevalent phenomenon observed in diverse cell types. The posttranscriptional movement and localization of mRNA provides an important mechanism to target certain proteins to specific cytoplasmic regions of their function. Recent technical advances have enabled real-time visualization of single mRNA molecules in living cells. Studies analyzing the motion of individual mRNAs have shed light on the complex RNA transport system. This chapter presents an overview of general approaches for single particle tracking and some methodologies that are used for single mRNA detection. PMID:20580973

  2. Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA.

    PubMed

    Granger, Kathy; Moore, Robert J; Davies, John K; Vaughan, Jill A; Stiles, Paula L; Stewart, David J; Tizard, Mark L V

    2004-05-01

    RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment. PMID:15063064

  3. Triticum mosaic poacevirus enlists P1 rather than HC-Pro to suppress RNA silencing-mediated host defense

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA silencing, or posttranscriptional gene silencing (PTGS) is one of the most important defense mechanisms employed by higher plants and animals to defend against viral infections. Plant viruses evolved by adopting divergent proteins, even within single virus families, to counter this host defense ...

  4. Pathogenesis mediated by proviral host factors involved in translation and replication of plant positive-strand RNA viruses.

    PubMed

    Hyodo, Kiwamu; Okuno, Tetsuro

    2016-04-01

    Viral pathogenesis comes from complex interactions between viruses and hosts. All the processes of viral infection, including translation of viral factors and replication of viral genomes, define viral pathogenesis; therefore, molecular insights into the mechanisms underlying viral replication strategies unambiguously pave the way for our comprehensive understanding of viral pathogenesis and disease outcome, as well as for developing new antiviral strategies against plant virus disease. Recent studies of plant positive-strand RNA [(+)RNA] viruses have advanced our understanding of co-opted host factors and their roles in viral translation and replication. It is becoming clear that plant (+)RNA viruses harness host factors involved in membrane trafficking and lipid metabolism to establish the viral replication complex (VRC). In this review, we aim to discuss the contribution of co-opted host factors in translation and genome replication of plant (+)RNA viruses mainly focusing on those involved in the biogenesis of the VRC, which may act as a central hub in almost all the processes of viral infection as well as viral pathogenesis. PMID:26651023

  5. A bromodomain-containing host protein mediates the nuclear importation of a satellite RNA of Cucumber mosaic virus.

    PubMed

    Chaturvedi, Sonali; Kalantidis, Kriton; Rao, A L N

    2014-02-01

    Replication of the satellite RNA (satRNA) of Cucumber Mosaic Virus is dependent on replicase proteins of helper virus (HV). However, we recently demonstrated that like with Potato spindle tuber viroid (PSTVd), a satRNA associated with Cucumber Mosaic Virus strain Q (Q-satRNA) has the propensity to localize in the nucleus and generate multimers that subsequently serve as templates for HV-dependent replication. But the mechanism regulating the nuclear importation of Q-satRNA is unknown. Here we show that the nuclear importation of Q-satRNA is mediated by a bromodomain-containing host protein (BRP1), which is also apparently involved in the nuclear localization of PSTVd. A comparative analysis of nuclear and cytoplasmic fractions from Nicotiana benthamiana plants coinfected with Q-satRNA and its HV confirmed the association of Q-satRNA but not HV with the nuclear compartment. A combination of the MS2-capsid protein-based RNA tagging assay and confocal microscopy demonstrated that the nuclear localization of Q-satRNA was completely blocked in transgenic lines of Nicotiana benthamiana (ph5.2nb) that are defective in BRP1 expression. This defect, however, was restored when the ph5.2nb lines of N. benthamiana were trans-complemented by ectopically expressed BRP1. The binding specificity of BRP1 with Q-satRNA was confirmed in vivo and in vitro by coimmunoprecipitation and electrophoretic mobility shift assays, respectively. Finally, infectivity assays involving coexpression of Q-satRNA and its HV in wild-type and ph5.2nb lines of N. benthamiana accentuated a biological role for BRP1 in the Q-satRNA infection cycle. The significance of these results in relation to a possible evolutionary relationship to viroids is discussed. PMID:24284314

  6. Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

    SciTech Connect

    Mayman, B.A.; Nishioka, Y.

    1985-01-01

    The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed.

  7. Virus and Host Mechanics Support Membrane Penetration and Cell Entry.

    PubMed

    Greber, Urs F

    2016-04-01

    Viruses are quasi-inert macromolecular assemblies. Their metastable conformation changes during entry into cells, when chemical and mechanical host cues expose viral membrane-interacting proteins. This leads to membrane rupture or fusion and genome uncoating. Importantly, virions tune their physical properties and enhance penetration and uncoating. For example, influenza virus softens at low pH to uncoat. The stiffness and pressure of adenovirus control uncoating and membrane penetration. Virus and host mechanics thus present new opportunities for antiviral therapy. PMID:26842477

  8. Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier

    PubMed Central

    Kuo, Lili; Godeke, Gert-Jan; Raamsman, Martin J. B.; Masters, Paul S.; Rottier, Peter J. M.

    2000-01-01

    Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells. PMID:10627550

  9. Necroptosis: The Trojan horse in cell autonomous antiviral host defense.

    PubMed

    Mocarski, Edward S; Guo, Hongyan; Kaiser, William J

    2015-05-01

    Herpesviruses suppress cell death to assure sustained infection in their natural hosts. Murine cytomegalovirus (MCMV) encodes suppressors of apoptosis as well as M45-encoded viral inhibitor of RIP activation (vIRA) to block RIP homotypic interaction motif (RHIM)-signaling and recruitment of RIP3 (also called RIPK3), to prevent necroptosis. MCMV and human cytomegalovirus encode a viral inhibitor of caspase (Casp)8 activation to block apoptosis, an activity that unleashes necroptosis. Herpes simplex virus (HSV)1 and HSV2 incorporate both RHIM and Casp8 suppression strategies within UL39-encoded ICP6 and ICP10, respectively, which are herpesvirus-conserved homologs of MCMV M45. Both HSV proteins sensitize human cells to necroptosis by blocking Casp8 activity while preventing RHIM-dependent RIP3 activation and death. In mouse cells, HSV1 ICP6 interacts with RIP3 and, surprisingly, drives necroptosis. Thus, herpesviruses have illuminated the contribution of necoptosis to host defense in the natural host as well as its potential to restrict cross-species infections in nonnatural hosts. PMID:25819165

  10. Initial adherence of EPEC, EHEC and VTEC to host cells

    PubMed Central

    Bardiau, Marjorie; Szalo, Mihai; Mainil, Jacques G.

    2010-01-01

    Initial adherence to host cells is the first step of the infection of enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC) and verotoxigenic Escherichia coli (VTEC) strains. The importance of this step in the infection resides in the fact that (1) adherence is the first contact between bacteria and intestinal cells without which the other steps cannot occur and (2) adherence is the basis of host specificity for a lot of pathogens. This review describes the initial adhesins of the EPEC, EHEC and VTEC strains. During the last few years, several new adhesins and putative colonisation factors have been described, especially in EHEC strains. Only a few adhesins (BfpA, AF/R1, AF/R2, Ral, F18 adhesins) appear to be host and pathotype specific. The others are found in more than one species and/or pathotype (EPEC, EHEC, VTEC). Initial adherence of EPEC, EHEC and VTEC strains to host cells is probably mediated by multiple mechanisms. PMID:20423697

  11. A white spot syndrome virus microRNA promotes the virus infection by targeting the host STAT

    PubMed Central

    Ren, Qian; Huang, Ying; He, Yaodong; Wang, Wen; Zhang, Xiaobo

    2015-01-01

    JAK/STAT pathway plays an important role in invertebrates during virus infection. However the microRNA (miRNA)-mediated regulation of JAK/STAT is not intensively investigated. Viral miRNAs, encoded by virus genome, have emerged as important regulators in the virus-host interactions. In this study, a WSSV (white spot syndrome virus)-encoded miRNA (WSSV-miR-22) was characterized in shrimp during virus infection. The results showed that the viral miRNA could promote WSSV infection in shrimp by targeting the host STAT gene. When the expression of JAK or STAT was knocked down by sequence-specific siRNA, the WSSV copies in shrimp were significantly increased, indicating that the JAK/STAT played positive roles in the antiviral immunity of shrimp. The further findings revealed that TEP1 and TEP2 were the effectors of JAK-STAT signaling pathway. The silencing of TEP1 or TEP2 led to an increase of WSSV copies in shrimp, showing TEP1 and TEP2 were involved in the shrimp immune response against virus infection. Therefore our study presented a novel viral miRNA-mediated JAK/STAT-TEP1/TEP2 signaling pathway in virus infection. PMID:26671453

  12. A white spot syndrome virus microRNA promotes the virus infection by targeting the host STAT.

    PubMed

    Ren, Qian; Huang, Ying; He, Yaodong; Wang, Wen; Zhang, Xiaobo

    2015-01-01

    JAK/STAT pathway plays an important role in invertebrates during virus infection. However the microRNA (miRNA)-mediated regulation of JAK/STAT is not intensively investigated. Viral miRNAs, encoded by virus genome, have emerged as important regulators in the virus-host interactions. In this study, a WSSV (white spot syndrome virus)-encoded miRNA (WSSV-miR-22) was characterized in shrimp during virus infection. The results showed that the viral miRNA could promote WSSV infection in shrimp by targeting the host STAT gene. When the expression of JAK or STAT was knocked down by sequence-specific siRNA, the WSSV copies in shrimp were significantly increased, indicating that the JAK/STAT played positive roles in the antiviral immunity of shrimp. The further findings revealed that TEP1 and TEP2 were the effectors of JAK-STAT signaling pathway. The silencing of TEP1 or TEP2 led to an increase of WSSV copies in shrimp, showing TEP1 and TEP2 were involved in the shrimp immune response against virus infection. Therefore our study presented a novel viral miRNA-mediated JAK/STAT-TEP1/TEP2 signaling pathway in virus infection. PMID:26671453

  13. Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase

    PubMed Central

    Zenkin, Nikolay; Severinov, Konstantin; Yuzenkova, Yulia

    2015-01-01

    Regulation of transcription elongation is based on response of RNA polymerase (RNAP) to various pause signals and is modulated by various accessory factors. Here we report that a 7 kDa protein p7 encoded by bacteriophage Xp10 acts as an elongation processivity factor of RNAP of host bacterium Xanthomonas oryzae, a major rice pathogen. Our data suggest that p7 stabilizes the upstream DNA duplex of the elongation complex thus disfavouring backtracking and promoting forward translocated states of the elongation complex. The p7-induced ‘pushing’ of RNAP and modification of RNAP contacts with the upstream edge of the transcription bubble lead to read-through of various types of pauses and termination signals and generally increase transcription processivity and elongation rate, contributing for transcription of an extremely long late genes operon of Xp10. Forward translocation was observed earlier upon the binding of unrelated bacterial elongation factor NusG, suggesting that this may be a general pathway of regulation of transcription elongation. PMID:26038312

  14. RNA-seq based transcriptomic analysis of single bacterial cells.

    PubMed

    Wang, Jiangxin; Chen, Lei; Chen, Zixi; Zhang, Weiwen

    2015-11-01

    Gene-expression heterogeneity among individual cells determines the fate of a bacterial population. Here we report the first bacterial single-cell RNA sequencing (RNA-seq), BaSiC RNA-seq, a method integrating RNA isolation, cDNA synthesis and amplification, and RNA-seq analysis of the whole transcriptome of single cyanobacterium Synechocystis sp. PCC 6803 cells which typically contain approximately 5-7 femtogram total RNA per cell. We applied the method to 3 Synechocystis single cells at 24 h and 3 single cells at 72 h after nitrogen-starvation stress treatment, as well as their bulk-cell controls under the same conditions, to determine the heterogeneity upon environmental stress. With 82-98% and 31-48% of all putative Synechocystis genes identified in single cells of 24 and 72 h, respectively, the results demonstrated that the method could achieve good identification of the transcripts in single bacterial cells. In addition, the preliminary results from nitrogen-starved cells also showed a possible increasing gene-expression heterogeneity from 24 h to 72 h after nitrogen starvation stress. Moreover, preliminary analysis of single-cell transcriptomic datasets revealed that genes from the "Mobile elements" functional category have the most significant increase of gene-expression heterogeneity upon stress, which was further confirmed by single-cell RT-qPCR analysis of gene expression in 24 randomly selected cells. PMID:26331465

  15. Probes for intracellular RNA imaging in live cells.

    PubMed

    Santangelo, Philip J; Alonas, Eric; Jung, Jeenah; Lifland, Aaron W; Zurla, Chiara

    2012-01-01

    RNA localization, dynamics, and regulation are becoming increasingly important to our basic understanding of gene expression and RNA virus pathogenesis. An improved understanding of these processes will be necessary in order to identify new drug targets, as well as to create models of gene expression networks. Much of this new understanding will likely come from imaging studies of RNA, which can generate the spatiotemporal information necessary to characterize RNA within the cellular milieu. Ideally, this would be performed imaging native, nonengineered RNAs, but the approaches for performing these experiments are still evolving. In order for them to reach their potential, it is critical that they have characteristics that allow for the tracking of RNA throughout their life cycle. This chapter presents an overview of RNA imaging methodologies, and focuses on a single RNA sensitive method, employing exogenous probes, for imaging, native, nonengineered RNA in live cells. PMID:22289464

  16. A Sense of Self: RIG-I's Tolerance to Host RNA.

    PubMed

    Schoggins, John W

    2015-07-21

    The innate immune sensor RIG-I recognizes viral RNA while avoiding unwanted activation by self RNA. In this issue of Immunity, Schuberth-Wagner et al. (2015) show that a histidine residue in the RNA binding pocket of RIG-I sterically excludes the cap1 structure of self RNA, thereby preventing downstream activation. PMID:26200004

  17. microRNA Response to Listeria monocytogenes Infection in Epithelial Cells

    PubMed Central

    Izar, Benjamin; Mannala, Gopala Krishna; Mraheil, Mobarak Abu; Chakraborty, Trinad; Hain, Torsten

    2012-01-01

    microRNAs represent a family of very small non-coding RNAs that control several physiologic and pathologic processes, including host immune response and cancer by antagonizing a number of target mRNAs. There is limited knowledge about cell expression and the regulatory role of microRNAs following bacterial infections. We investigated whether infection with a Gram-positive bacterium leads to altered expression of microRNAs involved in the host cell response in epithelial cells. Caco-2 cells were infected with Listeria monocytogenes EGD-e, a mutant strain (ΔinlAB or Δhly) or incubated with purified listeriolysin (LLO). Total RNA was isolated and microRNA and target gene expression was compared to the expression in non-infected cells using microRNA microarrays and qRT-PCR. We identified and validated five microRNAs (miR- 146b, miR-16, let-7a1, miR-145 and miR-155) that were significantly deregulated following listerial infection. We show that expression patterns of particular microRNAs strongly depend on pathogen localization and the presence of bacterial effector proteins. Strikingly, miR-155 which was shown to have an important role in inflammatory responses during infection was induced by wild-type bacteria, by LLO-deficient bacteria and following incubation with purified LLO. It was downregulated following ΔinlAB infection indicating a new potent role for internalins in listerial pathogenicity and miRNA regulation. Concurrently, we observed differences in target transcript expression of the investigated miRNAs. We provide first evidence that L. monocytogenes infection leads to deregulation of a set of microRNAs with important roles in host response. Distinct microRNA expression depends on both LLO and pathogen localization. PMID:22312311

  18. West Nile virus encodes a microRNA-like small RNA in the 3′ untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells

    PubMed Central

    Hussain, Mazhar; Torres, Shessy; Schnettler, Esther; Funk, Anneke; Grundhoff, Adam; Pijlman, Gorben P.; Asgari, Sassan

    2012-01-01

    West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3′ untranslated region (3′-UTR) of the flavivirus genome, in particular the terminal 3′ stem–loop (3′SL) fulfils multiple functions in virus replication and virus–host interactions. Using the Kunjin strain of WNV (WNVKUN), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3′SL. Transcription of WNVKUN pre-miRNA (3′SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNVKUN virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication. PMID:22080551

  19. Global translation variations in host cells upon attack of lytic and sublytic Staphylococcus aureus α-haemolysin.

    PubMed

    Clamer, Massimiliano; Tebaldi, Toma; Marchioretto, Marta; Bernabò, Paola; Bertini, Efrem; Guella, Graziano; Dalla Serra, Mauro; Quattrone, Alessandro; Viero, Gabriella

    2015-11-15

    Genome-wide analyses of translation can provide major contributions in our understanding of the complex interplay between virulent factors and host cells. So far, the activation of host translational control mechanisms by bacterial toxins, owing to specific recruitment of mRNAs, RNA-binding proteins (RBPs) and ncRNAs (non-coding RNAs), are far from being understood. In the present study, we characterize for the first time the changes experienced by the translational control system of host cells in response to the well-known Staphylococcus aureus α-haemolysin (AHL) under both sublytic and lytic conditions. By comparing variations occurring in the cellular transcriptome and translatome, we give evidence that global gene expression is primarily rewired at the translational level, with the contribution of the RBP ELAVL1 (HuR) in the sublytic response. These results reveal the importance of translational control during host-pathogen interaction, opening new approaches for AHL-induced diseases. PMID:26371376

  20. Hairpin dsRNA does not trigger RNA interference in Candida albicans cells.

    PubMed

    Staab, Janet F; White, Theodore C; Marr, Kieren A

    2011-01-01

    RNA interference/silencing mechanisms triggered by double-stranded RNA (dsRNA) have been described in many eukaryotes, including fungi. These mechanisms have in common small RNA molecules (siRNAs or microRNAs) originating from dsRNAs that, together with the effector protein Argonaute, mediate silencing. The genome of the fungal pathogen Candida albicans harbours a well-conserved Argonaute and a non-canonical Dicer, essential members of silencing pathways. Prototypical siRNAs are detected as members of the C. albicans transcriptome, which is potential evidence of RNA interference/silencing pathways in this organism. Surprisingly, expression of a dsRNA a hairpin ADE2 dsRNA molecule to interfere with the endogenous ADE2 mRNA did not result in down-regulation of the message or produce adenine auxotrophic strains. Cell free assays showed that the hairpin dsRNA was a substrate for the putative C. albicans Dicer, discounting the possibility that the nature of the dsRNA trigger affects silencing functionality. Our results suggested that unknown cellular events govern the functionality of siRNAs originating from transgenes in RNA interference/silencing pathways in C. albicans. PMID:20737430

  1. RNA imaging in living cells – methods and applications

    PubMed Central

    Urbanek, Martyna O; Galka-Marciniak, Paulina; Olejniczak, Marta; Krzyzosiak, Wlodzimierz J

    2014-01-01

    Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications. PMID:25483044

  2. Host-Cell Survival and Death During Chlamydia Infection

    PubMed Central

    Ying, Songmin; Pettengill, Matthew; Ojcius, David M.; Häcker, Georg

    2008-01-01

    Different Chlamydia trachomatis strains are responsible for prevalent bacterial sexually-transmitted disease and represent the leading cause of preventable blindness worldwide. Factors that predispose individuals to disease and mechanisms by which chlamydiae cause inflammation and tissue damage remain unclear. Results from recent studies indicate that prolonged survival and subsequent death of infected cells and their effect on immune effector cells during chlamydial infection may be important in determining the outcome. Survival of infected cells is favored at early times of infection through inhibition of the mitochondrial pathway of apoptosis. Death at later times displays features of both apoptosis and necrosis, but pro-apoptotic caspases are not involved. Most studies on chlamydial modulation of host-cell death until now have been performed in cell lines. The consequences for pathogenesis and the immune response will require animal models of chlamydial infection, preferably mice with targeted deletions of genes that play a role in cell survival and death. PMID:18843378

  3. Host cell kinases and the hepatitis C virus life cycle.

    PubMed

    Colpitts, Che C; Lupberger, Joachim; Doerig, Christian; Baumert, Thomas F

    2015-10-01

    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. PMID:25896387

  4. Recombinant host cells and media for ethanol production

    DOEpatents

    Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W

    2014-02-18

    Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

  5. Dissection of tumour and host cells from target organs of metastasis for testing gene expression directly ex vivo.

    PubMed Central

    Rocha, M.; Hexel, K.; Bucur, M.; Schirrmacher, V.; Umansky, V.

    1996-01-01

    We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells (lymphocytes, macrophages, endothelial cells) from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity (adhesion, costimulatory and cytotoxic molecules, cytokines, etc.) at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy. Images Figure 3 PMID:8883407

  6. Subcellular localization of host and viral proteins associated with tobamovirus RNA replication.

    PubMed

    Hagiwara, Yuka; Komoda, Keisuke; Yamanaka, Takuya; Tamai, Atsushi; Meshi, Tetsuo; Funada, Ryo; Tsuchiya, Tomohiro; Naito, Satoshi; Ishikawa, Masayuki

    2003-01-15

    Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A. PMID:12514140

  7. Integrated miRNA and mRNA transcriptomes of porcine alveolar macrophages (PAM cells) identifies strain-specific miRNA molecular signatures associated with H-PRRSV and N-PRRSV infection.

    PubMed

    Cong, Peiqing; Xiao, Shuqi; Chen, Yaosheng; Wang, Liangliang; Gao, Jintao; Li, Ming; He, Zuyong; Guo, Yunxue; Zhao, Guangyin; Zhang, Xiaoyu; Chen, Luxi; Mo, Delin; Liu, Xiaohong

    2014-09-01

    Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant viral diseases in swine, which causes large economic losses to the swine industry worldwide. There is considerable strain variation in PRRSV and two examples of this are the highly virulent Chinese-type PRRSV (H-PRRSV) and the classical North American type PRRSV (N-PRRSV), both with different pathogenesis. These differences may be due in part to genetic and phenotypic differences in virus replication, but also interaction with the host cell. MicroRNAs (miRNAs) are crucial regulators of gene expression and play vital roles in virus and host interactions. However, the regulation role of miRNAs during PRRSV infection has not been systematically investigated. In order to better understand the differential regulation roles of cellular miRNAs in the host response to PRRSV, miRNA expression and a global mRNA transcriptome profile was determined in primary cells infected with either H-PRRSV or N-PRRSV as multiple time points during the viral lifecycle. miRNA-mRNA interactome networks were constructed by integrating the differentially expressed miRNAs and inversely correlated target mRNAs. Using gene ontology and pathway enrichment analyses, cellular pathways associated with deregulated miRNAs were identified, including immune response, phagosome, autophagy, lysosome, autolysis, apoptosis and cell cycle regulation. To our knowledge, this is the first global analysis of strain-specific host miRNA molecular signatures associated with H- and N-PRRSV infection by integrating miRNA and mRNA transcriptomes and provides a new perspective on the contribution of miRNAs to the pathogenesis of PRRSV infection. PMID:24962047

  8. RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins.

    PubMed

    Moissiard, Guillaume; Voinnet, Olivier

    2006-12-19

    RNA silencing is an ancient mechanism of gene regulation with important antiviral roles in plants and insects. Although induction of RNA silencing by RNA viruses has been well documented in plants, the interactions between DNA viruses and the host silencing machinery remain poorly understood. We investigate this question with cauliflower mosaic virus (CaMV), a dsDNA virus that expresses its genome through the polycistronic 35S RNA, which carries an unusually extensive secondary structure known as translational leader. We show that CaMV-derived siRNAs accumulate in turnip- and Arabidopsis-infected plants and that the leader is a major, albeit not exclusive, source for those molecules. Biogenesis of leader-derived siRNA requires the coordinated and hierarchical action of the four Arabidopsis Dicer-like (DCL) proteins. Our study also uncovers a "facilitating" role exerted by the microRNA biosynthetic enzyme DCL1 on accumulation of DCL2-, DCL3-, and DCL4-dependent siRNAs derived from the 35S leader. This feature of DCL1 defines a small RNA biosynthetic pathway that might have relevance for endogenous gene regulation. Several leader-derived siRNAs were found to bear near-perfect sequence complementarity to Arabidopsis transcripts, and, using a sensor transgene, we provide direct evidence that at least one of those molecules acts as a bona fide siRNA in infected turnip. Extensive bioinformatics searches identified >100 transcripts potentially targeted by CaMV-derived siRNAs, several of which are effectively down-regulated during infection. The implications of virus-directed silencing of host gene expression are discussed. PMID:17164336

  9. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  10. Infection Strategies of Intestinal Parasite Pathogens and Host Cell Responses.

    PubMed

    Di Genova, Bruno M; Tonelli, Renata R

    2016-01-01

    Giardia lamblia, Cryptosporidium sp., and Entamoeba histolytica are important pathogenic intestinal parasites and are amongst the leading causes worldwide of diarrheal illness in humans. Diseases caused by these organisms, giardiasis, cryptosporidiosis, and amoebiasis, respectively, are characterized by self-limited diarrhea but can evolve to long-term complications. The cellular and molecular mechanisms underlying the pathogenesis of diarrhea associated with these three pathogens are being unraveled, with knowledge of both the strategies explored by the parasites to establish infection and the methods evolved by hosts to avoid it. Special attention is being given to molecules participating in parasite-host interaction and in the mechanisms implicated in the diseases' pathophysiologic processes. This review focuses on cell mechanisms that are modulated during infection, including gene transcription, cytoskeleton rearrangements, signal transduction pathways, and cell death. PMID:26973630

  11. Uropathogenic Escherichia coli Epigenetically Manipulate Host Cell Death Pathways.

    PubMed

    Zhang, Zhengguo; Wang, Ming; Eisel, Florian; Tchatalbachev, Svetlin; Chakraborty, Trinad; Meinhardt, Andreas; Bhushan, Sudhanshu

    2016-04-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in human. It is well established that UPEC can subvert innate immune responses, but the role of UPEC in interfering with host cell death pathways is not known. Here, we show that UPEC abrogates activation of the host cell prosurvival protein kinase B signaling pathway, which results in the activation of mammalian forkhead box O (FOXO) transcription factors. Although FOXOs were localized in the nucleus and showed increased DNA-binding activity, no change in the expression levels of FOXO target genes were observed. UPEC can suppress BIM expression induced by LY249002, which results in attenuation of caspase 3 activation and blockage of apoptosis. Mechanistically, BIM expression appears to be epigenetically silenced by a decrease in histone 4 acetylation at the BIM promoter site. Taken together, these results suggest that UPEC can epigenetically silence BIM expression, a molecular switch that prevents apoptosis. PMID:26621912

  12. Infection Strategies of Intestinal Parasite Pathogens and Host Cell Responses

    PubMed Central

    Di Genova, Bruno M.; Tonelli, Renata R.

    2016-01-01

    Giardia lamblia, Cryptosporidium sp., and Entamoeba histolytica are important pathogenic intestinal parasites and are amongst the leading causes worldwide of diarrheal illness in humans. Diseases caused by these organisms, giardiasis, cryptosporidiosis, and amoebiasis, respectively, are characterized by self-limited diarrhea but can evolve to long-term complications. The cellular and molecular mechanisms underlying the pathogenesis of diarrhea associated with these three pathogens are being unraveled, with knowledge of both the strategies explored by the parasites to establish infection and the methods evolved by hosts to avoid it. Special attention is being given to molecules participating in parasite–host interaction and in the mechanisms implicated in the diseases’ pathophysiologic processes. This review focuses on cell mechanisms that are modulated during infection, including gene transcription, cytoskeleton rearrangements, signal transduction pathways, and cell death. PMID:26973630

  13. Chlamydia trachomatis Inclusion Disrupts Host Cell Cytokinesis to Enhance Its Growth in Multinuclear Cells.

    PubMed

    Sun, He Song; Sin, Alex T-W; Poirier, Mathieu B; Harrison, Rene E

    2016-01-01

    Chlamydia trachomatis, the leading cause of bacterial sexually transmitted infections, disrupts cytokinesis and causes significant multinucleation in host cells. Here, we demonstrate that multinuclear cells that result from unsuccessful cell division contain significantly higher Golgi content, an important source of lipids for chlamydiae. Using immunofluorescence and fluorescent live cell imaging, we show that C. trachomatis in multinuclear cells indeed intercept Golgi-derived lipid faster than in mononuclear cells. Moreover, multinuclear cells enhance C. trachomatis inclusion growth and infectious particle formation. Together, these results indicate that C. trachomatis robustly position inclusions to the cell equator to disrupt host cell division in order to acquire host Golgi-derived lipids more quickly in multinucleated progeny cells. PMID:26084267

  14. Baculovirus Infection Influences Host Protein Expression in Two Established Insect Cell Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus–host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive...

  15. Bacterial-induced cell reprogramming to stem cell-like cells: new premise in host-pathogen interactions

    PubMed Central

    Hess, Samuel; Rambukkana, Anura

    2015-01-01

    Bacterial pathogens employ a myriad of strategies to alter host tissue cell functions for bacterial advantage during infection. Recent advances revealed a fusion of infection biology with stem cell biology by demonstrating developmental reprogramming of lineage committed host glial cells to progenitor/stem cell-like cells by an intracellular bacterial pathogen Mycobacterium leprae. Acquisition of migratory and immunomodulatory properties of such reprogrammed cells provides an added advantage for promoting bacterial spread. This presents a previously unseen sophistication of cell manipulation by hijacking the genomic plasticity of host cells by a human bacterial pathogen. The rationale for such extreme fate conversion of host cells may be directly linked to the exceedingly passive obligate life style of M. leprae with a degraded genome and host cell dependence for both bacterial survival and dissemination, particularly the use of host-derived stem cell-like cells as a vehicle for spreading infection without being detected by immune cells. Thus, this unexpected link between cell reprogramming and infection opens up a new premise in host-pathogen interactions. Furthermore, such bacterial ingenuity could also be harnessed for developing natural ways of reprogramming host cells for repairing damaged tissues from infection, injury and diseases. PMID:25541240

  16. Comprehensive Identification of RNA-Binding Domains in Human Cells.

    PubMed

    Castello, Alfredo; Fischer, Bernd; Frese, Christian K; Horos, Rastislav; Alleaume, Anne-Marie; Foehr, Sophia; Curk, Tomaz; Krijgsveld, Jeroen; Hentze, Matthias W

    2016-08-18

    Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells. PMID:27453046

  17. Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

    PubMed

    Abernathy, Emma; Gilbertson, Sarah; Alla, Ravi; Glaunsinger, Britt

    2015-08-12

    Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, SOX, that cleaves most cellular mRNAs. Cleaved fragments are subsequently degraded by the cellular 5'-3' mRNA exonuclease Xrn1, thereby suppressing cellular gene expression and facilitating viral evasion of host defenses. We reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering RNA Polymerase II (RNAPII) transcription in the nucleus. Measuring RNAPII recruitment to promoters and nascent mRNA synthesis revealed that the majority of affected genes are transcriptionally repressed in SOX-expressing cells. The transcriptional feedback does not occur in response to the initial viral endonuclease-induced cleavage, but instead to degradation of the cleaved fragments by cellular exonucleases. In particular, Xrn1 catalytic activity is required for transcriptional repression. Notably, viral mRNA transcription escapes decay-induced repression, and this escape requires Xrn1. Collectively, these results indicate that mRNA decay rates impact transcription and that gamma-herpesviruses use this feedback mechanism to facilitate viral gene expression. PMID:26211836

  18. A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements

    PubMed Central

    2016-01-01

    Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs) silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1) the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2) a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations. PMID:26681955

  19. A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements.

    PubMed

    Haase, Astrid D

    2016-01-01

    Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs) silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1) the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2) a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations. PMID:26681955

  20. B7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality.

    PubMed

    Veenstra, Rachelle G; Flynn, Ryan; Kreymborg, Katharina; McDonald-Hyman, Cameron; Saha, Asim; Taylor, Patricia A; Osborn, Mark J; Panoskaltsis-Mortari, Angela; Schmitt-Graeff, Annette; Lieberknect, Elisabeth; Murphy, William J; Serody, Jonathan S; Munn, David H; Freeman, Gordon J; Allison, James P; Mak, Tak W; van den Brink, Marcel; Zeiser, Robert; Blazar, Bruce R

    2015-05-21

    Members of the B7 family have been shown to be important for regulating immune responses by providing either positive or negative costimulatory signals. The function of B7-H3 has been controversial. We show that B7-H3 is upregulated in graft-versus-host disease (GVHD) target organs, including the colon, liver, and lung. Infusion of allogeneic donor T cells into B7-H3(-/-) vs wild-type (WT) recipients resulted in increased GVHD lethality associated with increased T-cell proliferation, colonic inflammatory cytokines, and destruction of epithelial barriers. Allogeneic B7-H3(-/-) vs WT donor T cells also had increased T-cell proliferation and GVHD lethality associated with increased proliferation and cytokine secretion in the spleen, intraepithelial lymphocyte inflammatory cytokines, and intestinal permeability. Both resting and activated regulatory T cells (Tregs) lack B7-H3 messenger RNA. Consistent with these data, GVHD was augmented in recipients of B7-H3(-/-) Treg-depleted grafts. In two delayed lymphocyte infusion (DLI) models, T cells lacking B7-H3 are capable of providing graft-versus-leukemia (GVL) effects. We conclude that B7-H3 is responsible for providing a negative costimulatory signal. Our studies provide support for developing and testing new therapies directed toward the B7-H3 pathway, including approaches to augment host B7-H3 early after bone marrow transplantation to prevent GVHD and to develop potent antagonistic antibodies later after transplant to facilitate DLI-mediated GVL without GVHD complications. PMID:25814530

  1. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection.

    PubMed

    Radoshitzky, Sheli R; Pegoraro, Gianluca; Chī, Xi Olì; D Ng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C; Cooper, Christopher L; Courier, Duane; Langan, David P; Underwood, Knashka; Kuehl, Kathleen A; Sun, Mei G; Caì, Yíngyún; Yú, Shu Qìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H; Bavari, Sina

    2016-03-01

    Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling. PMID:27031835

  2. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

    PubMed Central

    Radoshitzky, Sheli R.; Pegoraro, Gianluca; Chī, Xiǎolì; Dǒng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C.; Cooper, Christopher L.; Courier, Duane; Langan, David P.; Underwood, Knashka; Kuehl, Kathleen A.; Sun, Mei G.; Caì, Yíngyún; Yú, Shuǐqìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H.; Bavari, Sina

    2016-01-01

    Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1–PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling. PMID:27031835

  3. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

    PubMed Central

    Li, Wei; Liu, Liangyi; Gomez, Aurelie; Zhang, Jilu; Ramadan, Abdulraouf; Zhang, Qing; Choi, Sung W.; Zhang, Peng; Greenson, Joel K.; Liu, Chen; Jiang, Di; Virts, Elizabeth; Kelich, Stephanie L.; Chu, Hong Wei; Flynn, Ryan; Blazar, Bruce R.; Hanenberg, Helmut; Hanash, Samir; Paczesny, Sophie

    2016-01-01

    Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA– transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT. PMID:27195312

  4. MicroRNA-26a/b and their host genes synergistically regulate triacylglycerol synthesis by targeting the INSIG1 gene.

    PubMed

    Wang, Hui; Luo, Jun; Zhang, Tianying; Tian, Huibin; Ma, Yue; Xu, Huifen; Yao, Dawei; Loor, Juan J

    2016-05-01

    The microRNA-26 (miR-26) family is known to control adipogenesis in non-ruminants. The genomic loci of miR-26a and miR-26b have been localized in the introns of genes encoding for the proteins of the C-terminal domain RNA polymerase II polypeptide A small phosphatase (CTDSP) family. Insulin-induced gene 1 (INSIG1) encodes a protein with a key role in the regulation of lipogenesis in rodent liver. In the present study, we investigated the synergistic function of the miR-26 family and their host genes in goat mammary epithelial cells (GMEC). Downregulation of miR-26a/b and their host genes in GMEC decreased the expression of genes relate to fatty acid synthesis (PPARG, LXRA, SREBF1, FASN, ACACA, GPAM, LPIN1, DGAT1 and SCD1), triacylglycerol accumulation and unsaturated fatty acid synthesis. Luciferase reporter assays confirmed INSIG1 as a direct target of miR-26a/b. Furthermore, inhibition of the CTDSP family also downregulated the expression of INSIG1. Taken together, our findings highlight a functional association of miR-26a/b, their host genes and INSIG1, and provide new insights into the regulatory network controlling milk fat synthesis in GMEC. The data indicate that targeting this network via nutrition might be important for regulating milk fat synthesis in ruminants. PMID:27002347

  5. Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

    PubMed

    Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

    2016-09-01

    Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. PMID:27235586

  6. Cas9-mediated targeting of viral RNA in eukaryotic cells

    PubMed Central

    Price, Aryn A.; Sampson, Timothy R.; Ratner, Hannah K.; Grakoui, Arash; Weiss, David S.

    2015-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense. PMID:25918406

  7. Toward Rare Blood Cell Preservation for RNA Sequencing.

    PubMed

    Vickovic, Sanja; Ahmadian, Afshin; Lewensohn, Rolf; Lundeberg, Joakim

    2015-07-01

    Cancer is driven by various events leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues-a prerequisite for successful cell profiling. Although advances in RNA preservation have been made, some of the largest biobanks still do not employ RNA blood preservation as standard because of limitations in low blood-input volume and RNA stability over the whole gene body. Therefore, we have developed a robust protocol for blood preservation and long-term storage while maintaining RNA integrity. Furthermore, we explored the possibility of using the protocol for preserving rare cell samples, such as circulating tumor cells. The results of our study confirmed that gene expression was not impacted by the preservation procedure (r(2) > 0.88) or by long-term storage (r(2) = 0.95), with RNA integrity number values averaging over 8. Similarly, cell surface antigens were still available for antibody selection (r(2) = 0.95). Lastly, data mining for fusion events showed that it was possible to detect rare tumor cells among a background of other cells present in blood irrespective of fixation. Thus, the developed protocol would be suitable for rare blood cell preservation followed by RNA sequencing analysis. PMID:25989392

  8. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    SciTech Connect

    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna; Yang, Hanchun; Hu, Hongbo

    2012-08-01

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/{beta}-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome-lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  9. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    PubMed Central

    Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

    2015-01-01

    Summary Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. PMID:26748716

  10. An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

    PubMed Central

    Schreiber, Claire A.; Sakuma, Toshie; Izumiya, Yoshihiro; Holditch, Sara J.; Hickey, Raymond D.; Bressin, Robert K.; Basu, Upamanyu; Koide, Kazunori; Asokan, Aravind; Ikeda, Yasuhiro

    2015-01-01

    Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors. PMID:26244496

  11. B Cells in Chronic Graft versus Host Disease

    PubMed Central

    Sarantopoulos, Stefanie; Blazar, Bruce R.; Cutler, Corey; Ritz, Jerome

    2015-01-01

    Chronic graft versus host disease (cGVHD) continues to be a common complication of allogeneic hematopoietic stem cell transplantation (HSCT). Unlike acute GVHD, which is mediated almost entirely by donor T cells, the immune pathology of cGVHD is more complex and donor B cells have also been found to play an important role. Recent studies from several laboratories have enhanced our understanding of how donor B cells contribute to this clinical syndrome and this has led to new therapeutic opportunities. Here, Dr. Sarantopoulos reviews some of the important mechanisms responsible for persistent B cell activation and loss of B cell tolerance in patients with cGVHD. Dr. Blazar describes recent studies in preclinical models that have identified novel B cell directed agents that may be effective for prevention or treatment of cGVHD. Some B cell directed therapies have already been tested in patients with cGVHD and Dr. Cutler reviews the results of these studies documenting the potential efficacy of this approach. Supported by studies mechanistic studies in patients and preclinical models, new B cell directed therapies for cGVHD will now be evaluated in clinical trials. PMID:25452031

  12. Strain-specific differences in pili formation and the interaction of Corynebacterium diphtheriae with host cells

    PubMed Central

    2010-01-01

    Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates. Results Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. Conclusions The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains. PMID:20942914

  13. Ureaplasma parvum infection alters filamin a dynamics in host cells

    PubMed Central

    2011-01-01

    Background Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection. Methods We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI), and complicated UTI. One protein that was perturbed by infection (filamin A) was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1). BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA. Results Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A) that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P < 0.004; ANOVA, P < 0.02). This phenomenon was independent of clinical profile (asymptomatic vs. complicated UTI). We selected filamin A as a target for additional studies. In the BPH-1 model, we confirmed that U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine2152 (P ≤ 0.01), which correlated with impaired proteolysis of the protein and its normal intracellular distribution. Conclusion Filamin A dynamics were perturbed in both models of infection

  14. Umbilical Cord Mesenchymal Stem Cells Suppress Host Rejection

    PubMed Central

    Coulson-Thomas, Vivien Jane; Gesteira, Tarsis Ferreira; Hascall, Vincent; Kao, Winston

    2014-01-01

    Umbilical cord mesenchymal stem cells (UMSCs) have unique immunosuppressive properties enabling them to evade host rejection and making them valuable tools for cell therapy. We previously showed that human UMSCs survive xenograft transplantation and successfully correct the corneal clouding defects associated with the mouse model for the congenital metabolic disorder mucopolysaccharidosis VII. However, the precise mechanism by which UMSCs suppress the immune system remains elusive. This study aimed to determine the key components involved in the ability of the UMSCs to modulate the inflammatory system and to identify the inflammatory cells that are regulated by the UMSCs. Our results show that human UMSCs transplanted into the mouse stroma 24 h after an alkali burn suppress the severe inflammatory response and enable the recovery of corneal transparency within 2 weeks. Furthermore, we demonstrated in vitro that UMSCs inhibit the adhesion and invasion of inflammatory cells and also the polarization of M1 macrophages. UMSCs also induced the maturation of T-regulatory cells and led to inflammatory cell death. Moreover, UMSCs exposed to inflammatory cells synthesize a rich extracellular glycocalyx composed of the chondroitin sulfate-proteoglycan versican bound to a heavy chain (HC)-modified hyaluronan (HA) matrix (HC-HA). This matrix also contains TNFα-stimulated gene 6 (TSG6), the enzyme that transfers HCs to HA, and pentraxin-3, which further stabilizes the matrix. Our results, both in vivo and in vitro, show that this glycocalyx confers the ability for UMSCs to survive the host immune system and to regulate the inflammatory cells. PMID:24986866

  15. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infection by a variety of viruses alters the nuclear-cytoplasmic trafficking of certain host cell proteins. In our continued search for interacting factors, we reported the re-localization of RNA helicase A (RHA) from the nucleus to the cytoplasm in cells infected with foot-and-mouth disease virus ...

  16. Effect of Host Species on the Distribution of Mutational Fitness Effects for an RNA Virus

    PubMed Central

    Lalić, Jasna; Cuevas, José M.; Elena, Santiago F.

    2011-01-01

    Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multi-host parasites and thus for the emergence of new pathogens. Here we characterize the DMFE for a collection of twenty single-nucleotide substitution mutants of Tobacco etch potyvirus (TEV) across a set of eight host environments. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas the other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail. Within the Solanaceae hosts, the distribution contained an excess of deleterious mutations, whereas for the non-Solanaceae the fraction of beneficial mutations was significantly larger. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host. PMID:22125497

  17. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  18. Enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication

    PubMed Central

    Ilnytska, Olha; Santiana, Marianita; Hsu, Nai-Yun; Du, Wen-Li; Chen, Ying-Han; Viktorova, Ekaterina G.; Belov, Georgy; Brinker, Anita; Storch, Judith; Moore, Christopher; Dixon, Joseph L.; Altan-Bonnet, Nihal

    2013-01-01

    Cholesterol is a critical component of cellular membranes, regulating assembly and function of membrane-based protein/lipid complexes. Many RNA viruses, including enteroviruses, remodel host membranes to generate organelles with unique lipid blueprints on which they assemble replication complexes and synthesize viral RNA. Here we find that clathrin-mediated endocytosis (CME) is harnessed by enteroviruses to traffic cholesterol from the plasma membrane (PM) and extracellular medium to replication organelles where cholesterol then regulates viral polyprotein processing and facilitates genome synthesis. When CME is disrupted, cellular cholesterol pools are instead stored in lipid droplets; cholesterol cannot be trafficked to replication organelles; and replication is inhibited. In contrast, replication is stimulated in cholesterol-elevated cells like those lacking caveolins or those from Niemann-Pick disease patients. Our findings indicate cholesterol as a critical determinant for enteroviral replication and outline roles for the endocytic machinery in both the enteroviral lifecycle and host cell cholesterol homeostasis. PMID:24034614

  19. Next-Generation "-omics" Approaches Reveal a Massive Alteration of Host RNA Metabolism during Bacteriophage Infection of Pseudomonas aeruginosa.

    PubMed

    Chevallereau, Anne; Blasdel, Bob G; De Smet, Jeroen; Monot, Marc; Zimmermann, Michael; Kogadeeva, Maria; Sauer, Uwe; Jorth, Peter; Whiteley, Marvin; Debarbieux, Laurent; Lavigne, Rob

    2016-07-01

    As interest in the therapeutic and biotechnological potentials of bacteriophages has grown, so has value in understanding their basic biology. However, detailed knowledge of infection cycles has been limited to a small number of model bacteriophages, mostly infecting Escherichia coli. We present here the first analysis coupling data obtained from global next-generation approaches, RNA-Sequencing and metabolomics, to characterize interactions between the virulent bacteriophage PAK_P3 and its host Pseudomonas aeruginosa. We detected a dramatic global depletion of bacterial transcripts coupled with their replacement by viral RNAs over the course of infection, eventually leading to drastic changes in pyrimidine metabolism. This process relies on host machinery hijacking as suggested by the strong up-regulation of one bacterial operon involved in RNA processing. Moreover, we found that RNA-based regulation plays a central role in PAK_P3 lifecycle as antisense transcripts are produced mainly during the early stage of infection and viral small non coding RNAs are massively expressed at the end of infection. This work highlights the prominent role of RNA metabolism in the infection strategy of a bacteriophage belonging to a new characterized sub-family of viruses with promising therapeutic potential. PMID:27380413

  20. To translate, or not to translate: viral and host mRNA regulation by interferon-stimulated genes.

    PubMed

    Li, Melody M H; MacDonald, Margaret R; Rice, Charles M

    2015-06-01

    Type I interferon (IFN) is one of the first lines of cellular defense against viral pathogens. As a result of IFN signaling, a wide array of IFN-stimulated gene (ISG) products is upregulated to target different stages of the viral life cycle. We review recent findings implicating a subset of ISGs in translational regulation of viral and host mRNAs. Translation inhibition is mediated either by binding to viral RNA or by disrupting physiological interactions or levels of the translation complex components. In addition, many of these ISGs localize to translationally silent cytoplasmic granules, such as stress granules and processing bodies, and intersect with the microRNA (miRNA)-mediated silencing pathway to regulate translation of cellular mRNAs. PMID:25748385

  1. Visualization of RNA-Quadruplexes in Live Cells.

    PubMed

    Laguerre, Aurélien; Hukezalie, Kyle; Winckler, Pascale; Katranji, Fares; Chanteloup, Gaëtan; Pirrotta, Marc; Perrier-Cornet, Jean-Marie; Wong, Judy M Y; Monchaud, David

    2015-07-01

    Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells. PMID:26056849

  2. Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

    PubMed Central

    Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

    2016-01-01

    Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle. PMID:27180572

  3. Antisense RNA: Function and Fate of Duplex RNA in Cells of Higher Eukaryotes

    PubMed Central

    Kumar, Madhur; Carmichael, Gordon G.

    1998-01-01

    There is ample evidence that cells of higher eukaryotes express double-stranded RNA molecules (dsRNAs) either naturally or as the result of viral infection or aberrant, bidirectional transcriptional readthrough. These duplex molecules can exist in either the cytoplasmic or nuclear compartments. Cells have evolved distinct ways of responding to dsRNAs, depending on the nature and location of the duplexes. Since dsRNA molecules are not thought to exist naturally within the cytoplasm, dsRNA in this compartment is most often associated with viral infections. Cells have evolved defensive strategies against such molecules, primarily involving the interferon response pathway. Nuclear dsRNA, however, does not induce interferons and may play an important posttranscriptional regulatory role. Nuclear dsRNA appears to be the substrate for enzymes which deaminate adenosine residues to inosine residues within the polynucleotide structure, resulting in partial or full unwinding. Extensively modified RNAs are either rapidly degraded or retained within the nucleus, whereas transcripts with few modifications may be transported to the cytoplasm, where they serve to produce altered proteins. This review summarizes our current knowledge about the function and fate of dsRNA in cells of higher eukaryotes and its potential manipulation as a research and therapeutic tool. PMID:9841677

  4. Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

    PubMed Central

    Vilchez Larrea, Salomé C.; Schlesinger, Mariana; Kevorkian, María L.; Flawiá, Mirtha M.; Alonso, Guillermo D.; Fernández Villamil, Silvia H.

    2013-01-01

    Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease. PMID:23776710

  5. Influenza virus binds its host cell using multiple dynamic interactions

    PubMed Central

    Sieben, Christian; Kappel, Christian; Zhu, Rong; Wozniak, Anna; Rankl, Christian; Hinterdorfer, Peter; Grubmüller, Helmut; Herrmann, Andreas

    2012-01-01

    Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609–9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus–cell binding quantitatively at the molecular level. PMID:22869709

  6. Small Molecule-Mediated Cleavage of RNA in Living Cells

    PubMed Central

    Guan, Lirui

    2013-01-01

    Antisense oligonucleotides and small interfering RNAs (siRNAs) control gene expression by triggering the degradation of a mRNA via recruitment of RNase H or the RNA-induced silencing complex (RISC), respectively.[1] These approaches are hampered, however, by the poor cellular permeability of oligonucleotides. A small molecule approach to cleave RNA targets could obviate uptake issues. Several compounds can induce RNA cleavage in vitro,[2] however, to the best of our knowledge no small molecules have been previously described to cleave RNA in living cells. Herein, we describe the development of a potentially general approach to design small molecules that specifically cleave an RNA in a living cell, affecting biological function. Specifically, a designed, modularly assembled small molecule that binds the RNA that causes myotonic dystrophy type 1 (DM1)[3] was appended with a moiety that generates hydroxyl radicals upon irradiation. Cleavage of the transcript improves DM1-associated defects in cell culture, and compounds are non-toxic at an efficacious dose as determined by a MTT viability assay. This approach may allow for the site-specific cleavage and inactivation of other cellular RNAs.[4] Compounds that bind to and cleave RNA have the potential to serve as chemical genetics probes of function or lead therapeutics with spatial and temporal control. PMID:23280953

  7. PICSAR: Long Noncoding RNA in Cutaneous Squamous Cell Carcinoma.

    PubMed

    Luo, Yunhai; Morgan, Stefanie L; Wang, Kevin C

    2016-08-01

    It is increasingly evident that long noncoding RNAs may play the roles of both oncogenes and tumor suppressors during cancer development. A new study from Piipponen et al. provides evidence that a long noncoding RNA, PICSAR, promotes cutaneous squamous cell carcinoma development through activation of extracellular signal-regulated kinase signaling. Because specific inhibition of PICSAR suppresses tumor growth, this long noncoding RNA may serve as a useful diagnostic marker and therapeutic target for cutaneous squamous cell carcinoma. PMID:27450499

  8. RNA Polymerase II cluster dynamics predict mRNA output in living cells

    PubMed Central

    Cho, Won-Ki; Jayanth, Namrata; English, Brian P; Inoue, Takuma; Andrews, J Owen; Conway, William; Grimm, Jonathan B; Spille, Jan-Hendrik; Lavis, Luke D; Lionnet, Timothée; Cisse, Ibrahim I

    2016-01-01

    Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output. DOI: http://dx.doi.org/10.7554/eLife.13617.001 PMID:27138339

  9. Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos.

    PubMed

    Hong, Ni; Li, Mingyou; Zeng, Zhiqiang; Yi, Meisheng; Deng, Jiaorong; Gui, Jianfang; Winkler, Christoph; Schartl, Manfred; Hong, Yunhan

    2010-04-01

    Chimera formation is a powerful tool for analyzing pluripotency in vivo. It has been widely accepted that host cell lineages are generally accessible to embryonic stem (ES) cells with the actual contribution depending solely on the intrinsic pluripotency of transplanted donor cells. Here, we show in the fish medaka (Oryzias latipes) that the host accessibility to ES cell contribution exhibits dramatic differences. Specifically, of three albino host strains tested (i (1) , i (3) and af), only strain i (1) generated pigmented chimeras. Strikingly, this accessibility is completely lost in i (1) but acquired in i (3) after host gamma-irradiation. Host irradiation also differentially affected ES cell contribution to somatic organs and gonad. Therefore, the accessibility of various host cell lineages can vary considerably depending on host strains and cell lineages as well as on irradiation. Our findings underscore the importance of host genotypes for interpreting donor cell pluripotency and for improving ES-derived chimera production. PMID:20238480

  10. Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

    PubMed Central

    2010-01-01

    Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections. PMID:20663125

  11. Vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo.

    PubMed Central

    Black, B L; Lyles, D S

    1992-01-01

    Infection by vesicular stomatitis virus (VSV) results in a rapid inhibition of host cell transcription and translation. To determine whether the viral matrix (M) protein was involved in this inhibition of host cell gene expression, an M protein expression vector was cotransfected with a target gene vector, encoding the target gene, encoding chloramphenicol acetyltransferase (CAT). Expression of M protein caused a decrease in CAT activity in a gene dosage-dependent manner, and inhibition was apparent by 12 h posttransfection. The inhibitory effect of M protein was quite potent. The level of M protein required for a 10-fold inhibition of CAT activity was less than 1% of the level of M protein produced during the sixth hour of VSV infection. Northern (RNA) analysis of cotransfected cells showed that expression of M protein caused a reduction in the steady-state level of the vector-encoded mRNAs. Expression of both CAT and M mRNAs was reduced in cells cotransfected with a plasmid encoding M protein, indicating that expression of small amounts of M protein from plasmid DNA inhibits further expression of both M and CAT mRNAs. Nuclear runoff transcription analysis demonstrated that expression of M protein inhibited transcription of the target genes. This is the first report of a viral gene product which is capable of inhibiting transcription in vivo in the absence of any other viral component. Images PMID:1318397

  12. Bordetella pertussis adenylate cyclase inactivation by the host cell.

    PubMed Central

    Gilboa-Ron, A; Rogel, A; Hanski, E

    1989-01-01

    Bordetella pertussis produces a calmodulin-dependent adenylate cyclase (AC) which acts as a toxin capable of penetrating eukaryotic cells and generating high levels of intracellular cyclic AMP. Transfer of target cells into B. pertussis AC-free medium leads to a rapid decay in the intracellular AC activity, implying that the invasive enzyme is unstable in the host cytoplasm. We report here that treatment of human lymphocytes with a glycolysis inhibitor and an uncoupler of oxidative phosphorylation completely blocked the intracellular inactivation of B. pertussis AC. Lymphocyte lysates inactivated all forms of B. pertussis AC in the presence of exogenous ATP. This inactivation was associated with degradation of an 125I-labelled 200 kDa form of B. pertussis AC. It appears that ATP is required for the proteolytic pathway, but not as an energy source, since non-hydrolysable ATP analogues supported inactivation and complete degradation of the enzyme. The possibility that binding of ATP to B. pertussis AC renders it susceptible to degradation by the host cell protease is discussed. Images Fig. 2. Fig. 4. PMID:2554887

  13. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells.

    PubMed

    Lichinchi, Gianluigi; Gao, Shang; Saletore, Yogesh; Gonzalez, Gwendolyn Michelle; Bansal, Vikas; Wang, Yinsheng; Mason, Christopher E; Rana, Tariq M

    2016-01-01

    N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m(6)A in the immune system or its role in host-pathogen interactions. Here, we investigate the topology, dynamics and bidirectional influences of the viral-host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m(6)A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m(6)A for viral replication was demonstrated by silencing of the m(6)A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev response element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system. PMID:27572442

  14. Single-cell RNA-seq: advances and future challenges

    PubMed Central

    Saliba, Antoine-Emmanuel; Westermann, Alexander J.; Gorski, Stanislaw A.; Vogel, Jörg

    2014-01-01

    Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here—each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field. PMID:25053837

  15. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

    PubMed Central

    Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

    2015-01-01

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

  16. Two interferon-independent double-stranded RNA-induced host defense strategies suppress the common cold virus at warm temperature.

    PubMed

    Foxman, Ellen F; Storer, James A; Vanaja, Kiran; Levchenko, Andre; Iwasaki, Akiko

    2016-07-26

    Most strains of rhinovirus (RV), the common cold virus, replicate better at cool temperatures found in the nasal cavity (33-35 °C) than at lung temperature (37 °C). Recent studies found that although 37 °C temperature suppressed RV growth largely by engaging the type 1 IFN response in infected epithelial cells, a significant temperature dependence to viral replication remained in cells devoid of IFN induction or signaling. To gain insight into IFN-independent mechanisms limiting RV replication at 37 °C, we studied RV infection in human bronchial epithelial cells and H1-HeLa cells. During the single replication cycle, RV exhibited temperature-dependent replication in both cell types in the absence of IFN induction. At 37 °C, earlier signs of apoptosis in RV-infected cells were accompanied by reduced virus production. Furthermore, apoptosis of epithelial cells was enhanced at 37 °C in response to diverse stimuli. Dynamic mathematical modeling and B cell lymphoma 2 (BCL2) overexpression revealed that temperature-dependent host cell death could partially account for the temperature-dependent growth observed during RV amplification, but also suggested additional mechanisms of virus control. In search of a redundant antiviral pathway, we identified a role for the RNA-degrading enzyme RNAseL. Simultaneous antagonism of apoptosis and RNAseL increased viral replication and dramatically reduced temperature dependence. These findings reveal two IFN-independent mechanisms active in innate defense against RV, and demonstrate that even in the absence of IFNs, temperature-dependent RV amplification is largely a result of host cell antiviral restriction mechanisms operating more effectively at 37 °C than at 33 °C. PMID:27402752

  17. Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus.

    PubMed

    Sehgal, O P; Sinha, R C; Gellatly, D L; Ivanov, I; AbouHaidar, M G

    1993-04-01

    Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species. PMID:7682254

  18. Shrinkage of Genome Size in a Plant RNA Virus upon Transfer of an Essential Viral Gene into the Host Genome

    PubMed Central

    Tromas, Nicolas; Zwart, Mark P.; Forment, Javier; Elena, Santiago F.

    2014-01-01

    Nonretroviral integrated RNA viruses (NIRVs) are genes of nonretroviral RNA viruses found in the genomes of many eukaryotic organisms. NIRVs are thought to sometimes confer virus resistance, meaning that they could impact spread of the virus in the host population. However, a NIRV that is expressed may also impact the evolution of virus populations within host organisms. Here, we experimentally addressed the evolution of a virus in a host expressing a NIRV using Tobacco etch virus (TEV), a plant RNA virus, and transgenic tobacco plants expressing its replicase, NIb. We found that a virus missing the NIb gene, TEV-ΔNIb, which is incapable of autonomous replication in wild-type plants, had a higher fitness than the full-length TEV in the transgenic plants. Moreover, when the full-length TEV was evolved by serial passages in transgenic plants, we observed genomic deletions within NIb—and in some cases the adjacent cistrons—starting from the first passage. When we passaged TEV and TEV-ΔNIb in transgenic plants, we found mutations in proteolytic sites, but these only occurred in TEV-ΔNIb lineages, suggesting the adaptation of polyprotein processing to altered NIb expression. These results raise the possibility that NIRV expression can indeed induce the deletion of the corresponding genes in the viral genome, resulting in the formation of viruses that are replication defective in hosts that do not express the same NIRV. Moreover, virus genome evolution was contingent upon the deletion of the viral replicase, suggesting NIRV expression could also alter patterns of virus evolution. PMID:24558257

  19. Post-Transcriptional Control of LINE-1 Retrotransposition by Cellular Host Factors in Somatic Cells

    PubMed Central

    Pizarro, Javier G.; Cristofari, Gaël

    2016-01-01

    Long INterspersed Element-1 (LINE-1 or L1) retrotransposons form the only autonomously active family of transposable elements in humans. They are expressed and mobile in the germline, in embryonic stem cells and in the early embryo, but are silenced in most somatic tissues. Consistently, they play an important role in individual genome variations through insertional mutagenesis and sequence transduction, which occasionally lead to novel genetic diseases. In addition, they are reactivated in nearly half of the human epithelial cancers, contributing to tumor genome dynamics. The L1 element codes for two proteins, ORF1p and ORF2p, which are essential for its mobility. ORF1p is an RNA-binding protein with nucleic acid chaperone activity and ORF2p possesses endonuclease and reverse transcriptase activities. These proteins and the L1 RNA assemble into a ribonucleoprotein particle (L1 RNP), considered as the core of the retrotransposition machinery. The L1 RNP mediates the synthesis of new L1 copies upon cleavage of the target DNA and reverse transcription of the L1 RNA at the target site. The L1 element takes benefit of cellular host factors to complete its life cycle, however several cellular pathways also limit the cellular accumulation of L1 RNPs and their deleterious activities. Here, we review the known cellular host factors and pathways that regulate positively or negatively L1 retrotransposition at post-transcriptional level, in particular by interacting with the L1 machinery or L1 replication intermediates; and how they contribute to control L1 activity in somatic cells. PMID:27014690

  20. A deeply conserved, noncanonical miRNA hosted by ribosomal DNA

    PubMed Central

    Chak, Li-Ling; Mohammed, Jaaved; Lai, Eric C.; Tucker-Kellogg, Greg

    2015-01-01

    Advances in small RNA sequencing technologies and comparative genomics have fueled comprehensive microRNA (miRNA) gene annotations in humans and model organisms. Although new miRNAs continue to be discovered in recent years, these have universally been lowly expressed, recently evolved, and of debatable endogenous activity, leading to the general assumption that virtually all biologically important miRNAs have been identified. Here, we analyzed small RNAs that emanate from the highly repetitive rDNA arrays of Drosophila. In addition to endo-siRNAs derived from sense and antisense strands of the pre-rRNA sequence, we unexpectedly identified a novel, deeply conserved, noncanonical miRNA. Although this miRNA is widely expressed, this miRNA was not identified by previous studies due to bioinformatics filters removing such repetitive sequences. Deep-sequencing data provide clear evidence for specific processing with precisely defined 5′ and 3′ ends. Furthermore, we demonstrate that the mature miRNA species is incorporated in the effector complexes and has detectable trans regulatory activity. Processing of this miRNA requires Dicer-1, whereas the Drosha–Pasha complex is dispensable. The miRNA hairpin sequence is located in the internal transcribed spacer 1 region of rDNA and is highly conserved among Dipteran species that were separated from their common ancestor ∼100 million years ago. Our results suggest that biologically active miRNA genes may remain unidentified even in well-studied organisms. PMID:25605965

  1. Impaired CD98 signaling protects against graft-versus-host disease by increasing regulatory T cells.

    PubMed

    Nishio, Yoshiaki; Fujino, Masayuki; Cai, Songjie; Kitajima, Yuya; Saito, Taro; Tsumura, Hideki; Ito, Morihiro; Ito, Yasuhiko; Nagahara, Yukitoshi; Li, Xiao-Kang

    2016-03-01

    Graft-versus-host disease (GvHD) is a major barrier to the broader use of allogenic hematopoietic stem cell transplantation for non-malignant clinical applications. A murine model of C57BL/6 to B6D2F1 acute GvHD was employed with T lymphocytes harboring a deletion of the CD98 heavy chain (CD98hc(-/-)) as donor cells. The CD98hc(-/-) resulted in lower responses to alloantigen stimulation in a mixed leukocyte reaction assay, and prevented the mortality associated with disease progression. The percentage of donor CD8 T lymphocytes was significantly decreased, while the percentage of Foxp3-positive regulatory T cells (Tregs) in recipients was increased by CD98hc(-/-). Decreased expression of FAS, FASL, ICOS, ICOSL, PD-1 and PD-L1 by donor CD8 T cells, and mRNA expression of cytotoxic T cell-related cytokines in the recipients were shown in those with CD98hc(-/-). Fewer infiltrated cells are found in the lungs, liver, tongue and skin of recipients with CD98hc(-/-) compared with the wild type recipients. Taken together, our data indicate that T cell-specific deletion of CD98hc can contribute to the prevention of GvHD development due to the attenuation of lymphocyte migration and by increasing the generation of Treg cells. These findings are expected to make it possible to develop novel approaches for the prevention of GvHD. PMID:26836475

  2. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  3. The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

    PubMed

    Bannister, L H

    1979-04-11

    Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the

  4. Langerhans' cells are depleted in chronic graft versus host disease.

    PubMed Central

    Aractingi, S; Gluckman, E; Dauge-Geffroy, M C; Le Goué, C; Flahaut, A; Dubertret, L; Carosella, E

    1997-01-01

    AIMS: To measure Langerhans' cells in skin of patients treated by bone marrow transplantation who developed chronic graft versus host disease (GvHD); to determine whether the reduction in Langerhans' cells resulted directly from the GvHD or from other factors, such as the immunosuppressive regimens used in bone marrow transplant patients. PATIENTS AND METHODS: Lesional and nonlesional skin specimens from nine patients with lichen planus-like lesions and three patients with sclerodermoid lesions were studied. Control skin specimens were taken from three patients undergoing breast reduction surgery. The number of Langerhans' cells/mm2 and the area of Langerhans' cells as a percentage of total epidermis were measured by counting cells labelled with antihuman CD1a. RESULTS: A significant reduction in Langerhans' cell area and number were found in specimens with lesions (area 3.5%; number 507/mm2) compared with specimens without lesions (8.42%; 2375/mm2). In contrast, Langerhans' cell area and number in skin without lesions were similar to controls (10.26%; 2968/mm2). CONCLUSIONS: Langerhans' cells were significantly reduced in skin with lesions of chronic GvHD but not in skin without lesions from the same patient, suggesting that the reduction is a direct consequence of GvHD and not linked to immunosuppressive drugs or late effects of conditioning regimens. In long term bone marrow transplant recipients, Langerhans' cells are derived mainly from the donor cells; therefore, this result suggests the occurrence of autoreactive phenomenon in chronic GvHD. Images PMID:9215146

  5. Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host

    PubMed Central

    Lioy, Virginia S.; Goussard, Sylvie; Guerineau, Vincent; Yoon, Eun-Jeong; Courvalin, Patrice; Galimand, Marc; Grillot-Courvalin, Catherine

    2014-01-01

    In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution—ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness. PMID:24398977

  6. Immune Monitoring Using mRNA-Transfected Dendritic Cells.

    PubMed

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by mRNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA. PMID:27236804

  7. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

    PubMed Central

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros) of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized. PMID:26999188

  8. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells.

    PubMed

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3C(pro)s) of picornaviruses share similar spatial structures and it is becoming apparent that 3C(pro) plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3C(pro) are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3C(pro) can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3C(pro) and these essential factors, 3C(pro) is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3C(pro) are ongoing and a better understanding of the roles played by 3C(pro) may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3C(pro) is summarized. PMID:26999188

  9. Measles Virus Matrix Protein Inhibits Host Cell Transcription.

    PubMed

    Yu, Xuelian; Shahriari, Shadi; Li, Hong-Mei; Ghildyal, Reena

    2016-01-01

    Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription. PMID:27551716

  10. Measles Virus Matrix Protein Inhibits Host Cell Transcription

    PubMed Central

    Yu, Xuelian; Shahriari, Shadi; Li, Hong-Mei; Ghildyal, Reena

    2016-01-01

    Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription. PMID:27551716

  11. Innate Lymphoid Cells in Graft‐Versus‐Host Disease

    PubMed Central

    Mjösberg, J.

    2015-01-01

    Innate lymphoid cells (ILC) are lymphocytes lacking rearranged antigen receptors such as those expressed by T and B cells. ILC are important effector and regulatory cells of the innate immune system, controlling lymphoid organogenesis, tissue inflammation, and homeostasis. The family of ILC consists of cytotoxic NK cells and the more recently described noncytotoxic group 1, 2, and 3 ILC. The classification of noncytotoxic ILC—in many aspects—mirrors that of T helper cells, which is based on the expression of master transcription factors and signature cytokines specific for each subset. The IL‐22 producing RORγt+ ILC3 subset was recently found to be critical in the prevention of intestinal graft‐versus‐host disease (GVHD) following allogeneic hematopoietic cell transplantation (HCT) via strengthening the intestinal mucosal barrier. In this review, we summarize the current view of the immunological functions of human noncytotoxic ILC subsets and discuss the potentially beneficial features of IL‐22 producing ILC3 in improving allo‐HCT efficacy by attenuating susceptibility to GVHD. In addition, we explore the possibility of other ILC subsets playing a role in GVHD. PMID:26228632

  12. Distinct host cell proteins incorporated by SIV replicating in CD4+ T Cells from natural disease resistant versus non-natural disease susceptible hosts

    PubMed Central

    2010-01-01

    Background Enveloped viruses including the simian immunodeficiency virus (SIV) replicating within host cells acquire host proteins upon egress from the host cells. A number of studies have catalogued such host proteins, and a few have documented the potential positive and negative biological functions of such host proteins. The studies conducted herein utilized proteomic analysis to identify differences in the spectrum of host proteins acquired by a single source of SIV replicating within CD4+ T cells from disease resistant sooty mangabeys and disease susceptible rhesus macaques. Results While a total of 202 host derived proteins were present in viral preparations from CD4+ T cells from both species, there were 4 host-derived proteins that consistently and uniquely associated with SIV replicating within CD4+ T cells from rhesus macaques but not sooty mangabeys; and, similarly, 28 host-derived proteins that uniquely associated with SIV replicating within CD4+ T cells from sooty mangabeys, but not rhesus macaques. Of interest was the finding that of the 4 proteins uniquely present in SIV preparations from rhesus macaques was a 26 S protease subunit 7 (MSS1) that was shown to enhance HIV-1 'tat" mediated transactivation. Among the 28 proteins found in SIV preparations from sooty mangabeys included several molecules associated with immune function such as CD2, CD3ε, TLR4, TLR9 and TNFR and a bioactive form of IL-13. Conclusions The finding of 4 host proteins that are uniquely associated with SIV replicating within CD4+ T cells from disease susceptible rhesus macaques and 28 host proteins that are uniquely associated with SIV replicating within CD4+ T cells from disease resistant sooty mangabeys provide the foundation for determining the potential role of each of these unique host-derived proteins in contributing to the polarized clinical outcome in these 2 species of nonhuman primates. PMID:21162735

  13. Post-transcriptional RNA Regulons Affecting Cell Cycle and Proliferation

    PubMed Central

    Blackinton, Jeff G.

    2014-01-01

    The cellular growth cycle is initiated and maintained by punctual, yet agile, regulatory events involving modifications of cell cycle proteins as well as coordinated gene expression to support cyclic checkpoint decisions. Recent evidence indicates that post-transcriptional partitioning of messenger RNA subsets by RNA-binding proteins help physically localize, temporally coordinate, and efficiently translate cell cycle proteins. This dynamic organization of mRNAs encoding cell cycle components contributes to the overall economy of the cell cycle consistent with the post-transcriptional RNA regulon model of gene expression. This review examines several recent studies demonstrating the coordination of mRNA subsets encoding cell cycle proteins during nuclear export and subsequent coupling to protein synthesis, and discusses evidence for mRNA coordination of p53 targets and the DNA damage response pathway. We consider how these observations may connect to upstream and downstream post-transcriptional coordination and coupling of splicing, export, localization, and translation. Published examples from yeast, nematode, insect, and mammalian systems are discussed, and we consider genetic evidence supporting the conclusion that dysregulation of RNA regulons may promote pathogenic states of growth such as carcinogenesis. PMID:24882724

  14. RNA helicases

    PubMed Central

    Ranji, Arnaz

    2010-01-01

    RNA helicases serve multiple roles at the virus-host interface. In some situations, RNA helicases are essential host factors to promote viral replication; however, in other cases they serve as a cellular sensor to trigger the antiviral state in response to viral infection. All family members share the conserved ATP-dependent catalytic core linked to different substrate recognition and protein-protein interaction domains. These flanking domains can be shuffled between different helicases to achieve functional diversity. This review summarizes recent studies, This review summarizes recent studies of RNA helicases in virus biology. First, RNA helicases are catalysts of progressive RNA-protein rearrangements that begin at gene transcription and culminate in release of infectious virus. Second, RNA helicases can act as a scaffold for alternative protein-protein interactions that can defeat the antiviral state. The mounting fundamental understanding of RNA helicases is being used to develop selective and efficacious drugs against human and animal pathogens. The analysis of RNA helicases in virus model systems continues to provide insights into virology, cell biology and immunology and has provided fresh perspective to continue unraveling the complexity of virus-host interactions. PMID:21173576

  15. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

    PubMed

    Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

    2016-01-01

    Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

  16. Role of Exosome Shuttle RNA in Cell-to-Cell Communication.

    PubMed

    Zhang, Wei; Peng, Peng; Shen, Keng

    2016-08-01

    There are several ways that transpire in cell-to-cell communication,with or without cell contact. Exosomes play an important role in cell-to-cell communication,which do not need cell contact,as that can result in a relatively long-distance influence. Exosome contains RNA components including mRNA and micro-RNA,which are protected by exosomes rigid membranes. This allows those components be passed long distance through the circulatory system. The mRNA components are far different from their donor cells,and the micro-RNA components may reflect the cell they originated. In this article we review the role of exosomes in cell-to-cell communication,with particular focus on their potentials in both diagnostic and therapeutic applications. PMID:27594165

  17. Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated

    PubMed Central

    Yang, Chunfu; Starr, Tregei; Song, Lihua; Carlson, John H.; Sturdevant, Gail L.; Beare, Paul A.; Whitmire, William M.

    2015-01-01

    ABSTRACT Chlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1 to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene. PMID:26556273

  18. A novel bacteriophage-encoded RNA polymerase binding protein inhibits transcription initiation and abolishes transcription termination by host RNA polymerase.

    PubMed

    Nechaev, Sergei; Yuzenkova, Yulia; Niedziela-Majka, Anita; Heyduk, Tomasz; Severinov, Konstantin

    2002-06-28

    Xp10 is a lytic bacteriophage of Xanthomonas oryzae, a Gram-negative bacterium that causes rice blight. We purified an Xp10 protein, p7, that binds to and inhibits X. oryzae RNA polymerase (RNAP). P7 is a novel 73 amino acid-long protein; it does not bind to and hence does not affect transcription by Escherichia coli RNAP. Analysis of E. coli/X. oryzae RNAP hybrids locates the p7 binding site to the largest X. oryzae RNAP subunit, beta'. Binding of p7 to X. oryzae RNAP holoenzyme prevents large conformational change that places the sigma subunit region 4 into the correct position for interaction with the -35 promoter element. As a result, open promoter complex formation on the -10/-35 class promoters is inhibited. Inhibition of promoter complex formation on the extended -10 class promoters is less efficient. The p7 protein also abolishes factor-independent transcription termination by X. oryzae RNAP by preventing the release of nascent RNA at terminators. Further physiological and mechanistic studies of this novel transcription factor should provide additional insights into its biological role and the processes of promoter recognition and transcription termination. PMID:12079331

  19. Genome-Wide Analysis of miRNA-mRNA Interactions in Marrow Stromal Cells

    PubMed Central

    Balakrishnan, Ilango; Yang, Xiaodong; Brown, Joseph; Ramakrishnan, Aravind; Torok–Storb, Beverly; Kabos, Peter; Hesselberth, Jay R.; Pillai, Manoj M.

    2014-01-01

    Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) located within the bone marrow. Both secreted and cell-surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA-mRNA interactions are indirect and have high false-positive and negative rates. In this report, a genome-wide biochemical technique (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation or HITS-CLIP) was used to generate unbiased genome-wide maps of miRNA-mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome-wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME. PMID:24038734

  20. Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand confer antitumor immunity.

    PubMed

    Fujii, Shin-ichiro; Goto, Akira; Shimizu, Kanako

    2009-04-30

    The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of alpha-galactosylceramide (alpha-GalCer)-loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with alpha-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)-mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with alpha-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases. PMID:19164596

  1. Expression of the vault RNA protects cells from undergoing apoptosis

    PubMed Central

    Amort, Melanie; Nachbauer, Birgit; Tuzlak, Selma; Kieser, Arnd; Schepers, Aloys; Villunger, Andreas; Polacek, Norbert

    2015-01-01

    Non-protein-coding RNAs are a functionally versatile class of transcripts exerting their biological roles on the RNA level. Recently, we demonstrated that the vault complex-associated RNAs (vtRNAs) are significantly upregulated in Epstein–Barr virus (EBV)-infected human B cells. Very little is known about the function(s) of the vtRNAs or the vault complex. Here, we individually express latent EBV-encoded proteins in B cells and identify the latent membrane protein 1 (LMP1) as trigger for vtRNA upregulation. Ectopic expression of vtRNA1-1, but not of the other vtRNA paralogues, results in an improved viral establishment and reduced apoptosis, a function located in the central domain of vtRNA1-1. Knockdown of the major vault protein has no effect on these phenotypes revealing that vtRNA1-1 and not the vault complex contributes to general cell death resistance. This study describes a NF-κB-mediated role of the non-coding vtRNA1-1 in inhibiting both the extrinsic and intrinsic apoptotic pathways. PMID:25952297

  2. Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

    SciTech Connect

    Nitschke, Matthias; Korte, Thomas; Ter-Avetisyan, Gohar; Tuennemann, Gisela; Cardoso, M. Cristina; Veit, Michael Herrmann, Andreas

    2008-08-01

    Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.

  3. Bacterium-Generated Nitric Oxide Hijacks Host Tumor Necrosis Factor Alpha Signaling and Modulates the Host Cell Cycle In Vitro

    PubMed Central

    Mocca, Brian

    2012-01-01

    In mammalian cells, nitric oxide (NO·) is an important signal molecule with concentration-dependent and often controversial functions of promoting cell survival and inducing cell death. An inducible nitric oxide synthase (iNOS) in various mammalian cells produces higher levels of NO· from l-arginine upon infections to eliminate pathogens. In this study, we reveal novel pathogenic roles of NO· generated by bacteria in bacterium-host cell cocultures using Moraxella catarrhalis, a respiratory tract disease-causing bacterium, as a biological producer of NO·. We recently demonstrated that M. catarrhalis cells that express the nitrite reductase (AniA protein) can produce NO· by reducing nitrite. Our study suggests that, in the presence of pathophysiological levels of nitrite, this opportunistic pathogen hijacks host cell signaling and modulates host gene expression through its ability to produce NO· from nitrite. Bacterium-generated NO· significantly increases the secretion of tumor necrosis factor alpha (TNF-α) and modulates the expression of apoptotic proteins, therefore triggering host cell programmed death partially through TNF-α signaling. Furthermore, our study reveals that bacterium-generated NO· stalls host cell division and directly results in the death of dividing cells by reducing the levels of an essential regulator of cell division. This study provides unique insight into why NO· may exert more severe cytotoxic effects on fast growing cells, providing an important molecular basis for NO·-mediated pathogenesis in infections and possible therapeutic applications of NO·-releasing molecules in tumorigenesis. This study strongly suggests that bacterium-generated NO· can play important pathogenic roles during infections. PMID:22636782

  4. Effective delivery of chemotherapeutic nanoparticles by depleting host Kupffer cells.

    PubMed

    Ohara, Yusuke; Oda, Tatsuya; Yamada, Keiichi; Hashimoto, Shinji; Akashi, Yoshimasa; Miyamoto, Ryoichi; Kobayashi, Akihiko; Fukunaga, Kiyoshi; Sasaki, Ryoko; Ohkohchi, Nobuhiro

    2012-11-15

    Although chemotherapeutic nanoparticles would confer various advantages, the majority of administrated nanoparticles are known to be spoiled by the reticuloendothelial system (RES). Intending to more effectively deliver therapeutic nanoparticles to target regions in vivo, host RES, especially Kupffer cells in the liver, have been depleted ahead of drug administration. To demonstrate this hypothesis, clodronate liposomes were preinjected into BALB/c nude mice for depletion of Kupffer cells 2 days before, and pegylated liposomal doxorubicin (Doxil) at the doses of 1.25, 2.5 and 5.0 mg/kg was administered. As a result, doxorubicin accumulation in the liver was decreased from 36 to 26% injected dose/organ by the Kupffer cells depletion, and consequently, the plasma concentration of doxorubicin was significantly enhanced threefold (from 11 to 33 μg/mL) on day 1 at 1.25 mg/kg-dose group. Doxorubicin accumulation in the tumor was increased from 0.78 to 3.0 μg/g-tissue on day 3, and tumor growth inhibition by Doxil was significantly boosted (tumor volumes from 751 to 482 mm(3) on day 24) by the Kupffer cells depletion. In conclusion, Kupffer cells depletion by clodronate liposomes enhanced the plasma concentration and antitumor effects of Doxil, and would be widely applicable for various clinical cancer chemotherapies using nanoparticles. PMID:22362271

  5. Mast cell tryptases and chymases in inflammation and host defense

    PubMed Central

    Caughey, George H.

    2008-01-01

    Summary Tryptases and chymases are the major proteins stored and secreted by mast cells. The types, amounts and properties of these serine peptidases vary by mast cell subtype, tissue, and mammal of origin. Membrane-anchored γ-tryptases are tryptic, prostasin-like, type I peptidases that remain membrane-attached upon release and act locally. Soluble tryptases, including their close relatives, mastins, form inhibitor-resistant oligomers that act more remotely. Befitting their greater destructive potential, chymases are quickly inhibited after release, although some gain protection by associating with proteoglycans. Most chymase-like enzymes, including mast cell cathepsin G, hydrolyze chymotryptic substrates, an uncommon capability in the proteome. Some rodent chymases, however, have mutations resulting in elastolytic activity. Secreted tryptases and chymases promote inflammation, matrix destruction, and tissue remodeling by several mechanisms, including destroying pro-coagulant, matrix, growth and differentiation factors, and activating proteinase-activated receptors, urokinase, metalloproteinases, and angiotensin. They also modulate immune responses by hydrolyzing chemokines and cytokines. At least one chymase protects mice from intestinal worms. Tryptases and chymases also can oppose inflammation by inactivating allergens and neuropeptides causing inflammation and bronchoconstriction. Thus, like mast cells themselves, mast cell serine peptidases play multiple roles in host defense and any accounting of benefit versus harm is necessarily context-specific. PMID:17498057

  6. Herpesvirus saimiri MicroRNAs Preferentially Target Host Cell Cycle Regulators

    PubMed Central

    Guo, Yang Eric; Oei, Theresa

    2015-01-01

    ABSTRACT In latently infected marmoset T cells, Herpesvirus saimiri (HVS) expresses six microRNAs (known as miR-HSURs [H. saimiri U-rich RNAs]). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover the functions of miR-HSURs, we identified mRNA targets in infected cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs that map to the host genome but few in the HVS genome. Gene ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional coactivator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER)-localized chaperone facilitating maturation of major histocompatibility complex class I (MHC-I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell cycle progression, leading to reduced phosphorylation of its substrate, cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency. IMPORTANCE Viruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, the functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic gammaherpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better

  7. Long noncoding RNA derived from CD244 signaling epigenetically controls CD8+ T-cell immune responses in tuberculosis infection.

    PubMed

    Wang, Yang; Zhong, Huiling; Xie, Xiaodan; Chen, Crystal Y; Huang, Dan; Shen, Ling; Zhang, Hui; Chen, Zheng W; Zeng, Gucheng

    2015-07-21

    Molecular mechanisms for T-cell immune responses modulated by T cell-inhibitory molecules during tuberculosis (TB) infection remain unclear. Here, we show that active human TB infection up-regulates CD244 and CD244 signaling-associated molecules in CD8(+) T cells and that blockade of CD244 signaling enhances production of IFN-γ and TNF-α. CD244 expression/signaling in TB correlates with high levels of a long noncoding RNA (lncRNA)-BC050410 [named as lncRNA-AS-GSTT1(1-72) or lncRNA-CD244] in the CD244(+)CD8(+) T-cell subpopulation. CD244 signaling drives lncRNA-CD244 expression via sustaining a permissive chromatin state in the lncRNA-CD244 locus. By recruiting polycomb protein enhancer of zeste homolog 2 (EZH2) to infg/tnfa promoters, lncRNA-CD244 mediates H3K27 trimethylation at infg/tnfa loci toward repressive chromatin states and inhibits IFN-γ/TNF-α expression in CD8(+) T cells. Such inhibition can be reversed by knock down of lncRNA-CD244. Interestingly, adoptive transfer of lncRNA-CD244-depressed CD8(+) T cells to Mycobacterium tuberculosis (MTB)-infected mice reduced MTB infection and TB pathology compared with lncRNA-CD244-expressed controls. Thus, this work uncovers previously unidentified mechanisms in which T cell-inhibitory signaling and lncRNAs regulate T-cell responses and host defense against TB infection. PMID:26150504

  8. Long noncoding RNA derived from CD244 signaling epigenetically controls CD8+ T-cell immune responses in tuberculosis infection

    PubMed Central

    Wang, Yang; Zhong, Huiling; Xie, Xiaodan; Chen, Crystal Y.; Huang, Dan; Shen, Ling; Zhang, Hui; Chen, Zheng W.; Zeng, Gucheng

    2015-01-01

    Molecular mechanisms for T-cell immune responses modulated by T cell-inhibitory molecules during tuberculosis (TB) infection remain unclear. Here, we show that active human TB infection up-regulates CD244 and CD244 signaling-associated molecules in CD8+ T cells and that blockade of CD244 signaling enhances production of IFN-γ and TNF-α. CD244 expression/signaling in TB correlates with high levels of a long noncoding RNA (lncRNA)-BC050410 [named as lncRNA-AS-GSTT1(1-72) or lncRNA-CD244] in the CD244+CD8+ T-cell subpopulation. CD244 signaling drives lncRNA-CD244 expression via sustaining a permissive chromatin state in the lncRNA-CD244 locus. By recruiting polycomb protein enhancer of zeste homolog 2 (EZH2) to infg/tnfa promoters, lncRNA-CD244 mediates H3K27 trimethylation at infg/tnfa loci toward repressive chromatin states and inhibits IFN-γ/TNF-α expression in CD8+ T cells. Such inhibition can be reversed by knock down of lncRNA-CD244. Interestingly, adoptive transfer of lncRNA-CD244–depressed CD8+ T cells to Mycobacterium tuberculosis (MTB)-infected mice reduced MTB infection and TB pathology compared with lncRNA-CD244–expressed controls. Thus, this work uncovers previously unidentified mechanisms in which T cell-inhibitory signaling and lncRNAs regulate T-cell responses and host defense against TB infection. PMID:26150504

  9. Classification of low quality cells from single-cell RNA-seq data.

    PubMed

    Ilicic, Tomislav; Kim, Jong Kyoung; Kolodziejczyk, Aleksandra A; Bagger, Frederik Otzen; McCarthy, Davis James; Marioni, John C; Teichmann, Sarah A

    2016-01-01

    Single-cell RNA sequencing (scRNA-seq) has broad applications across biomedical research. One of the key challenges is to ensure that only single, live cells are included in downstream analysis, as the inclusion of compromised cells inevitably affects data interpretation. Here, we present a generic approach for processing scRNA-seq data and detecting low quality cells, using a curated set of over 20 biological and technical features. Our approach improves classification accuracy by over 30 % compared to traditional methods when tested on over 5,000 cells, including CD4+ T cells, bone marrow dendritic cells, and mouse embryonic stem cells. PMID:26887813

  10. NK cell regulation of CD4 T cell-mediated graft-versus-host disease.

    PubMed

    Noval Rivas, Magali; Hazzan, Marc; Weatherly, Kathleen; Gaudray, Florence; Salmon, Isabelle; Braun, Michel Y

    2010-06-15

    CD3-negative NK cells are granular lymphocytes capable of producing inflammatory cytokines and killing malignant, infected, or stressed cells. We have recently observed a new role for NK cells in the control of the proliferation of CD4 T cells under persistent antigenic stimulation. Monoclonal anti-male CD4 T cells transferred into Rag2-/- male recipients did not expand or were rapidly eliminated. Remarkably, T cells transferred into NK cell-deficient Rag2-/- Il-2Rgammac-/- male hosts expanded extensively and mediated tissue lesions usually observed in chronic graft-versus-host disease (GVHD). T cell failure to proliferate and to induce chronic GVHD was the result of NK cell activity, because depletion of the recipient's NK1.1+ cells by Ab treatment induced T cell expansion and chronic GVHD. T cells under chronic Ag stimulation upregulated ligands of the activating receptor NKG2D, and regulatory activity of NK cells was inhibited by the injection of Abs directed to NKG2D. On the contrary, blocking NKG2A inhibitory receptors did not increase NK cell regulatory activity. Finally, we show that NK regulation of T cell expansion did not involve perforin-mediated lytic activity of NK cells, but depended on T cell surface expression of a functional Fas molecule. These results highlight the potential role played by NK cells in controlling the Ag-specific CD4+ T cells responsible for chronic GVHD. PMID:20488796

  11. Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs.

    PubMed Central

    Kwong, A D; Frenkel, N

    1987-01-01

    The herpes simplex virus virion contains a function that mediates the shutoff of host-protein synthesis and the degradation of host mRNA. Viral mutants affected in this function (vhs mutants) have previously been derived. Cells infected with these mutants exhibit a more stable synthesis of host as well as the immediate early (alpha)-viral proteins. We now show that a function associated with purified virions of the wild-type virus reduces the half-life of host and alpha mRNAs, whereas purified vhs-1 mutant virions lack this activity. The functional half-life of many early (beta)- and late (gamma)-viral mRNAs is also prolonged in mutant virus infections. These studies suggest that the wild-type virion brings into cells a function that indiscriminately reduces the half-life of both host and viral transcripts and that the early translational shutoff of the host is a consequence of this function. This function may facilitate rapid transitions in the expression of groups of genes that are transcriptionally turned on at different times after infection. Images PMID:3031658

  12. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  13. Inhibition of host cell catalase by Mycoplasma pneumoniae: a possible mechanism for cell injury.

    PubMed Central

    Almagor, M; Yatziv, S; Kahane, I

    1983-01-01

    This study demonstrates that viable Mycoplasma pneumoniae cells inhibit catalase activity in several types of intact human cells as well as in solution. Human erythrocyte catalase was inhibited up to 72%, and the inhibition of catalase in human cultured skin fibroblasts, lung carcinoma epithelial cells, and ciliated epithelial cells from human nasal polyps ranged between 75 and 80%. UV light-killed mycoplasmas failed to inhibit catalase activity both in intact cells and in vitro. After M. pneumoniae infection of human cultured skin fibroblasts, the level of malonyldialdehyde, an indicator for membrane lipid peroxidation, was 3.5 times higher than in control fibroblasts. Virulent M. pneumoniae completely inhibited catalase activity in solution, whereas the nonvirulent strains had a lesser ability to inhibit catalase activity. These findings suggest that as a result of host cell catalase inhibition by M. pneumoniae, the toxicity of the hydrogen peroxide generated by the microorganism and the affected cell is enhanced, thereby inducing host cell damage. PMID:6407999

  14. A Whole-Genome RNA Interference Screen for Human Cell Factors Affecting Myxoma Virus Replication

    PubMed Central

    Teferi, Wondimagegnehu M.; Dodd, Kristopher; Maranchuk, Rob; Favis, Nicole

    2013-01-01

    Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes (“hits”) and nonsignificant genes (“nonhits”) of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G1, or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G1/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-d-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy. PMID

  15. Plant RNA processing: soybean pre-mRNA in a pea cell-free extract

    SciTech Connect

    Schuler, M.A.; Hanley, B.A.

    1987-05-01

    Using a pea cell-free extract they have demonstrated the splicing of an SP6 fusion transcript containing an intron derived from the soybean seed storage protein ..beta..-subunit gene. Intron 115 from the conglycinin gene was cloned into a SP6 vector and transcribed using standard recombinant DNA techniques. Incubation of radioactively labeled fusion transcripts in the cell-free system produced a number of products which were identified by primer extension and S1 nuclease analysis. All the products are linear RNA molecules. Lariat intermediates, similar to those found in the yeast and HeLa cell RNA processing systems, have not been detected. The linear RNA products detected in their plant in vitro processing system have various portions of the intron removed which suggests that alternative splice sites are used in processing of this plant intron due to activation of cryptic splice sites or creation of splice sites in the fusion construction. The kinetics of the reactions and parameters of the extract are similar to those determined for the HeLa cell system. Sucrose gradient analysis has demonstrated that the plant RNA products sedimented in a 30S particle, similar in size to that found for the spliceosome of the HeLa cell system.

  16. A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions.

    PubMed

    Delincé, Matthieu J; Bureau, Jean-Baptiste; López-Jiménez, Ana Teresa; Cosson, Pierre; Soldati, Thierry; McKinney, John D

    2016-08-16

    The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements. PMID:27425421

  17. The long non-coding RNA expression profile of Coxsackievirus A16 infected RD cells identified by RNA-seq.

    PubMed

    Shi, Yingying; Tu, Huilin; Chen, Xiong; Zhang, Yingying; Chen, Liujun; Liu, Zhongchun; Sheng, Jiqun; Han, Song; Yin, Jun; Peng, Biwen; He, Xiaohua; Liu, Wanhong

    2016-04-01

    Coxsackievirus A16 (CVA16) is one of major pathogens of hand, foot and mouth disease (HFMD) in children. Long non-coding RNAs (IncRNAs) have been implicated in various biological processes, but they have not been associated with CVA16 infection. In this study, we comprehensively characterized the landscape of IncRNAs of normal and CVA16 infected rhabdomyosarcoma (RD) cells using RNA-Seq to investigate the functional relevance of IncRNAs. We showed that a total of 760 IncRNAs were upregulated and 1210 IncRNAs were downregulated. Out of these dysregulated IncRNAs, 43.64% were intergenic, 22.31% were sense, 15.89% were intronic, 8.67% were bidirectional, 5.59% were antisense, 3.85% were sRNA host IncRNAs and 0.05% were enhancer. Six dysregulated IncRNAs were validated by quantitative PCR assays and the secondary structures of these IncRNAs were projected. Moreover, we conducted a bioinformatics analysis of an IncRNAs (ENST00000602478) to elucidate the diversity of modification and functions of IncRNAs. In summary, the current study compared the dysregulated IncRNAs profile upon CVA16 challenge and illustrated the intricate relationship between coding and IncRNAs transcripts. These results may not only provide a complete picture of transcription in CVA16 infected cells but also provide novel molecular targets for treatments of HFMD. PMID:27060091

  18. RNA computers in live cells: results and prospects

    PubMed Central

    Benenson, Yaakov

    2009-01-01

    Summary Man-made molecular “computers” that operate inside live cells will enable unprecedented level of control over cellular physiology. A promising approach to building these computers uses RNA molecules and RNA-based regulation. RNA naturally lends itself to create “digital” molecular networks that embody standardized (normal) forms of logic functions. The network’s inputs, that may or may not be inverted by single-input NOT logic gates, feed into multi-input AND gates whose outputs are in turn integrated in a multi-input OR gate. Below I review recent steps that have been taken toward implementing these networks with allosteric riboswitches and ribozymes in bacteria and yeast, and RNAi in mammalian cells. I also propose how to co-opt recently-discovered additional RNA regulation mechanisms into future construction efforts. PMID:19720518

  19. Biomimetic RNA Silencing Nanocomplexes Overcome Multidrug Resistance in Cancer Cells**

    PubMed Central

    Wang, Zhongliang; Wang, Zhe; Liu, Dingbin; Yan, Xuefeng; Wang, Fu; Niu, Gang

    2015-01-01

    RNA interference (RNAi) is an RNA-dependent gene silencing approach controlled by RNA-induced silencing complex (RISC). Here we represent a synthetic RISC-mimic nanocomplex, which can actively cleave its target RNA in a sequence-specific manner. With high enzymatic stability and efficient self-delivery to target cells, the designed nanocomplex can selectively and potently induce gene silencing without cytokine activation. The nanocomplexes targeting to multidrug resistance are able to not only bypass P-glycoprotein (Pgp) transporter due to their nano-size effect, but also effectively suppress the Pgp expression, thus resulting in successful restoration of drug sensitivity of OVCAR8/ADR cells to Pgp-transportable cytotoxic agents. This nanocomplex approach has the potential for both functional genomics and cancer therapy. PMID:24446433

  20. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

    PubMed Central

    Ortega, Álvaro D.; Quereda, Juan J.; Pucciarelli, M. Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of “intact” intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections. PMID:25429360

  1. Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host.

    PubMed

    Tsetsarkin, Konstantin A; Liu, Guangping; Kenney, Heather; Hermance, Meghan; Thangamani, Saravanan; Pletnev, Alexander G

    2016-01-01

    Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates. PMID:27620807

  2. Transcription of the genome of adenovirus type 12. I. Viral mRNA in abortively infected and transformed cells.

    PubMed Central

    Ortin, J; Doerfler, W

    1975-01-01

    In baby hamster kidney (BKH-21) cells abortively infected with adenovirus type 12, polysome-associated, virus-specific RNA could be detected starting 5 to 7 h after infection. The amount of this RNA reached a maximum between 10 to 12 h after infection and continued to be synthesized at a reduced level until late in infection (48 to 50 h.). In BHK-21 cells transformed by adenovirus type 12 (HB cells), 0.26% of the polysome-associated mRNA was virus specific. The size of the virus-specific mRNA isolated from polysomes of BHK-21 cells abortively infected with, or transformed by adenovirus type 12 was determined by electrophoresis in polyacrylamide gels in 98% formamide, i.e., under conditions which eliminated secondary structure or aggregation of RNA. In abortively infected hamster cells viral mRNA size classes of molecular weights 0.9 times 10-6 and 0.65 times 10-6 to 0.67 times 10-6 were predominant. A minor fraction of 1.5 times 10-6 daltons was consistently found and increased with time after infection. Late after infection (24 to 26 h), viral mRNA of 1.9 times 10-6 daltons was also observed. The size distribution of adenovirus type 12-specific mRNA from transformed hamster cells (HB line) was very similar to that in abortively infected cells, except that the relative amount of the viral mRNA fraction of 1.5 times 10-6 daltons was much higher. It is uncertain whether the viral mRNA of high-molecular-weight represents mixed transcripts derived from integrated viral genomes and adjacent host genes. PMID:1167602

  3. Host volatiles mediate cell invasion of honey bee brood cells by Varroa destructor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A female Varroa destructor mite parasitizes capped bee brood by invading the cell of a late 5th instar larvae just before the cell is capped, usually by transfer from a worker bee to the new larval host. Female mites must rely on chemical cues to successfully locate and transfer to an appropriate ag...

  4. Smooth Muscle Enriched Long Noncoding RNA (SMILR) Regulates Cell Proliferation

    PubMed Central

    Ballantyne, Margaret D.; Pinel, Karine; Dakin, Rachel; Vesey, Alex T.; Diver, Louise; Mackenzie, Ruth; Garcia, Raquel; Welsh, Paul; Sattar, Naveed; Hamilton, Graham; Joshi, Nikhil; Dweck, Marc R.; Miano, Joseph M.; McBride, Martin W.; Newby, David E.; McDonald, Robert A.

    2016-01-01

    Background— Phenotypic switching of vascular smooth muscle cells from a contractile to a synthetic state is implicated in diverse vascular pathologies, including atherogenesis, plaque stabilization, and neointimal hyperplasia. However, very little is known about the role of long noncoding RNA (lncRNA) during this process. Here, we investigated a role for lncRNAs in vascular smooth muscle cell biology and pathology. Methods and Results— Using RNA sequencing, we identified >300 lncRNAs whose expression was altered in human saphenous vein vascular smooth muscle cells following stimulation with interleukin-1α and platelet-derived growth factor. We focused on a novel lncRNA (Ensembl: RP11-94A24.1), which we termed smooth muscle–induced lncRNA enhances replication (SMILR). Following stimulation, SMILR expression was increased in both the nucleus and cytoplasm, and was detected in conditioned media. Furthermore, knockdown of SMILR markedly reduced cell proliferation. Mechanistically, we noted that expression of genes proximal to SMILR was also altered by interleukin-1α/platelet-derived growth factor treatment, and HAS2 expression was reduced by SMILR knockdown. In human samples, we observed increased expression of SMILR in unstable atherosclerotic plaques and detected increased levels in plasma from patients with high plasma C-reactive protein. Conclusions— These results identify SMILR as a driver of vascular smooth muscle cell proliferation and suggest that modulation of SMILR may be a novel therapeutic strategy to reduce vascular pathologies. PMID:27052414

  5. OsRDR6 plays role in host defense against double-stranded RNA virus, Rice Dwarf Phytoreovirus

    PubMed Central

    Hong, Wei; Qian, Dan; Sun, Runhong; Jiang, Lin; Wang, Yu; Wei, Chunhong; Zhang, Zhongkai; Li, Yi

    2015-01-01

    RNAi is a major antiviral defense response in plant and animal model systems. RNA-dependent RNA polymerase 6 (RDR6) is an essential component of RNAi, which plays an important role in the resistance against viruses in the model plants. We found previously that rice RDR6 (OsRDR6) functioned in the defense against Rice stripe virus (RSV), and Rice Dwarf Phytoreovirus (RDV) infection resulted in down-regulation of expression of RDR6. Here we report our new findings on the function of OsRDR6 against RDV. Our result showed that down-regulation of OsRDR6 through the antisense (OsRDR6AS) strategy increased rice susceptibility to RDV infection while over-expression of OsRDR6 had no effect on RDV infection. The accumulation of RDV vsiRNAs was reduced in the OsRDR6AS plants. In the OsRDR6 over-expressed plants, the levels of OsRDR6 RNA transcript and protein were much higher than that in the control plants. Interestingly, the accumulation level of OsRDR6 protein became undetectable after RDV infection. This finding indicated that the translation and/or stability of OsRDR6 protein were negatively impacted upon RDV infection. This new finding provides a new light on the function of RDR6 in plant defense response and the cross-talking between factors encoded by host plant and double-stranded RNA viruses. PMID:26165755

  6. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    PubMed Central

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jose R.

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Delivered RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1, and OPR) in the hemi-biotrophic fungus F. oxysporum f. sp. conglutinans. Expression of double stranded RNA (dsRNA) molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75, 83, and 72% reduction for FOW2, FRP1, and OPR, respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30–50% survival and OPR between 45 and 70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants. PMID:25654075

  7. Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

    PubMed

    Ohmura, Mitsuyo; Hishiki, Takako; Yamamoto, Takehiro; Nakanishi, Tsuyoshi; Kubo, Akiko; Tsuchihashi, Kenji; Tamada, Mayumi; Toue, Sakino; Kabe, Yasuaki; Saya, Hideyuki; Suematsu, Makoto

    2015-04-30

    CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue. PMID:25461272

  8. Investigations of host defence in patients with sickle cell disease.

    PubMed

    Boghossian, S H; Wright, G; Webster, A D; Segal, A W

    1985-03-01

    Parameters of host defence were investigated in 30 patients with sickle cell disease (SCD). A newly devised perfusion system was used to study the kinetics in whole blood of leucocyte adherence, phagocytosis, killing and solubilization of a mixture of Staph. aureus and Str. pneumoniae, and secretion of lactoferrin. A skin window technique was used to examine the accumulation of leucocytes at inflammatory foci and their subsequent rate of movement through a filter. Serum concentrations of C3, C4, total haemolytic complement and immunoglobulins were also measured. The rate of neutrophil migration into filters was slightly reduced in patients with SCD. The proportion of monocytes that emigrated from the skin windows and their rate of migration were markedly diminished. The adhesion of neutrophils and their ability to kill staphylococci were also reduced, particularly in patients of the haemoglobin (Hb) SS and Hb S-beta-thalassaemia genotypes. Neutrophil function was mostly impaired in patients with the greatest frequency of bacterial infection. The rate of clearance of pneumococci was related to the concentration of type specific immunoglobulin G but not M. Serum concentrations of immunoglobulins and complement were normal. We were unable to define a defect of host defence of sufficient magnitude to explain the susceptibility of these patients to severe infection. PMID:3882140

  9. MicroRNA-mediated immune modulation as a therapeutic strategy in host-implant integration.

    PubMed

    Ong, Siew-Min; Biswas, Subhra K; Wong, Siew-Cheng

    2015-07-01

    The concept of implanting an artificial device into the human body was once the preserve of science fiction, yet this approach is now often used to replace lost or damaged biological structures in human patients. However, assimilation of medical devices into host tissues is a complex process, and successful implant integration into patients is far from certain. The body's immediate response to a foreign object is immune-mediated reaction, hence there has been extensive research into biomaterials that can reduce or even ablate anti-implant immune responses. There have also been attempts to embed or coat anti-inflammatory drugs and pro-regulatory molecules onto medical devices with the aim of preventing implant rejection by the host. In this review, we summarize the key immune mediators of medical implant reaction, and we evaluate the potential of microRNAs to regulate these processes to promote wound healing, and prolong host-implant integration. PMID:26024977

  10. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    PubMed Central

    2012-01-01

    incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells. Conclusions Profiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN. PMID:22889230

  11. Full-Length mRNA-Seq from single cell levels of RNA and individual circulating tumor cells

    PubMed Central

    Ramsköld, Daniel; Luo, Shujun; Wang, Yu-Chieh; Li, Robin; Deng, Qiaolin; Faridani, Omid R.; Daniels, Gregory A.; Khrebtukova, Irina; Loring, Jeanne F.; Laurent, Louise C.; Schroth, Gary P.; Sandberg, Rickard

    2012-01-01

    In the last decade, genome-wide transcriptome analyses have been routinely used to monitor tissue-, disease- and cell type-specific gene expression, but it has been technically challenging to generate expression profiles from single cells. Here we describe a novel and robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which significantly enhances detailed analyses of alternative transcript isoforms and identification of SNPs. We have determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series. Applying Smart-Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including new candidate biomarkers for melanoma circulating tumor cells. Importantly, our protocol can easily be utilized for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells. PMID:22820318

  12. Inkjet printing of silk nest arrays for cell hosting.

    PubMed

    Suntivich, Rattanon; Drachuk, Irina; Calabrese, Rossella; Kaplan, David L; Tsukruk, Vladimir V

    2014-04-14

    An inkjet printing approach is presented for the facile fabrication of microscopic arrays of biocompatible silk "nests" capable of hosting live cells for prospective biosensors. The patterning of silk fibroin nests were constructed by the layer-by-layer (LbL) assembly of silk polyelectrolytes chemically modified with poly-(l-lysine) and poly-(l-glutamic acid) side chains. The inkjet-printed silk circular regions with a characteristic "nest" shape had diameters of 70-100 μm and a thickness several hundred nanometers were stabilized by ionic pairing and by the formation of the silk II crystalline secondary structure. These "locked-in" silk nests remained anchored to the substrate during incubation in cell growth media to provide a biotemplated platform for printing-in, immobilization, encapsulation and growth of cells. The process of inkjet-assisted printing is versatile and can be applied on any type of substrate, including rigid and flexible, with scalability and facile formation. PMID:24605757

  13. Differential expression and interaction of host factors augment HIV-1 gene expression in neonatal mononuclear cells

    SciTech Connect

    Sundaravaradan, Vasudha; Mehta, Roshni; Harris, David T.; Zack, Jerome A.; Ahmad, Nafees

    2010-04-25

    We have previously shown a higher level of HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells (CBMC) compared with adult blood cells (PBMC), which could be due to differential expression of host factors. We performed the gene expression profile of CBMC and PBMC and found that 8013 genes were expressed at higher levels in CBMC than PBMC and 8028 genes in PBMC than CBMC, including 1181 and 1414 genes upregulated after HIV-1 infection in CBMC and PBMC, respectively. Several transcription factors (NF-kappaB, E2F, HAT-1, TFIIE, Cdk9, Cyclin T1), signal transducers (STAT3, STAT5A) and cytokines (IL-1beta, IL-6, IL-10) were upregulated in CBMC than PBMC, which are known to influence HIV-1 replication. In addition, a repressor of HIV-1 transcription, YY1, was down regulated in CBMC than PBMC and several matrix metalloproteinase (MMP-7, -12, -14) were significantly upregulated in HIV-1 infected CBMC than PBMC. Furthermore, we show that CBMC nuclear extracts interacted with a higher extent to HIV-1 LTR cis-acting sequences, including NF-kappaB, NFAT, AP1 and NF-IL6 compared with PBMC nuclear extracts and retroviral based short hairpin RNA (shRNA) for STAT3 and IL-6 down regulated their own and HIV-1 gene expression, signifying that these factors influenced differential HIV-1 gene expression in CBMC than PBMC.

  14. Host cell Golgi anti-apoptotic protein (GAAP) and growth of Chlamydia pneumoniae.

    PubMed

    Markkula, Eveliina; Hulkkonen, Jaakko; Penttilä, Tuula; Puolakkainen, Mirja

    2013-01-01

    Chlamydia pneumoniae protein CPn0809 is a type three secretion system substrate, the exact function of which in infection pathogenesis has remained unknown. In this study, we identified by yeast two-hybrid screening a potential host cell interaction partner of CPn0809, Golgi anti-apoptotic protein (GAAP), a conserved protein found in eukaryotic cells. GAAP gene is expressed at relatively constant levels and its expression remained stable also after C. pneumoniae infection. The interaction between GAAP and C. pneumoniae was suggested by transfection studies. GAAP knock-down by siRNA in infected A549 cells resulted in an increased number of C. pneumoniae genomes and growth of the bacteria as judged by quantitative PCR and inclusion counts, respectively. Silencing of GAAP did not make the A549 cells more susceptible to apoptosis per se, and infection with C. pneumoniae prevented staurosporin-induced apoptosis also in transfected cultures. Taken together, the proposed interaction between C. pneumoniae and GAAP modulates bacterial growth in A549 cells. PMID:23000903

  15. Fire blight disease reactome: RNA-seq transcriptional profile of apple host plant defense responses to Erwinia amylovora pathogen infection

    PubMed Central

    Kamber, Tim; Buchmann, Jan P.; Pothier, Joël F.; Smits, Theo H. M.; Wicker, Thomas; Duffy, Brion

    2016-01-01

    The molecular basis of resistance and susceptibility of host plants to fire blight, a major disease threat to pome fruit production globally, is largely unknown. RNA-sequencing data from challenged and mock-inoculated flowers were analyzed to assess the susceptible response of apple to the fire blight pathogen Erwinia amylovora. In presence of the pathogen 1,080 transcripts were differentially expressed at 48 h post inoculation. These included putative disease resistance, stress, pathogen related, general metabolic, and phytohormone related genes. Reads, mapped to regions on the apple genome where no genes were assigned, were used to identify potential novel genes and open reading frames. To identify transcripts specifically expressed in response to E. amylovora, RT-PCRs were conducted and compared to the expression patterns of the fire blight biocontrol agent Pantoea vagans strain C9-1, another apple pathogen Pseudomonas syringae pv. papulans, and mock inoculated apple flowers. This led to the identification of a peroxidase superfamily gene that was lower expressed in response to E. amylovora suggesting a potential role in the susceptibility response. Overall, this study provides the first transcriptional profile by RNA-seq of the host plant during fire blight disease and insights into the response of susceptible apple plants to E. amylovora. PMID:26883568

  16. Fire blight disease reactome: RNA-seq transcriptional profile of apple host plant defense responses to Erwinia amylovora pathogen infection.

    PubMed

    Kamber, Tim; Buchmann, Jan P; Pothier, Joël F; Smits, Theo H M; Wicker, Thomas; Duffy, Brion

    2016-01-01

    The molecular basis of resistance and susceptibility of host plants to fire blight, a major disease threat to pome fruit production globally, is largely unknown. RNA-sequencing data from challenged and mock-inoculated flowers were analyzed to assess the susceptible response of apple to the fire blight pathogen Erwinia amylovora. In presence of the pathogen 1,080 transcripts were differentially expressed at 48 h post inoculation. These included putative disease resistance, stress, pathogen related, general metabolic, and phytohormone related genes. Reads, mapped to regions on the apple genome where no genes were assigned, were used to identify potential novel genes and open reading frames. To identify transcripts specifically expressed in response to E. amylovora, RT-PCRs were conducted and compared to the expression patterns of the fire blight biocontrol agent Pantoea vagans strain C9-1, another apple pathogen Pseudomonas syringae pv. papulans, and mock inoculated apple flowers. This led to the identification of a peroxidase superfamily gene that was lower expressed in response to E. amylovora suggesting a potential role in the susceptibility response. Overall, this study provides the first transcriptional profile by RNA-seq of the host plant during fire blight disease and insights into the response of susceptible apple plants to E. amylovora. PMID:26883568

  17. Development of an Insect Vector Cell Culture and RNA Interference System To Investigate the Functional Role of Fijivirus Replication Protein

    PubMed Central

    Jia, Dongsheng; Chen, Hongyan; Zheng, Ailing; Chen, Qian; Liu, Qifei; Xie, Lianhui

    2012-01-01

    An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins. PMID:22398296

  18. [Synthetic RNA technologies to control functions of mammalian cells].

    PubMed

    Saito, Hirohide

    2015-01-01

    We recently succeeded in producing nanostructures made of RNA-protein (RNP) complexes. We show that RNA and the ribosomal protein L7Ae can form a triangular-like nanostructure that consists of three L7Ae proteins, which form the apices of the triangle, bound to one RNA scaffold. This shape is created through a 60° kink introduced into the RNA structure on L7Ae binding. By varying the size of the RNA scaffold we could in turn alter the overall size of the triangular nanostructure. Several functions can be added to this nanostructure by the introduction of effector proteins fused to L7Ae. The design and construction of functional RNP nanostructures that detect specific cancer cells are discussed herein. In parallel, we developed synthetic RNP translational switches to control production levels of particular proteins depending on certain input(s) within the intracellular environment. The RNP-binding module was successfully incorporated into mRNA to generate functional RNP switches. The designed ON/OFF translational switches detect expression of the trigger factor and repress or activate expression of a desired protein (e.g., apoptosis regulator) in target mammalian cells. Taken together, RNP-binding module could be employed for constructing designer genetic switches and functional nanostructures to regulate cellular processes. PMID:25759049

  19. Discovery of Novel dsRNA Viral Sequences by In Silico Cloning and Implications for Viral Diversity, Host Range and Evolution

    PubMed Central

    Liu, Huiquan; Fu, Yanping; Xie, Jiatao; Cheng, Jiasen; Ghabrial, Said A.; Li, Guoqing; Yi, Xianhong; Jiang, Daohong

    2012-01-01

    Genome sequence of viruses can contribute greatly to the study of viral evolution, diversity and the interaction between viruses and hosts. Traditional molecular cloning methods for obtaining RNA viral genomes are time-consuming and often difficult because many viruses occur in extremely low titers. DsRNA viruses in the families, Partitiviridae, Totiviridae, Endornaviridae, Chrysoviridae, and other related unclassified dsRNA viruses are generally associated with symptomless or persistent infections of their hosts. These characteristics indicate that samples or materials derived from eukaryotic organisms used to construct cDNA libraries and EST sequencing might carry these viruses, which were not easily detected by the researchers. Therefore, the EST databases may include numerous unknown viral sequences. In this study, we performed in silico cloning, a procedure for obtaining full or partial cDNA sequence of a gene by bioinformatics analysis, using known dsRNA viral sequences as queries to search against NCBI Expressed Sequence Tag (EST) database. From this analysis, we obtained 119 novel virus-like sequences related to members of the families, Endornaviridae, Chrysoviridae, Partitiviridae, and Totiviridae. Many of them were identified in cDNA libraries of eukaryotic lineages, which were not known to be hosts for these viruses. Furthermore, comprehensive phylogenetic analysis of these newly discovered virus-like sequences with known dsRNA viruses revealed that these dsRNA viruses may have co-evolved with respective host supergroups over a long evolutionary time while potential horizontal transmissions of viruses between different host supergroups also is possible. We also found that some of the plant partitiviruses may have originated from fungal viruses by horizontal transmissions. These findings extend our knowledge of the diversity and possible host range of dsRNA viruses and offer insight into the origin and evolution of relevant viruses with their hosts. PMID

  20. Stable tRNA precursors in HeLa cells.

    PubMed Central

    Harada, F; Matsubara, M; Kato, N

    1984-01-01

    Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors. Images PMID:6514577

  1. Host cell death due to enteropathogenic Escherichia coli has features of apoptosis.

    PubMed

    Crane, J K; Majumdar, S; Pickhardt, D F

    1999-05-01

    Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen. PMID:10225923

  2. MicroRNA (miRNA) expression is regulated by butyrate induced epigenetic modulation of gene expression in bovine cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present evidence that butyrate induced histone acetylation regulates miRNA expression. MicroRNA expression microarray profiling revealed that 35 miRNA transcripts are significantly (p <0.05) differentially expressed after cells were treated with 10 mM butyrate. Among them, 11 transcripts are dif...

  3. A generic RNA-pulsed dendritic cell vaccine strategy for renal cell carcinoma

    PubMed Central

    Geiger, Christiane; Regn, Sybille; Weinzierl, Andreas; Noessner, Elfriede; Schendel, Dolores J

    2005-01-01

    We present a generic dendritic cell (DC) vaccine strategy for patients with renal cell carcinoma (RCC) based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs). Instead of preparing RNA from tumor tissue of each individual RCC patient, we propose to substitute RNA prepared from a well characterized highly immunogenic RCC cell line (RCC-26 tumor cells) as a generic source of TAAs for loading of DCs. We demonstrate here that efficient RNA transfer can be achieved using lipofection of immature DCs, which are subsequently matured with a cytokine cocktail to express high levels of MHC and costimulatory molecules as well as the chemokine receptor CCR7. Neither RNA itself nor the lipid component impacted on the phenotype or the cytokine secretion of mature DCs. Following RNA loading, DCs derived from HLA-A2-positive donors were able to activate effector-memory cytotoxic T lymphocytes (CTLs) specific for a TAA ligand expressed by the RCC-26 cell line. CTL responses to RNA-loaded DCs reached levels comparable to those stimulated directly by the RCC-26 tumor cells. Furthermore, DCs expressing tumor cell RNA primed naïve T cells, yielding T cell lines with cytotoxicity and cytokine secretion after contact with RCC tumor cells. RCC-26 cell lines are available as good manufacturing practice (GMP)-certified reagents enabling this source of RNA to be easily standardized and adapted for clinical testing. In addition, well defined immune monitoring tools, including the use of RNA expressing B cell lines, are available. Thus, this DC vaccine strategy can be directly compared with an ongoing gene therapy trial using genetically-engineered variants of the RCC-26 cell line as vaccines for RCC patients with metastatic disease. PMID:16045799

  4. The effect of enterohemorrhagic E. coli infection on the cell mechanics of host cells.

    PubMed

    Chen, Yin-Quan; Su, Pin-Tzu; Chen, Yu-Hsuan; Wei, Ming-Tzo; Huang, Chien-Hsiu; Osterday, Kathryn; del Álamo, Juan C; Syu, Wan-Jr; Chiou, Arthur

    2014-01-01

    Enterohaemorrhagic E. coli (EHEC) is a type of human pathogenic bacteria. The main virulence characteristics of EHEC include the formation of attaching and effacing lesions (A/E lesions) and the production of one or more Shiga-like toxins, which may induce human uremic complications. When EHEC infects host cells, it releases translocated intimin receptor (Tir) and effector proteins inside the host cells, inducing the rearrangement and accumulation of the F-actin cytoskeleton, a phenotype leading to the formation of pedestals in the apical cell surface, and the growth of stress fibers at the base of the cells. To examine the effect of EHEC infection on cell mechanics, we carried out a series of experiments to examine HeLa cells with and without EHEC infection to quantify the changes in (1) focal adhesion area, visualized by anti-vinculin staining; (2) the distribution and orientation of stress fibers; and (3) the intracellular viscoelasticity, via directional video particle tracking microrheology. Our results indicated that in EHEC-infected HeLa cells, the focal adhesion area increased and the actin stress fibers became thicker and more aligned. The cytoskeletal reorganization induced by EHEC infection mediated a dramatic increase in the cytoplasmic elastic shear modulus of the infected cells, and a transition in the viscoelastic behavior of the cells from viscous-like to elastic-like. These changes in mechanobiological characteristics might modulate the attachments between EHEC and the host cell to withstand exfoliation, and between the host cell and the extracellular matrix, and might also alter epithelial integrity. PMID:25369259

  5. Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus

    SciTech Connect

    Macnab, J.C.M.; Adams, R.L.P.; Rinaldi, A.; Orr, A.; Clark, L.

    1988-04-01

    Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.

  6. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    SciTech Connect

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  7. Sets of RNA Repeated Tags and Hybridization-Sensitive Fluorescent Probes for Distinct Images of RNA in a Living Cell

    PubMed Central

    Kubota, Takeshi; Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

    2010-01-01

    Background Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. Methodology/Principal Findings Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs. Conclusions/Significance A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging. PMID:20885944

  8. Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines.

    PubMed

    Hoshina, Shiho; Sekizuka, Tsuyoshi; Kataoka, Michiyo; Hasegawa, Hideki; Hamada, Hiromichi; Kuroda, Makoto; Katano, Harutaka

    2016-01-01

    Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. PMID:27611973

  9. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    PubMed

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states. PMID:24299736

  10. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation.

    PubMed

    Abdallah, Abdallah M; Bestebroer, Jovanka; Savage, Nigel D L; de Punder, Karin; van Zon, Maaike; Wilson, Louis; Korbee, Cees J; van der Sar, Astrid M; Ottenhoff, Tom H M; van der Wel, Nicole N; Bitter, Wilbert; Peters, Peter J

    2011-11-01

    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. PMID:21957139

  11. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    NASA Astrophysics Data System (ADS)

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jimmy

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1 and OPR) in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

  12. Novel Virus Discovery and Genome Reconstruction from Field RNA Samples Reveals Highly Divergent Viruses in Dipteran Hosts

    PubMed Central

    Bass, David; Moureau, Gregory; Tang, Shuoya; McAlister, Erica; Culverwell, C. Lorna; Glücksman, Edvard; Wang, Hui; Brown, T. David K.; Gould, Ernest A.; Harbach, Ralph E.; de Lamballerie, Xavier; Firth, Andrew E.

    2013-01-01

    We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected. PMID:24260463

  13. Tobamovirus-resistant tobacco generated by RNA interference directed against host genes.

    PubMed

    Asano, Momoko; Satoh, Rena; Mochizuki, Atsuko; Tsuda, Shinya; Yamanaka, Takuya; Nishiguchi, Masamichi; Hirai, Katsuyuki; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

    2005-08-15

    Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants. PMID:16081069

  14. Cell-penetrating peptide-siRNA conjugate loaded YSA-modified nanobubbles for ultrasound triggered siRNA delivery.

    PubMed

    Xie, Xiangyang; Yang, Yanfang; Lin, Wen; Liu, Hui; Liu, Hong; Yang, Yang; Chen, Ying; Fu, Xudong; Deng, Jianping

    2015-12-01

    Due to the absence of effective in vivo delivery systems, the employment of small interference RNA (siRNA) in the clinic has been hindered. In this paper, a new siRNA targeting system for EphA2-positive tumors was developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, a CPP-siRNA conjugate (CPP-siRNA) was entrapped in an ephrin mimetic peptide (YSA peptide)-modified NB (CPP-siRNA/YSA-NB) and the penetration of the CPP-siRNA was temporally masked; local ultrasound stimulation triggered the release of CPP-siRNA from the NBs and activated its penetration. Subsequent research demonstrated that the CPP-siRNA/YSA-NBs had particle sizes of approximately 200 nm and a siRNA entrapment efficiency of more than 85%. The in vitro release results showed that over 90% of the encapsulated CPP-siRNA released from the NBs in the presence of ultrasound, while less than 1.5% of that (30 min) released without ultrasound. Cell experiments showed a the higher CPP-siRNA cellular uptake of CPP-siRNA/YSA-NB among the various formulations in human breast adenocarcinoma cells (MCF-7, EphA2 positive cells). Additionally, after systemic administration in mice, CPP-siRNA/YSA-NB accumulated in the tumor, augmented c-Myc silencing and delayed tumor progression. In conclusion, the application of CPP-siRNA/YSA-NB with ultrasound may provide a strategy for the selective and efficient delivery of siRNA. PMID:26492155

  15. Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses

    PubMed Central

    Nagy, Peter D.; Pogany, Judit; Xu, Kai

    2016-01-01

    Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes. PMID:26950140

  16. Translational Control of UIS4 Protein of the Host-Parasite Interface Is Mediated by the RNA Binding Protein Puf2 in Plasmodium berghei Sporozoites

    PubMed Central

    Silva, Patrícia A. G. C.; Guerreiro, Ana; Santos, Jorge M.; Braks, Joanna A. M.; Janse, Chris J.; Mair, Gunnar R.

    2016-01-01

    UIS4 is a key protein component of the host-parasite interface in the liver stage of the rodent malaria parasite Plasmodium berghei and required for parasite survival after invasion. In the infectious sporozoite, UIS4 protein has variably been shown to be translated but also been reported to be translationally repressed. Here we show that uis4 mRNA translation is regulated by the P. berghei RNA binding protein Pumilio-2 (PbPuf2 or Puf2 from here on forward) in infectious salivary gland sporozoites in the mosquito vector. Using RNA immunoprecipitation we show that uis4 mRNA is bound by Puf2 in salivary gland sporozoites. In the absence of Puf2, uis4 mRNA translation is de-regulated and UIS4 protein expression upregulated in salivary gland sporozoites. Here, using RNA immunoprecipitation, we reveal the first Puf2-regulated mRNA in this parasite. PMID:26808677

  17. MicroRNA-7a regulates pancreatic β cell function

    PubMed Central

    Latreille, Mathieu; Hausser, Jean; Stützer, Ina; Zhang, Quan; Hastoy, Benoit; Gargani, Sofia; Kerr-Conte, Julie; Pattou, Francois; Zavolan, Mihaela; Esguerra, Jonathan L.S.; Eliasson, Lena; Rülicke, Thomas; Rorsman, Patrik; Stoffel, Markus

    2014-01-01

    Dysfunctional microRNA (miRNA) networks contribute to inappropriate responses following pathological stress and are the underlying cause of several disease conditions. In pancreatic β cells, miRNAs have been largely unstudied and little is known about how specific miRNAs regulate glucose-stimulated insulin secretion (GSIS) or impact the adaptation of β cell function to metabolic stress. In this study, we determined that miR-7 is a negative regulator of GSIS in β cells. Using Mir7a2 deficient mice, we revealed that miR-7a2 regulates β cell function by directly regulating genes that control late stages of insulin granule fusion with the plasma membrane and ternary SNARE complex activity. Transgenic mice overexpressing miR-7a in β cells developed diabetes due to impaired insulin secretion and β cell dedifferentiation. Interestingly, perturbation of miR-7a expression in β cells did not affect proliferation and apoptosis, indicating that miR-7 is dispensable for the maintenance of endocrine β cell mass. Furthermore, we found that miR-7a levels are decreased in obese/diabetic mouse models and human islets from obese and moderately diabetic individuals with compensated β cell function. Our results reveal an interconnecting miR-7 genomic circuit that regulates insulin granule exocytosis in pancreatic β cells and support a role for miR-7 in the adaptation of pancreatic β cell function in obesity and type 2 diabetes. PMID:24789908

  18. Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence

    PubMed Central

    Gravelat, Fabrice N.; Ejzykowicz, Daniele E.; Chiang, Lisa Y.; Chabot, Josée C.; Urb, Mirjam; Macdonald, K. Denyese; al-Bader, Nadia; Filler, Scott G.; Sheppard, Donald C.

    2010-01-01

    In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared to A. nidulans. The A. fumigatus ΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatus ΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis. PMID:19889083

  19. Cutaneous graft-versus-host disease after hematopoietic stem cell transplant - a review*

    PubMed Central

    Villarreal, Cesar Daniel Villarreal; Alanis, Julio Cesar Salas; Pérez, Jose Carlos Jaime; Candiani, Jorge Ocampo

    2016-01-01

    Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplants (allo-HSCT) associated with significant morbidity and mortality. The earliest and most common manifestation is cutaneous graft-versus-host disease. This review focuses on the pathophysiology, clinical features, prevention and treatment of cutaneous graft-versus-host disease. We discuss various insights into the disease's mechanisms and the different treatments for acute and chronic skin graft-versus-host disease. PMID:27438202

  20. How Stem Cells Speak with Host Immune Cells in Inflammatory Brain Diseases

    PubMed Central

    Pluchino, Stefano; Cossetti, Chiara

    2014-01-01

    Advances in stem cell biology have raised great expectations that diseases and injuries of the central nervous system (CNS) may be ameliorated by the development of non-hematopoietic stem cell medicines. Yet, the application of adult stem cells as CNS therapeutics is challenging and the interpretation of some of the outcomes ambiguous. In fact, the initial idea that stem cell transplants work only via structural cell replacement has been challenged by the observation of consistent cellular signaling between the graft and the host. Cellular signaling is the foundation of coordinated actions and flexible responses, and arises via networks of exchanging and interacting molecules that transmit patterns of information between cells. Sustained stem cell graft-to-host communication leads to remarkable trophic effects on endogenous brain cells and beneficial modulatory actions on innate and adaptive immune responses in vivo, ultimately promoting the healing of the injured CNS. Among a number of adult stem cell types, mesenchymal stem cells (MSCs) and neural stem/precursor cells (NPCs) are being extensively investigated for their ability to signal to the immune system upon transplantation in experimental CNS diseases. Here, we focus on the main cellular signaling pathways that grafted MSCs and NPCs use to establish a therapeutically relevant cross talk with host immune cells, while examining the role of inflammation in regulating some of the bidirectionality of these communications. We propose that the identification of the players involved in stem cell signaling might contribute to the development of innovative, high clinical impact therapeutics for inflammatory CNS diseases. PMID:23633288

  1. Light-activated RNA interference in human embryonic stem cells.

    PubMed

    Huang, Xiao; Hu, Qirui; Braun, Gary B; Pallaoro, Alessia; Morales, Demosthenes P; Zasadzinski, Joseph; Clegg, Dennis O; Reich, Norbert O

    2015-09-01

    We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine. PMID:26086448

  2. Dual RNA-Sequencing of Eucalyptus nitens during Phytophthora cinnamomi Challenge Reveals Pathogen and Host Factors Influencing Compatibility

    PubMed Central

    Meyer, Febé E.; Shuey, Louise S.; Naidoo, Sitha; Mamni, Thandekile; Berger, Dave K.; Myburg, Alexander A.; van den Berg, Noëlani; Naidoo, Sanushka

    2016-01-01

    Damage caused by Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction. PMID:26973660

  3. Dual RNA-Sequencing of Eucalyptus nitens during Phytophthora cinnamomi Challenge Reveals Pathogen and Host Factors Influencing Compatibility.

    PubMed

    Meyer, Febé E; Shuey, Louise S; Naidoo, Sitha; Mamni, Thandekile; Berger, Dave K; Myburg, Alexander A; van den Berg, Noëlani; Naidoo, Sanushka

    2016-01-01

    Damage caused by Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction. PMID:26973660

  4. Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

    PubMed Central

    Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

    2015-01-01

    ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and

  5. BHK cell proteins that bind to the 3' stem-loop structure of the West Nile virus genome RNA.

    PubMed Central

    Blackwell, J L; Brinton, M A

    1995-01-01

    The first 83 3' nucleotides of the genome RNA of the flavivirus West Nile encephalitis virus (WNV) form a stable stem-loop (SL) structure which is followed in the genome by a smaller SL. These 3' structures are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and as such might specifically bind to cellular or viral proteins. Cellular proteins from uninfected and WNV-infected BHK-21 S100 cytoplasmic extracts formed three distinct complexes with the WNV plus-strand 3' SL [(+)3'SL] RNA in a gel mobility shift assay. Subsequent competitor gel shift analyses showed that two of these RNA-protein complexes, complexes 1 and 2, contained cell proteins that specifically bound to the WNV (+)3'SL RNA. UV-induced cross-linking and Northwestern blotting analyses detected WNV (+)3'SL RNA-binding proteins of 56, 84, and 105 kDa. When the S100 cytoplasmic extracts were partially purified by ion-exchange chromatography, a complex that comigrated with complex 1 was detected in fraction 19, while a complex that comigrated with complex 2 was detected in fraction 17. UV-induced cross-linking experiments indicated that an 84-kDa cell protein in fraction 17 and a 105-kDa protein in fraction 19 bound specifically to the WNV (+)3'SL RNA. In addition to binding to the (+)3'SL RNA, the 105-kDa protein bound to the SL structure located at the 3' end of the WNV minus-strand RNA. Initial mapping studies indicated that the 84- and 105-kDa proteins bind to different regions of the (+)3'SL RNA. The 3'-terminal SL RNA of another flavivirus, dengue virus type 3, specifically competed with the WNV (+)3'SL RNA in gel shift assays, suggesting that the host proteins identified in this study are flavivirus specific. PMID:7637011

  6. Live cell immunogold labelling of RNA polymerase II

    PubMed Central

    Orlov, Igor; Schertel, Andreas; Zuber, Guy; Klaholz, Bruno; Drillien, Robert; Weiss, Etienne; Schultz, Patrick; Spehner, Danièle

    2015-01-01

    Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range. PMID:25662860

  7. MicroRNA profiles in various hepatocellular carcinoma cell lines

    PubMed Central

    Morishita, Asahiro; Iwama, Hisakazu; Fujihara, Shintaro; Sakamoto, Teppei; Fujita, Koji; Tani, Joji; Miyoshi, Hisaaki; Yoneyama, Hirohito; Himoto, Takashi; Masaki, Tsutomu

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortality worldwide. Although surgery is considered the most effective treatment for patients with HCC, its indication is restricted by limited criteria and a high relapse rate following surgery; therefore, systemic chemotherapy is required for patients with advanced-stage HCC to prolong their survival. MicroRNAs (miRNAs) are endogenous non-coding RNAs of 18–22 nucleotides in length. It has been reported that aberrant expression of miRNAs is a feature shared by various types of human cancer. Previous studies have indicated that the modulation of non-coding RNAs, particularly miRNAs, may be a valuable therapeutic target for HCC. The aim of the present study was to elucidate the miRNA profiles associated with differentiation and hepatitis B virus (HBV) infection observed in HCC cell lines. The human Alex, Hep3B, HepG2, HuH1, HuH7, JHH1, JHH2, JHH5, JHH6, HLE, HLF and Li-7 HCC cell lines were used for an miRNA array. Replicate data were analyzed following their classification into: i) Poorly- and well-differentiated human HCC cells and ii) HBV-positive and -negative human HCC cells. Out of the 1,719 miRNAs, 4 were found to be significantly upregulated and 52 significantly downregulated in the poorly-differentiated cells, as compared with the well-differentiated cells. Conversely, in the HBV-positive cells 125 miRNAs were found to be upregulated and 2 downregulated, as compared with the HBV-negative cells. Unsupervised hierarchical clustering analysis with Pearson's correlation revealed that the miRNA expression levels were clustered both together and separately in each group. In conclusion, miRNA profile characterization based on various parameters may be a novel approach to determine the etiology of HCC.

  8. Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.

    PubMed

    Thiel, William H; Esposito, Carla L; Dickey, David D; Dassie, Justin P; Long, Matthew E; Adam, Joshua; Streeter, Jennifer; Schickling, Brandon; Takapoo, Maysam; Flenker, Katie S; Klesney-Tait, Julia; Franciscis, Vittorio de; Miller, Francis J; Giangrande, Paloma H

    2016-04-01

    Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-β phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease. PMID:26732878

  9. Stochastic mRNA synthesis in mammalian cells.

    PubMed

    Raj, Arjun; Peskin, Charles S; Tranchina, Daniel; Vargas, Diana Y; Tyagi, Sanjay

    2006-10-01

    Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise. PMID:17048983

  10. Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive

    PubMed Central

    Olarerin-George, Anthony O.; Hogenesch, John B.

    2015-01-01

    Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of nutrients and inducing global changes in gene expression. Over the last two decades, sentinel testing has revealed wide-ranging contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data from 9395 rodent and primate samples from 884 series in the NCBI Sequence Read Archive. We found 11% of these series were contaminated (defined as ≥100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-mapped reads aligned to ribosomal RNA. This was unexpected given 37% of contaminated series used poly(A)-selection for mRNA enrichment. Lastly, we examined the relationship between mycoplasma contamination and host gene expression in a single cell RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. In all, this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality. PMID:25712092

  11. Bacterial effectors target the plant cell nucleus to subvert host transcription

    PubMed Central

    Canonne, Joanne; Rivas, Susana

    2012-01-01

    In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) directly target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells. PMID:22353865

  12. Exploring the RNA World in Hematopoietic Cells Through the Lens of RNA-Binding Proteins

    PubMed Central

    Yuan, Joan; Muljo, Stefan A.

    2013-01-01

    Summary The discovery of microRNAs has renewed interest in post-transcriptional modes of regulation, fueling an emerging view of a rich RNA world within our cells that deserves further exploration. Much work has gone into elucidating genetic regulatory networks that orchestrate gene expression programs and direct cell fate decisions in the hematopoietic system. However, the focus has been to elucidate signaling pathways and transcriptional programs. To bring us one step closer to reverse engineering the molecular logic of cellular differentiation, it will be necessary to map post-transcriptional circuits as well and integrate them in the context of existing network models. In this regard, RNA-binding proteins (RBPs) may rival transcription factors as important regulators of cell fates and represent a tractable opportunity to connect the RNA world to the proteome. ChIP-seq has greatly facilitated genome-wide localization of DNA-binding proteins, helping us to understand genomic regulation at a systems level. Similarly, technological advances such as CLIP-seq allow transcriptome-wide mapping of RBP binding sites, aiding us to unravel post-transcriptional networks. Here, we review RBP-mediated post-transcriptional regulation, paying special attention to findings relevant to the immune system. As a prime example, we highlight the RBP Lin28B, which acts as a heterochronic switch between fetal and adult lymphopoiesis. PMID:23550653

  13. Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

    PubMed

    Hegde, Shrilakshmi; Gabriel, Cordula; Kragl, Martin; Chopra-Dewasthaly, Rohini

    2015-10-01

    Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae's consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host-pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner. PMID:26187893

  14. Real-time kinetics of restriction–modification gene expression after entry into a new host cell

    PubMed Central

    Mruk, Iwona; Blumenthal, Robert M.

    2008-01-01

    Most type II restriction–modification (R–M) systems produce separate restriction endonuclease (REase) and methyltransferase (MTase) proteins. After R–M system genes enter a new cell, protective MTase must appear before REase to avoid host chromosome cleavage. The basis for this apparent temporal regulation is not well understood. PvuII and some other R–M systems appear to achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator/repressor (the ‘C’ protein C.PvuII). To test this model, bacteriophage M13 was used to introduce the PvuII genes into a bacterial population in a relatively synchronous manner. REase mRNA and activity appeared ∼10 min after those of the MTase, but never rose if there was an inactivating pvuIIC mutation. Infection with recombinant M13pvuII phage had little effect on cell growth, relative to infection with parental M13. However, infection of cells pre-expressing C.PvuII led to cessation of growth. This study presents the first direct demonstration of delayed REase expression, relative to MTase, when type II R–M genes enter a new host cell. Surprisingly, though the C and REase genes are cotranscribed, the pvuIIC portion of the mRNA was more abundant than the pvuIIR portion after stable establishment of the R–M system. PMID:18334533

  15. Survival and persistence of fecal host-specific Bacteroidales cells and their DNA assessed by PMA-qPCR

    NASA Astrophysics Data System (ADS)

    Bae, S.; Bombardelli, F.; Wuertz, S.

    2008-12-01

    Understanding and managing microbial pollutions in water is one of the foremost challenges of establishing effective managements and remediation strategies to impaired water bodies polluted by uncharacterized fecal sources. Quantitative microbial source tracking (MST) approaches using fecal Bacteroidales and quantitative PCR (qPCR) assays to measure gene copies of host-specific 16S rRNA genetic markers are promising because they can allow for identifying and quantifying fecal loadings from a particular animal host and understanding the fate and transport of host-specific Bacteroidales over a range of conditions in water bodies. Similar to the case of traditional fecal indicator bacteria, a relatively long persistence of target DNA may hamper applied MST studies, if genetic markers cannot be linked to recent fecal pollution in water. We report a successful approach to removing the qPCR signal derived from free DNA and dead host-specific Bacteroidales cells by selectively binding the DNA and consequently inhibiting PCR amplification using light- activated propidium monoazide (PMA). Optimal PMA-qPCR conditions were determined as 100 µM of PMA concentration and a 10-min light exposure time at different solids concentrations in order to mimic a range of water samples. Under these conditions, PMA-qPCR resulted in the selective exclusion of DNA from heat- treated cells of non-culturable Bacteroidales in human feces and wastewater influent and effluent samples. Also, the persistence of feces-derived host-specific Bacteroidales DNA and their cells (determined by universal, human-, cow- and dog-specific Bacteroidales qPCR assays) in seawater was investigated in microcosms at environmental conditions. The average T99 (two log reduction) value for host-specific viable Bacteroidales cells was 28 h, whereas that for total host-specific Bacteroidales DNA was 177 h. Natural sunlight did not have a strong influence on the fate of fecal Bacteroidales cells and their DNA, presumably

  16. Identifying Francisella tularensis genes required for growth in host cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical Abstract: Francisella tularensis is a highly virulent Gram negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cel...

  17. α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

    PubMed Central

    Grula, Marjori A.; Buller, Patricia L.; Weaver, Robert F.

    1981-01-01

    [3H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II. PMID:16789208

  18. The mechanism of HCV entry into host cells.

    PubMed

    Douam, Florian; Lavillette, Dimitri; Cosset, François-Loïc

    2015-01-01

    Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus classified within the Flaviviridae family and is a major cause of liver disease worldwide. HCV life cycle and propagation are tightly linked to several aspects of lipid metabolism. HCV propagation depends on and also shapes several aspects of lipid metabolism such as cholesterol uptake and efflux through different lipoprotein receptors during its entry into cells, lipid metabolism modulating HCV genome replication, lipid droplets acting as a platform for recruitment of viral components, and very low density lipoprotein assembly pathway resulting in incorporation of neutral lipids and apolipoproteins into viral particles. During the first steps of infection, HCV enters hepatocytes through a multistep and slow process. The initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I, a major receptor of high-density lipoprotein, the CD81 tetraspanin, and the tight junction proteins Claudin-1 and Occludin. This tight concert of receptor interactions ultimately leads to uptake and cellular internalization of HCV through a process of clathrin-dependent endocytosis. Over the years, the identification of the HCV entry receptors and cofactors has led to a better understanding of HCV entry and of the narrow tropism of HCV for the liver. Yet, the role of the two HCV envelope glycoproteins, E1 and E2, remains ill-defined, particularly concerning their involvement in the membrane fusion process. Here, we review the current knowledge and advances addressing the mechanism of HCV cell entry within hepatocytes and we highlight the challenges that remain to be addressed. PMID:25595801

  19. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    SciTech Connect

    Zhang, Lei; Zhang, Qing; Yang, Yu; Wu, Chuanfang

    2014-02-14

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.

  20. Spatially resolved, highly multiplexed RNA profiling in single cells

    PubMed Central

    Chen, Kok Hao; Boettiger, Alistair N.; Moffitt, Jeffrey R.; Wang, Siyuan; Zhuang, Xiaowei

    2015-01-01

    Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 – 1000 unique RNA species in hundreds of individual cells. Correlation analysis of the ~104 – 106 pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins. PMID:25858977

  1. Identification and Analysis of Novel Viral and Host Dysregulated MicroRNAs in Variant Pseudorabies Virus-Infected PK15 Cells

    PubMed Central

    Liu, Fei; Zheng, Hao; Tong, Wu; Li, Guo-Xin; Tian, Qing; Liang, Chao; Li, Li-Wei; Zheng, Xu-Chen; Tong, Guang-Zhi

    2016-01-01

    Pseudorabies (PR) is one of the most devastating diseases in the pig industry. To identify changes in microRNA (miRNA) expression and post-transcriptional regulatory responses to PRV infection in porcine kidney epithelial (PK15) cells, we sequenced a small RNA (sRNA) library prepared from infected PK15 cells and compared it to a library prepared from uninfected cells using Illumina deep sequencing. Here we found 25 novel viral miRNAs by high-throughput sequencing and 20 of these miRNAs were confirmed through stem-loop RT-qPCR. Intriguingly, unlike the usual miRNAs encoded by the α-herpesviruses, which are found clustered in the large latency transcript (LLT), these novel viral miRNAs are throughout the PRV genome like β-herpesviruses. Viral miRNAs are predicted to target multiple genes and form a complex regulatory network. GO analysis on host targets of viral miRNAs were involved in complex cellular processes, including the metabolic pathway, biological regulation, stimulus response, signaling process and immune response. Moreover, 13 host miRNAs were expressed with significant difference after infection with PRV: 8 miRNAs were up-regulated and 5 miRNAs were down-regulated, which may affect viral replication in host cell. Our results provided new insight into the characteristic of miRNAs in response to PRV infection, which is significant for further study of these miRNAs function. PMID:26998839

  2. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  3. Characterization of the stage(s) in the virus replication cycle at which the host-cell specificity of the feline parvovirus subgroup is regulated in canine cells.

    PubMed

    Horiuchi, M; Ishiguro, N; Goto, H; Shinagawa, M

    1992-08-01

    Feline panleukopenia virus (FPLV), mink enteritis virus (MEV), and canine parvovirus (CPV) are classified as a host-range variants. They show different host-range specificity in vivo and host-cell specificity in vitro. For instance, FPLV and MEV cannot grow or can grow only inefficiently in canine cell lines such as MDCK and the canine fibroma cell line A72. Here we have studied the mechanism(s) by which the different cell tropism is mediated in vitro. When FPLV or MEV was inoculated to A72 cells, viral DNA replicated slightly, few viral-antigen-positive cells were detected, and the culture fluid contained the threshold level of infectivity. On the other hand, when an infectious molecular clone of MEV (pMEV) was introduced into A72 cells, viral DNA replicated efficiently, and the culture fluid of pMEV-transfected cells contained much higher infectivities than that of MEV-infected cells. In spite of the restrictive growth in A72 cells, MEV could bind to A72 cells as efficiently as CPV. No detectable viral RNA was produced in MEV-infected A72 cells. In contrast, efficient viral transcription occurred in pMEV-transfected A72 cells. These results suggest that the restrictive infections of MEV and FPLV in A72 cells are not mediated by the attachment of the virus to the cells or by the events occurring after the viral transcription. It appears to be caused by the stage(s) in the virus replication cycle, which exists between a postadsorptional step required for virus penetration and the initiation of viral transcription. PMID:1322591

  4. Efficient siRNA Delivery Using Novel Cell-Penetrating Peptide-siRNA Conjugate-Loaded Nanobubbles and Ultrasound.

    PubMed

    Xie, Xiangyang; Lin, Wen; Li, Mingyuan; Yang, Yang; Deng, Jianping; Liu, Hui; Chen, Ying; Fu, Xudong; Liu, Hong; Yang, Yanfang

    2016-06-01

    Because of the absence of tolerable and effective carriers for in vivo delivery, the applications of small interfering RNA (siRNA) in the clinic for therapeutic purposes have been limited. In this study, development of a novel siRNA delivery system based on ultrasound-sensitive nanobubbles (NBs, nano-sized echogenic liposomes) and cell-permeable peptides (CPPs) is described. A CPP-siRNA conjugate was entrapped in an NB, (CPP-siRNA)-NB, and the penetration of CPP-siRNA was temporally masked; local ultrasound stimulation triggered the release of CPP-siRNA from the NBs and activated its penetration. Subsequent research revealed that the (CPP-siRNA)-NBs had a mean particle size of 201 ± 2.05 nm and a siRNA entrapment efficiency >85%. In vitro release results indicated that >90% of the encapsulated CPP-siRNA was released from NBs in the presence of ultrasound, whereas <1.5% (30 min) was released in the absence of ultrasound. Cell experiments indicated higher cellular CPP-siRNA uptake of (CPP-siRNA)-NBs with ultrasound among the various formulations in human breast adenocarcinoma cells (HT-1080). Additionally, after systemic administration in mice, (CPP-siRNA)-NBs accumulated in the tumor, augmented c-myc silencing and delayed tumor progression. In conclusion, the application of (CPP-siRNA)-NBs with ultrasound may constitute an approach to selective targeted delivery of siRNA. PMID:27012462

  5. Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures

    DOEpatents

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2014-09-30

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  6. Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures

    DOEpatents

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2016-04-19

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  7. Host cell reactivation studies with epidermal cells of mice sensitive and resistant to carcinogenesis

    SciTech Connect

    Strickland, J.E.; Strickland, A.G.

    1984-03-01

    Primary epidermal cells from AKR, BALB/c, CD-1, and SENCAR mice, listed in order of least to most sensitive to epidermal carcinogenesis by initiation and promotion protocols, were found to be equally competent to ''reactivate'' herpes simplex virus type 1 irradiated by germicidal ultraviolet radiation. Nontumorigenic BALB/c epidermal cell lines selected in vitro for resistance to terminal differentiation after in vivo or in vitro treatment with initiating doses of carcinogens showed virus survival curves similar to those of primary cells. Similarly, primary cultures which were allowed to grow to confluency following a single treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml) retained normal host cell reactivation. Host cell reactivation studies with mouse dermal fibroblasts could not be done because of the failure of the herpes simplex virus to infect these cells and produce plaques. These results demonstrate that survival of ultraviolet light-damaged virus in primary epidermal cells in culture is unrelated to whether the cells are derived from mice sensitive or resistant to epidermal carcinogenesis. Furthermore, virus survival is not changed by tumor promoter treatment or by treatment with initiating doses of carcinogens which results in differentiation-resistant cells.

  8. Roles of microRNA on cancer cell metabolism

    PubMed Central

    2012-01-01

    Advanced studies of microRNAs (miRNAs) have revealed their manifold biological functions, including control of cell proliferation, cell cycle and cell death. However, it seems that their roles as key regulators of metabolism have drawn more and more attention in the recent years. Cancer cells display increased metabolic autonomy in comparison to non-transformed cells, taking up nutrients and metabolizing them in pathways that support growth and proliferation. MiRNAs regulate cell metabolic processes through complicated mechanisms, including directly targeting key enzymes or transporters of metabolic processes and regulating transcription factors, oncogenes / tumor suppressors as well as multiple oncogenic signaling pathways. MiRNAs like miR-375, miR-143, miR-14 and miR-29b participate in controlling cancer cell metabolism by regulating the expression of genes whose protein products either directly regulate metabolic machinery or indirectly modulate the expression of metabolic enzymes, serving as master regulators, which will hopefully lead to a new therapeutic strategy for malignant cancer. This review focuses on miRNA regulations of cancer cell metabolism,including glucose uptake, glycolysis, tricarboxylic acid cycle and insulin production, lipid metabolism and amino acid biogenesis, as well as several oncogenic signaling pathways. Furthermore, the challenges of miRNA-based strategies for cancer diagnosis, prognosis and therapeutics have been discussed. PMID:23164426

  9. Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission

    PubMed Central

    Schmidt, Nora; Hennig, Thomas; Serwa, Remigiusz A.; Marchetti, Magda

    2015-01-01

    ABSTRACT Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium, we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways in which virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as of mechanisms governing host DNA synthesis. IMPORTANCE We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus-induced paracrine

  10. Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

    PubMed Central

    Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W.

    2015-01-01

    Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases. PMID:25923429

  11. From microbiology to cell biology: when an intracellular bacterium becomes part of its host cell.

    PubMed

    McCutcheon, John P

    2016-08-01

    Mitochondria and chloroplasts are now called organelles, but they used to be bacteria. As they transitioned from endosymbionts to organelles, they became more and more integrated into the biochemistry and cell biology of their hosts. Work over the last 15 years has shown that other symbioses show striking similarities to mitochondria and chloroplasts. In particular, many sap-feeding insects house intracellular bacteria that have genomes that overlap mitochondria and chloroplasts in terms of size and coding capacity. The massive levels of gene loss in some of these bacteria suggest that they, too, are becoming highly integrated with their host cells. Understanding these bacteria will require inspiration from eukaryotic cell biology, because a traditional microbiological framework is insufficient for understanding how they work. PMID:27267617

  12. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

    PubMed

    Mamta; Reddy, K R K; Rajam, M V

    2016-02-01

    Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera. PMID:26659592

  13. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.

    PubMed

    Ramakrishna, Suresh; Kwaku Dad, Abu-Bonsrah; Beloor, Jagadish; Gopalappa, Ramu; Lee, Sang-Kyung; Kim, Hyongbum

    2014-06-01

    RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system represent an efficient tool for genome editing. RGENs consist of two components: Cas9 protein and guide RNA. Plasmid-mediated delivery of these components into cells can result in uncontrolled integration of the plasmid sequence into the host genome, and unwanted immune responses and potential safety problems that can be caused by the bacterial sequences. Furthermore, this delivery method requires transfection tools. Here we show that simple treatment with cell-penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs leads to endogenous gene disruptions in human cell lines. The Cas9 protein was conjugated to CPP via a thioether bond, whereas the guide RNA was complexed with CPP, forming condensed, positively charged nanoparticles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the modified Cas9 and guide RNA, leads to efficient gene disruptions with reduced off-target mutations relative to plasmid transfections, resulting in the generation of clones containing RGEN-induced mutations. Our CPP-mediated RGEN delivery process provides a plasmid-free and additional transfection reagent-free method to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing. PMID:24696462

  14. Establishment of Myotis myotis Cell Lines - Model for Investigation of Host-Pathogen Interaction in a Natural Host for Emerging Viruses

    PubMed Central

    He, Xiaocui; Korytář, Tomáš; Zhu, Yaqing; Pikula, Jiří; Bandouchova, Hana; Zukal, Jan; Köllner, Bernd

    2014-01-01

    Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a

  15. Establishment of Myotis myotis cell lines--model for investigation of host-pathogen interaction in a natural host for emerging viruses.

    PubMed

    He, Xiaocui; Korytář, Tomáš; Zhu, Yaqing; Pikula, Jiří; Bandouchova, Hana; Zukal, Jan; Köllner, Bernd

    2014-01-01

    Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a

  16. MicroRNA-Offset RNA Alters Gene Expression and Cell Proliferation

    PubMed Central

    Zhao, Jin; Schnitzler, Gavin R.; Iyer, Lakshmanan K.; Aronovitz, Mark J.; Baur, Wendy E.; Karas, Richard H.

    2016-01-01

    MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We demonstrate that endogenous or over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. We report that there is a “seed region” of moR-21 as well as a “seed match region” in the target gene 3’UTR that are indispensable for moR-21-mediated gene down-regulation. We further demonstrate that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. Taken together, these findings provide the first evidence that microRNA offset RNA alters gene expression and is biologically active. PMID:27276022

  17. Identification of a novel RNA giant nuclear body in cancer cells

    PubMed Central

    Gan, Xiaoxian; Gan, Yichao; Zheng, Weiwei; Kim, Byung-Wook; Xu, Xiaohua; Lu, Xiaoya; Dong, Qi; Zheng, Shu; Huang, Wendong; Xu, Rongzhen

    2016-01-01

    Constitutive synthesis of oncogenic mRNAs is essential for maintaining the uncontrolled growth of cancer cells. However, little is known about how these mRNAs are exported from the nucleus to the cytoplasm. Here, we report the identification of a RNA giant nuclear body (RNA-GNB) that is abundant in cancer cells but rare in normal cells. The RNA-GNB contains a RNA core surrounded by a protein shell. We identify 782 proteins from cancer-associated RNA-GNBs, 40% of which are involved in the nuclear mRNA trafficking. RNA-GNB is required for cell proliferation, and its abundance is positively associated with tumor burden and outcome of therapies. Our findings suggest that the RNA-GNB is a novel nuclear RNA trafficking organelle that may contribute to the nuclear mRNA exporting and proliferation of cancer cells. PMID:26678034

  18. Cell-free Circulating miRNA Biomarkers in Cancer

    PubMed Central

    Mo, Meng-Hsuan; Chen, Liang; Fu, Yebo; Wang, Wendy; Fu, Sidney W.

    2012-01-01

    Considerable attention and an enormous amount of resources have been dedicated to cancer biomarker discovery and validation. However, there are still a limited number of useful biomarkers available for clinical use. An ideal biomarker should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. Commonly used circulating biomarkers are proteins in serum, most of which require labor-intensive analysis hindered by low sensitivity in early tumor detection. Since the deregulation of microRNA (miRNA) is associated with cancer development and progression, profiling of circulating miRNAs has been used in a number of studies to identify novel minimally invasive miRNA biomarkers. In this review, we discuss the origin of the circulating cell-free miRNAs and their carriers in blood. We summarize the clinical use and function of potentially promising miRNA biomarkers in a variety of different cancers, along with their downstream target genes in tumor initiation and development. Additionally, we analyze some technical challenges in applying miRNA biomarkers to clinical practice. PMID:23074383

  19. A Systems Survey of Progressive Host-Cell Reorganization during Rotavirus Infection.

    PubMed

    Green, Victoria A; Pelkmans, Lucas

    2016-07-13

    Pathogen invasion is often accompanied by widespread alterations in cellular physiology, which reflects the hijacking of host factors and processes for pathogen entry and replication. Although genetic perturbation screens have revealed the complexity of host factors involved for numerous pathogens, it has remained challenging to temporally define the progression of events in host cell reorganization during infection. We combine high-confidence genome-scale RNAi screening of host factors required for rotavirus infection in human intestinal cells with an innovative approach to infer the trajectory of virus infection from fixed cell populations. This approach reveals a comprehensive network of host cellular processes involved in rotavirus infection and implicates AMPK in initiating the development of a rotavirus-permissive environment. Our work provides a powerful approach that can be generalized to order complex host cellular requirements along a trajectory of cellular reorganization during pathogen invasion. PMID:27414499

  20. Host cellular microRNA involvement in the control of hepatitis B virus gene expression and replication

    PubMed Central

    Mizuguchi, Yoshiaki; Takizawa, Toshihiro; Uchida, Eiji

    2015-01-01

    A large number of studies have demonstrated that the synergistic collaboration of a number of microRNAs (miRNAs), their growth factors and their downstream agents is required for the initiation and completion of pathogenesis in the liver. miRNAs are thought to exert a profound effect on almost every aspect of liver biology and pathology. Accumulating evidence indicates that several miRNAs are involved in the hepatitis B virus (HBV) life cycle and infectivity, in addition to HBV-associated liver diseases including fibrosis, cirrhosis and hepatocellular carcinoma (HCC). In turn, HBV can modulate the expression of several cellular miRNAs, thus promoting a favorable environment for its replication and survival. In this review, we focused on the involvement of host cellular miRNAs that are directly and indirectly associated with HBV RNA or HBV associated transcription factors. Exploring different facets of the interactions among miRNA, HBV and HCV infections, and the carcinogenesis and progress of HCC, could facilitate the development of novel and effective treatment approaches for liver disease. PMID:25866606

  1. Elimination of allogeneic multipotent stromal cells by host macrophages in different models of regeneration

    PubMed Central

    Arutyunyan, Irina; Elchaninov, Andrey; Fatkhudinov, Timur; Makarov, Andrey; Kananykhina, Evgeniya; Usman, Natalia; Bolshakova, Galina; Glinkina, Valeria; Goldshtein, Dmitry; Sukhikh, Gennady

    2015-01-01

    Allogeneic multipotent stromal cells were previously thought to be poorly recognized by host immune system; the prolonged survival in host environments was explained by their immune privileged status. As long as the concept is currently reconsidered, the routes of elimination of allogeneic multipotent stromal cells by host immunity must be taken into account. This is necessary for correct comprehension of their therapeutic action. The study was focused upon survival of umbilical cord-derived allogeneic multipotent stromal cells in different rat models of tissue regeneration induced by partial hepatectomy or by critical limb ischemia. The observations were carried out by means of vital labeling of the cells with PKH26 prior to injection, in combination with differential immunostaining of host macrophages with anti-CD68 antibody. According to the results, allogeneic multipotent stromal cells are specifically eliminated by host immune system; the efficacy can reach 100%. Massive clearance of transplanted cells by host macrophages is accompanied by appropriation of the label by the latter, and this is a pronounced case of misleading presentation of exogenous label by host cells. The study emphasizes the role of macrophages in host response and also the need of additional criteria for correct data interpretation. PMID:26191137

  2. Elimination of allogeneic multipotent stromal cells by host macrophages in different models of regeneration.

    PubMed

    Arutyunyan, Irina; Elchaninov, Andrey; Fatkhudinov, Timur; Makarov, Andrey; Kananykhina, Evgeniya; Usman, Natalia; Bolshakova, Galina; Glinkina, Valeria; Goldshtein, Dmitry; Sukhikh, Gennady

    2015-01-01

    Allogeneic multipotent stromal cells were previously thought to be poorly recognized by host immune system; the prolonged survival in host environments was explained by their immune privileged status. As long as the concept is currently reconsidered, the routes of elimination of allogeneic multipotent stromal cells by host immunity must be taken into account. This is necessary for correct comprehension of their therapeutic action. The study was focused upon survival of umbilical cord-derived allogeneic multipotent stromal cells in different rat models of tissue regeneration induced by partial hepatectomy or by critical limb ischemia. The observations were carried out by means of vital labeling of the cells with PKH26 prior to injection, in combination with differential immunostaining of host macrophages with anti-CD68 antibody. According to the results, allogeneic multipotent stromal cells are specifically eliminated by host immune system; the efficacy can reach 100%. Massive clearance of transplanted cells by host macrophages is accompanied by appropriation of the label by the latter, and this is a pronounced case of misleading presentation of exogenous label by host cells. The study emphasizes the role of macrophages in host response and also the need of additional criteria for correct data interpretation. PMID:26191137

  3. Listeria monocytogenes Exploits Normal Host Cell Processes to Spread from Cell to Cell✪

    PubMed Central

    Robbins, Jennifer R.; Barth, Angela I.; Marquis, Hélène; de Hostos, Eugenio L.; Nelson, W. James; Theriot, Julie A.

    1999-01-01

    The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1–2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy. PMID:10491395

  4. Analyses of the radiation of birnaviruses from diverse host phyla and of their evolutionary affinities with other double-stranded RNA and positive strand RNA viruses using robust structure-based multiple sequence alignments and advanced phylogenetic methods

    PubMed Central

    2013-01-01

    Background Birnaviruses form a distinct family of double-stranded RNA viruses infecting animals as different as vertebrates, mollusks, insects and rotifers. With such a wide host range, they constitute a good model for studying the adaptation to the host. Additionally, several lines of evidence link birnaviruses to positive strand RNA viruses and suggest that phylogenetic analyses may provide clues about transition. Results We characterized the genome of a birnavirus from the rotifer Branchionus plicalitis. We used X-ray structures of RNA-dependent RNA polymerases and capsid proteins to obtain multiple structure alignments that allowed us to obtain reliable multiple sequence alignments and we employed “advanced” phylogenetic methods to study the evolutionary relationships between some positive strand and double-stranded RNA viruses. We showed that the rotifer birnavirus genome exhibited an organization remarkably similar to other birnaviruses. As this host was phylogenetically very distant from the other known species targeted by birnaviruses, we revisited the evolutionary pathways within the Birnaviridae family using phylogenetic reconstruction methods. We also applied a number of phylogenetic approaches based on structurally conserved domains/regions of the capsid and RNA-dependent RNA polymerase proteins to study the evolutionary relationships between birnaviruses, other double-stranded RNA viruses and positive strand RNA viruses. Conclusions We show that there is a good correlation between the phylogeny of the birnaviruses and that of their hosts at the phylum level using the RNA-dependent RNA polymerase (genomic segment B) on the one hand and a concatenation of the capsid protein, protease and ribonucleoprotein (genomic segment A) on the other hand. This correlation tends to vanish within phyla. The use of advanced phylogenetic methods and robust structure-based multiple sequence alignments allowed us to obtain a more accurate picture (in terms of

  5. Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host.

    PubMed

    Gaimster, Hannah; Summers, David

    2015-03-01

    Regulation by non-coding RNAs was found to be widespread among plasmids and other mobile elements of bacteria well before its ubiquity in the eukaryotic world was suspected. As an increasing number of examples was characterised, a common mechanism began to emerge. Non-coding RNAs, such as CopA and Sok from plasmid R1, or RNAI from ColE1, exerted regulation by refolding the secondary structures of their target RNAs or modifying their translation. One regulatory RNA that seemed to swim against the tide was Rcd, encoded within the multimer resolution site of ColE1. Required for high fidelity maintenance of the plasmid in recombination-proficient hosts, Rcd was found to have a protein target, elevating indole production by stimulating tryptophanase. Rcd production is up-regulated in dimer-containing cells and the consequent increase in indole is part of the response to the rapid accumulation of dimers by over-replication (known as the dimer catastrophe). It is proposed that indole simultaneously inhibits cell division and plasmid replication, stopping the catastrophe and allowing time for the resolution of dimers to monomers. The idea of a plasmid-mediated cell division checkpoint, proposed but then discarded in the 1980s, appears to be enjoying a revival. PMID:25446541

  6. Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    PubMed Central

    Gan, Huachen; Hao, Qin; Idell, Steven; Tang, Hua

    2015-01-01

    Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β−NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis. PMID:26643317

  7. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation.

    PubMed

    Cheng, Xiaofei; Deng, Ping; Cui, Hongguang; Wang, Aiming

    2015-11-01

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. PMID:26351203

  8. Prostacyclin-producing human mesenchymal cells target H19 lncRNA to augment endogenous progenitor function in hindlimb ischaemia

    PubMed Central

    Deng, Yuxiao; Yang, Zhongwei; Terry, Toya; Pan, Su; Woodside, Darren G.; Wang, Jingxiong; Ruan, Kehe; Willerson, James T.; Dixon, Richard A. F.; Liu, Qi

    2016-01-01

    Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy to enhance the paracrine effects of hMSCs. In a mouse hindlimb ischaemia model, we examine the effects of hMSCs in which a novel triple-catalytic enzyme is introduced to stably produce prostacyclin (PGI2-hMSCs). We show that PGI2-hMSCs facilitate perfusion recovery and enhance running capability as compared with control hMSCs or iloprost (a stable PGI2 analogue). Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner. Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions. Together, our data reveal the novel ability of PGI2-hMSCs to stimulate host regenerative processes and improve physical function by regulating long noncoding RNA in resident progenitor cells. PMID:27080438

  9. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle.

    PubMed

    Catta-Preta, Carolina M C; Brum, Felipe L; da Silva, Camila C; Zuma, Aline A; Elias, Maria C; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M

    2015-01-01

    Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

  10. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

    PubMed Central

    Catta-Preta, Carolina M. C.; Brum, Felipe L.; da Silva, Camila C.; Zuma, Aline A.; Elias, Maria C.; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M.

    2015-01-01

    Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

  11. A host cell membrane microdomain is a critical factor for organelle discharge by Toxoplasma gondii.

    PubMed

    Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Matsubara, Ryuma; Aonuma, Hiroka; Nagamune, Kisaburo

    2016-10-01

    Host cell microdomains are involved in the attachment, entry, and replication of intracellular microbial pathogens. Entry into the host cell of Toxoplasma gondii and the subsequent survival of this protozoan parasite are tightly coupled with the proteins secreted from organelle called rhoptry. The rhoptry proteins are rapidly discharged into clusters of vesicles, called evacuoles, which are then delivered to parasitophorous vacuoles (PVs) or nucleus. In this study, we examined the roles of two host cell microdomain components, cholesterol and glycosylphosphatidylinositol (GPI), in evacuole formation. The acute depletion of cholesterol from the host cell plasma membrane blocked evacuole formation but not invasion. Whereas the lack of host cell GPI also altered evacuole formation but not invasion, instead inducing excess evacuole formation. The latter effect was not influenced by the evacuole-inhibiting effects of host cell cholesterol depletion, indicating the independent roles of host GPI and cholesterol in evacuole formation. In addition, the excess formation of evacuoles resulted in the enhanced recruitment of host mitochondria and endoplasmic reticulum to PVs, which in turn stimulated the growth of the parasite. PMID:27217289

  12. Human immunodeficiency virus and host cell lipids. Interesting pathways in research for a new HIV therapy.

    PubMed

    Raulin, Jeanine

    2002-01-01

    It has been reported in the literature that biological membranes arising from HIV-induced cell fusion, as well as syncytium formation between infected and non-infected cells and those involved in transduction, viral DNA nuclear import and virion budding from the host cell, are all made of proteins, a phospholipid (P) bilayer and cholesterol (C). However, the P/C molar ratio is higher in the retroviral envelope than in the plasma membrane where they originate, and higher than in the nuclear envelope. Mechanisms are described which elucidate this puzzling fact, as well as cholesterol-dependent leakage and pore formation during cell fusion. Fatty acylation of viral and host cell proteins is required to direct them to membranes. Detergent-insoluble microdomains enriched in cholesterol and sphingolipids, termed either DIGs (detergent-insoluble glycolipid-enriched complexes), DRMs (detergent resistant membranes), TIFFs (Triton-insoluble floating fractions) or GEMs (glycolipid-enriched membranes), function as platforms for attachment of proteins in the process of signal transduction. HIV-SUgp120 (HIV-surface glycoprotein), T-cell receptor (TCR)-CD4+ and co-receptors promote aggregation of these lipid "rafts" which concentrate the Src family tyrosine kinases SFKs (PTK, Lyn, Fyn, Lck), GPI (glycosyl phosphatidylinositol)-anchored proteins, and phosphatidylinositol kinases PI(3)K and PI(4)K, inducing cell signalling. HIV-SUgp120 transduces the activation signal and provokes the formation of polyunsaturated fatty acid (PUFA) metabolites, i.e. the prostaglandin PGE2 suppressor of immune function and inhibitor of cytotoxic T-lymphocyte (CTL) proliferation, while PGB2 activates SFKs and increases mRNA expression, as well as NFkappaB (nuclear transcription factor) translocation to nucleus. HIV nuclear import, DNA integration, chromatin template capacity may be mediated by the lipid environment. The lipid-enriched microdomains from which HIV-1 buds, may explain the high level of

  13. RNA-Based Vaccines in Cancer Immunotherapy

    PubMed Central

    McNamara, Megan A.; Nair, Smita K.; Holl, Eda K.

    2015-01-01

    RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy. PMID:26665011

  14. A novel betapartitivirus RnPV6 from Rosellinia necatrix tolerates host RNA silencing but is interfered by its defective RNAs.

    PubMed

    Chiba, Sotaro; Lin, Yu-Hsin; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2016-07-01

    The family Partitiviridae comprises of five genera with bi-segmented dsRNA genomes that accommodate members infecting plants, fungi or protists. All partitiviruses with only a few exceptions cause asymptomatic infections. We report the characterization of a novel betapartitivirus termed Rosellinia necatrix partitivirus 6 (RnPV6) from a field isolate of a plant pathogenic fungus, white root rot fungus. RnPV6 has typical partitivirus features: dsRNA1 and dsRNA2 are 2462 and 2499bps in length encoding RNA-dependent RNA polymerase and capsid protein. Purified particles are spherical with a diameter of 30nm. Taking advantage of infectivity as virions, RnPV6 was introduced into a model filamentous fungal host, chestnut blight fungus to investigate virus/host interactions. Unlike other partitiviruses tested previously, RnPV6 induced profound phenotypic alterations with symptoms characterized by a reduced growth rate and enhanced pigmentation and was tolerant to host RNA silencing. In addition, a variety of defective RNAs derived from dsRNA1 appear after virion transfection. These sub-viral RNAs were shown to interfere with RnPV6 replication, at least for that of cognate segment dsRNA1. Presence of these sub-viral elements resulted in reduced symptom expression by RnPV6, suggesting their nature as defective-interfering RNAs. The features of RnPV6 are similar to but distinct from those of a previously reported alphapartitivirus, Rosellinia necatrix partitivirus 2 that is susceptible to RNA silencing. PMID:26494168

  15. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    PubMed Central

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  16. PIWI-interacting RNA (piRNA) signatures in human cardiac progenitor cells.

    PubMed

    Vella, Serena; Gallo, Alessia; Lo Nigro, Antonio; Galvagno, Daniele; Raffa, Giuseppe Maria; Pilato, Michele; Conaldi, Pier Giulio

    2016-07-01

    Cardiac progenitors, such as cardiospheres and cardiosphere-derived cells, represent an attractive cell source for cardiac regeneration. The PIWI-interacting RNAs, piRNAs, are an intriguing class of small non-coding RNAs, implicated in the regulation of epigenetic state, maintenance of genomic integrity and stem cell functions. Although non-coding RNAs are an exploiting field in cardiovascular research, the piRNA signatures of cardiac progenitors has not been evaluated yet.We profiled, through microarrays, 15,311 piRNAs expressed in cardiospheres, cardiosphere-derived cells and cardiac fibroblasts. Results showed a set of differentially expressed piRNAs (fold change ≥2, p<0.01): 641 piRNAs were upregulated and 1,301 downregulated in the cardiospheres compared to cardiosphere-derived cells, while 255 and 708 piRNAs resulted up- and down-regulated, respectively, if compared to cardiac fibroblasts. We also identified 181 piRNAs that are overexpressed and 129 are downregulated in cardiosphere-derived cells respect to cardiac fibroblasts.Bioinformatics analysis showed that the deregulated piRNAs were mainly distributed on few chromosomes, suggesting that piRNAs are organized in discrete genomic clusters.Furthermore, the bioinformatics search showed that the most upregulated piRNAs target transposons, especially belonged to LINE-1 class, as validated by qRT-PCR. This reduction is also associated to an activation of AKT signaling, which is beneficial for cardiac regeneration.The present study is the first to show a highly consistent piRNA expression pattern for human cardiac progenitors, likely responsible of their different regenerative power. Moreover, this piRNome analysis may provide new methods for characterize cardiac progenitors and may shed new light on the understanding the complex molecular mechanisms of cardiac regeneration. PMID:27131603

  17. Tracking single mRNA molecules in live cells

    NASA Astrophysics Data System (ADS)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  18. Stage-specific regulation of natural killer cell homeostasis and response against viral infection by microRNA-155

    PubMed Central

    Zawislak, Carolyn L.; Beaulieu, Aimee M.; Loeb, Gabriel B.; Karo, Jenny; Canner, David; Bezman, Natalie A.; Lanier, Lewis L.; Rudensky, Alexander Y.; Sun, Joseph C.

    2013-01-01

    Natural killer (NK) cells function in the recognition and destruction of host cells infected with pathogens. Many regulatory mechanisms govern the potent responses of NK cells, both at the cellular and molecular level. Ablation of microRNA (miRNA) processing enzymes demonstrated that miRNAs play critical roles in NK cell differentiation and function; however, the role of individual miRNAs requires further investigation. Using mice containing a targeted deletion of microRNA-155 (miR-155), we observed defects in NK cell maintenance and maturation at steady state, as well as in homeostatic proliferation in lymphopenic mice. In addition, we discovered that miR-155 is up-regulated in activated NK cells during mouse cytomegalovirus (MCMV) infection in response to signals from the proinflammatory cytokines IL-12 and IL-18 and through signal transducer and activator of transcription 4 (STAT4) signaling. Although miR-155 was found to be dispensable for cytotoxicity and cytokine production when triggered through activating receptors, NK cells lacking miR-155 exhibited severely impaired effector and memory cell numbers in both lymphoid and nonlymphoid tissues after MCMV infection. We demonstrate that miR-155 differentially targets Noxa and suppressor of cytokine signaling 1 (SOCS1) in NK cells at distinct stages of homeostasis and activation. NK cells constitutively expressing Noxa and SOCS1 exhibit profound defects in expansion during the response to MCMV infection, suggesting that their regulation by miR-155 promotes antiviral immunity. PMID:23572582

  19. Cellular Aspects of Shigella Pathogenesis: Focus on the Manipulation of Host Cell Processes

    PubMed Central

    Killackey, Samuel A.; Sorbara, Matthew T.; Girardin, Stephen E.

    2016-01-01

    Shigella is a Gram-negative bacterium that is responsible for shigellosis. Over the years, the study of Shigella has provided a greater understanding of how the host responds to bacterial infection, and how bacteria have evolved to effectively counter the host defenses. In this review, we provide an update on some of the most recent advances in our understanding of pivotal processes associated with Shigella infection, including the invasion into host cells, the metabolic changes that occur within the bacterium and the infected cell, cell-to-cell spread mechanisms, autophagy and membrane trafficking, inflammatory signaling and cell death. This recent progress sheds a new light into the mechanisms underlying Shigella pathogenesis, and also more generally provides deeper understanding of the complex interplay between host cells and bacterial pathogens in general. PMID:27066460

  20. Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells

    PubMed Central

    Sladitschek, Hanna L.

    2016-01-01

    MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level. PMID:27152616

  1. Graft-infiltrating host dendritic cells play a key role in organ transplant rejection.

    PubMed

    Zhuang, Quan; Liu, Quan; Divito, Sherrie J; Zeng, Qiang; Yatim, Karim M; Hughes, Andrew D; Rojas-Canales, Darling M; Nakao, A; Shufesky, William J; Williams, Amanda L; Humar, Rishab; Hoffman, Rosemary A; Shlomchik, Warren D; Oberbarnscheidt, Martin H; Lakkis, Fadi G; Morelli, Adrian E

    2016-01-01

    Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection. PMID:27554168

  2. Graft-infiltrating host dendritic cells play a key role in organ transplant rejection

    PubMed Central

    Zhuang, Quan; Liu, Quan; Divito, Sherrie J.; Zeng, Qiang; Yatim, Karim M.; Hughes, Andrew D.; Rojas-Canales, Darling M.; Nakao, A.; Shufesky, William J.; Williams, Amanda L.; Humar, Rishab; Hoffman, Rosemary A.; Shlomchik, Warren D.; Oberbarnscheidt, Martin H.; Lakkis, Fadi G.; Morelli, Adrian E.

    2016-01-01

    Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection. PMID:27554168

  3. MicroRNA-196b promotes cell proliferation and suppress cell differentiation in vitro

    SciTech Connect

    Cao, Donglin Hu, Liangshan; Lei, Da; Fang, Xiaolin; Zhang, Zhihong; Wang, Ting; Lin, Maorui; Huang, Jiwei; Yang, Huawen; Zhou, Xuan; Zhong, Limei

    2015-01-30

    Highlights: • miRNA-196b increases proliferation and blocks differentiation of progenitor cell. • miRNA-196b inhibits apoptosis and increases viability of cells lines. • Forced expression of miR-196b blocks the differentiation of THP1 induced by PMA. - Abstract: MicroRNA-196b (miR-196b) is frequently amplified and aberrantly overexpressed in acute leukemias. To investigate the role of miR-196b in acute leukemias, it has been observed that forced expression of this miRNA increases proliferation and inhibits apoptosis in human cell lines. More importantly, we show that this miRNA can significantly increase the colony-forming capacity of mouse normal bone marrow progenitor cells alone, as well as partially blocking the cells from differentiation. Taken together, our studies suggest that miRNA-196b may play an essential role in the development of MLL-associated leukemias through inhibiting cell differentiation and apoptosis, while promoting cell proliferation.

  4. Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

    PubMed Central

    Agris, P F

    1975-01-01

    A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells. PMID:1057159

  5. A Sequential Model of Host Cell Killing and Phagocytosis by Entamoeba histolytica

    PubMed Central

    Sateriale, Adam; Huston, Christopher D.

    2011-01-01

    The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model. PMID:21331284

  6. Structural alterations of the ribosomal RNA genes in leukemic cells.

    PubMed

    Smirnova, I A

    1992-01-01

    Cloned 6.7 kb EcoR1 fragment of mice rDNA was used as a hybridization probe for rDNA structure analysis in mice, rat and calf haemopoietic tumor and normal cells. EcoR1, BglII and Pst1 restriction fragment length polymorphism (RFLP) was found in neoplastic rDNA and was not revealed in normal ones. The rRNA gene rearrangements were observed not only in spacer region but in coding sequences of the genes. Leukemic cells reveal also rDNA amplification. A role of genetic rearrangements of rDNA for mechanisms of carcinogenesis is suggested. PMID:1342066

  7. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference.

    PubMed

    Nijhof, Ard M; Taoufik, Amar; de la Fuente, José; Kocan, Katherine M; de Vries, Erik; Jongejan, Frans

    2007-05-01

    The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis. PMID:17196597

  8. Host parasite communications-Messages from helminths for the immune system: Parasite communication and cell-cell interactions.

    PubMed

    Coakley, Gillian; Buck, Amy H; Maizels, Rick M

    2016-07-01

    Helminths are metazoan organisms many of which have evolved parasitic life styles dependent on sophisticated manipulation of the host environment. Most notably, they down-regulate host immune responses to ensure their own survival, by exporting a range of immuno-modulatory mediators that interact with host cells and tissues. While a number of secreted immunoregulatory parasite proteins have been defined, new work also points to the release of extracellular vesicles, or exosomes, that interact with and manipulate host gene expression. These recent results are discussed in the overall context of how helminths communicate effectively with the host organism. PMID:27297184

  9. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    PubMed Central

    Macaulay, Iain C.; Svensson, Valentine; Labalette, Charlotte; Ferreira, Lauren; Hamey, Fiona; Voet, Thierry; Teichmann, Sarah A.; Cvejic, Ana

    2016-01-01

    Summary The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment. PMID:26804912

  10. A piRNA-like small RNA interacts with and modulates p-ERM proteins in human somatic cells

    PubMed Central

    Mei, Yuping; Wang, Yuyan; Kumari, Priti; Shetty, Amol Carl; Clark, David; Gable, Tyler; MacKerell, Alexander D.; Ma, Mark Z.; Weber, David J.; Yang, Austin J.; Edelman, Martin J.; Mao, Li

    2015-01-01

    PIWI-interacting RNAs (piRNAs) are thought to silence transposon and gene expression during development. However, the roles of piRNAs in somatic tissues are largely unknown. Here we report the identification of 555 piRNAs in