Involvement of Cryptosporidium parvum Cdg7_FLc_1000 RNA in the Attenuation of Intestinal Epithelial Cell Migration via Trans-Suppression of Host Cell SMPD3.

PubMed

Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Mathy, Nicholas W; Strauss-Soukup, Juliane K; Chen, Xian-Ming

2017-12-27

Intestinal infection by Cryptosporidium parvum causes inhibition of epithelial turnover, but underlying mechanisms are unclear. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using in vitro and in vivo models of intestinal cryptosporidiosis, we report here that host delivery of parasite Cdg7_FLc_1000 RNA results in inhibition of epithelial cell migration through suppression of the gene encoding sphingomyelinase 3 (SMPD3). Delivery of Cdg7_FLc_1000 into infected cells promotes the histone methyltransferase G9a-mediated H3K9 methylation in the SMPD3 locus. The DNA-binding transcriptional repressor, PR domain zinc finger protein 1, is required for the assembly of Cdg7_FLc_1000 into the G9a complex and associated with the enrichment of H3K9 methylation at the gene locus. Pathologically, nuclear transfer of Cryptosporidium parvum Cdg7_FLc_1000 RNA is involved in the attenuation of intestinal epithelial cell migration via trans-suppression of host cell SMPD3. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

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

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

PubMed

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

2016-02-03

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.

Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Mingyuan, E-mail: hanming@umich.edu; Ke, Hanz

Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversionmore » of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of {sub 126}-LQxxLxxxGL-{sub 135}. In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine

A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

PubMed Central

Hsu, Shih-Feng; Su, Wen-Chi; Jeng, King-Song

2015-01-01

ABSTRACT Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication. IMPORTANCE Investigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from

Engineering host-derived resistance against plant parasites through RNA interference: challenges and opportunities.

PubMed

Runo, Steven

2011-01-01

RNA interference (RNAi) has rapidly advanced to become a powerful genetic tool and holds promise to revolutionizing agriculture by providing a strategy for controlling a wide array of crop pests. Numerous studies document RNAi efficacy in achieving silencing in viruses, insects, nematodes and weeds parasitizing crops. In general, host derived pest resistance through RNAi is achieved by genetically transforming host plants with double stranded RNA constructs targeted at essential parasite genes leading to generation of small interfering RNAs (siRNAs). Small interfering RNAs formed in the host are then delivered to the parasite and transported to target cells. Delivery can be oral - worms and insects, viral infections, viruses - or through a vascular connections - parasitic plants, while delivery to target cells is by cell to cell systemic movement of the silencing signal. Despite the overall optimism in generating pest resistant crops through RNAi-mediated silencing, some hurdles have recently begun to emerge. Presently, the main challenge is delivery of sufficient siRNAs, in the right cells, and at the right time to mount; a strong, durable, and broad-spectrum posttranscriptional gene silencing (PTGS) signal. This review highlights the novel strategies available for improving host derived RNAi resistance in downstream applied agriculture.

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

Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus.

PubMed

Han, Mingyuan; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan

2017-05-01

Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL- 135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. Copyright © 2017 Elsevier Inc. All rights reserved.

Alternative Polyadenylation Allows Differential Negative Feedback of Human miRNA miR-579 on Its Host Gene ZFR

PubMed Central

Hinske, Ludwig Christian; Galante, Pedro A. F.; Limbeck, Elisabeth; Möhnle, Patrick; Parmigiani, Raphael B.; Ohno-Machado, Lucila; Camargo, Anamaria A.; Kreth, Simone

2015-01-01

About half of the known miRNA genes are located within protein-coding host genes, and are thus subject to co-transcription. Accumulating data indicate that this coupling may be an intrinsic mechanism to directly regulate the host gene’s expression, constituting a negative feedback loop. Inevitably, the cell requires a yet largely unknown repertoire of methods to regulate this control mechanism. We propose APA as one possible mechanism by which negative feedback of intronic miRNA on their host genes might be regulated. Using in-silico analyses, we found that host genes that contain seed matching sites for their intronic miRNAs yield longer 32UTRs with more polyadenylation sites. Additionally, the distribution of polyadenylation signals differed significantly between these host genes and host genes of miRNAs that do not contain potential miRNA binding sites. We then transferred these in-silico results to a biological example and investigated the relationship between ZFR and its intronic miRNA miR-579 in a U87 cell line model. We found that ZFR is targeted by its intronic miRNA miR-579 and that alternative polyadenylation allows differential targeting. We additionally used bioinformatics analyses and RNA-Seq to evaluate a potential cross-talk between intronic miRNAs and alternative polyadenylation. CPSF2, a gene previously associated with alternative polyadenylation signal recognition, might be linked to intronic miRNA negative feedback by altering polyadenylation signal utilization. PMID:25799583

RNA mobility in parasitic plant - host interactions.

PubMed

Westwood, James H; Kim, Gunjune

2017-04-03

The parasitic plant Cuscuta exchanges mRNAs with its hosts. Systemic mobility of mRNAs within plants is well documented, and has gained increasing attention as studies using grafted plant systems have revealed new aspects of mobile mRNA regulation and function. But parasitic plants take this phenomenon to a new level by forming seamless connections to a wide range of host species, and raising questions about how mRNAs might function after transfer to a different species. Cuscuta and other parasitic plant species also take siRNAs from their hosts, indicating that multiple types of RNA are capable of trans-specific movement. Parasitic plants are intriguing systems for studying RNA mobility, in part because such exchange opens new possibilities for control of parasitic weeds, but also because they provide a fresh perspective into understanding roles of RNAs in inter-organismal communication.

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

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

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

PubMed Central

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

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. Copyright © 2016. Published by Elsevier B.V.

MiRNA-124 is a link between measles virus persistent infection and cell division of human neuroblastoma cells.

PubMed

Naaman, Hila; Rall, Glenn; Matullo, Christine; Veksler-Lublinsky, Isana; Shemer-Avni, Yonat; Gopas, Jacob

2017-01-01

Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in rare cases, the virus can persistently infect cells within the central nervous system. Although some of the factors that allow MV to persist are known, the contribution of host cell-encoded microRNAs (miRNA) have not been described. MiRNAs are a class of noncoding RNAs transcribed from genomes of all multicellular organisms and some viruses, which regulate gene expression in a sequence-specific manner. We have studied the contribution of host cell-encoded miRNAs to the establishment of MV persistent infection in human neuroblastoma cells. Persistent MV infection was accompanied by differences in the expression profile and levels of several host cell-encoded microRNAs as compared to uninfected cells. MV persistence infection of a human neuroblastoma cell line (UKF-NB-MV), exhibit high miRNA-124 expression, and reduced expression of cyclin dependent kinase 6 (CDK6), a known target of miRNA-124, resulting in slower cell division but not cell death. By contrast, acute MV infection of UKF-NB cells did not result in increased miRNA-124 levels or CDK6 reduction. Ectopic overexpression of miRNA-124 affected cell viability only in UKF-NB-MV cells, causing cell death; implying that miRNA-124 over expression can sensitize cells to death only in the presence of MV persistent infection. To determine if miRNA-124 directly contributes to the establishment of MV persistence, UKF-NB cells overexpressing miRNA-124 were acutely infected, resulting in establishment of persistently infected colonies. We propose that miRNA-124 triggers a CDK6-dependent decrease in cell proliferation, which facilitates the establishment of MV persistence in neuroblastoma cells. To our knowledge, this is the first report to describe the role of a specific miRNA in MV persistence.

Generation of a stable cell line for constitutive miRNA expression.

PubMed

Lieber, Diana

2013-01-01

miRNAs have in recent years emerged as novel players in virus-host interactions. While individual miRNAs are capable of regulating many targets simultaneously, not much is known about the role of distinct host or viral miRNAs in the context of infection. Analysis of the function of a miRNA is often hampered by the complexity of virus-host interactions and the enormous changes in the host cell during infection. Many viral miRNAs as for example from Kaposi sarcoma-associated Herpesvirus (KSHV) are probably exclusively expressed in latent infection. This might lead to a steady-state situation with offense and defense mechanisms counteracting each other. Cellular miRNAs involved in defense against pathogens on the other hand might be suppressed in infection. A cell culture system allowing for constitutive expression of individual miRNAs at high levels is a useful tool to enhance miRNA-specific functions and to uncouple viral miRNA function from other infection-related mechanisms. Here, a protocol is described to generate stable cell lines for constitutive expression of single cellular or viral miRNA precursors in absence of infection. The procedure comprises cloning of the precursor sequence, generation of the lentiviral expression vector, transduction of the cells of interest, selection for polyclonal cell lines, and isolation of monoclonal cell lines by limiting dilution.

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

PubMed Central

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

Rate of novel host invasion affects adaptability of evolving RNA virus lineages.

PubMed

Morley, Valerie J; Mendiola, Sandra Y; Turner, Paul E

2015-08-22

Although differing rates of environmental turnover should be consequential for the dynamics of adaptive change, this idea has been rarely examined outside of theory. In particular, the importance of RNA viruses in disease emergence warrants experiments testing how differing rates of novel host invasion may impact the ability of viruses to adaptively shift onto a novel host. To test whether the rate of environmental turnover influences adaptation, we experimentally evolved 144 Sindbis virus lineages in replicated tissue-culture environments, which transitioned from being dominated by a permissive host cell type to a novel host cell type. The rate at which the novel host 'invaded' the environment varied by treatment. The fitness (growth rate) of evolved virus populations was measured on each host type, and molecular substitutions were mapped via whole genome consensus sequencing. Results showed that virus populations more consistently reached high fitness levels on the novel host when the novel host 'invaded' the environment more gradually, and gradual invasion resulted in less variable genomic outcomes. Moreover, virus populations that experienced a rapid shift onto the novel host converged upon different genotypes than populations that experienced a gradual shift onto the novel host, suggesting a strong effect of historical contingency. © 2015 The Author(s).

RNA mobility in parasitic plant – host interactions

PubMed Central

Kim, Gunjune

2017-01-01

ABSTRACT The parasitic plant Cuscuta exchanges mRNAs with its hosts. Systemic mobility of mRNAs within plants is well documented, and has gained increasing attention as studies using grafted plant systems have revealed new aspects of mobile mRNA regulation and function. But parasitic plants take this phenomenon to a new level by forming seamless connections to a wide range of host species, and raising questions about how mRNAs might function after transfer to a different species. Cuscuta and other parasitic plant species also take siRNAs from their hosts, indicating that multiple types of RNA are capable of trans-specific movement. Parasitic plants are intriguing systems for studying RNA mobility, in part because such exchange opens new possibilities for control of parasitic weeds, but also because they provide a fresh perspective into understanding roles of RNAs in inter-organismal communication. PMID:28277936

MicroRNA-Based Attenuation of Influenza Virus across Susceptible Hosts.

PubMed

Waring, Barbara M; Sjaastad, Louisa E; Fiege, Jessica K; Fay, Elizabeth J; Reyes, Ismarc; Moriarity, Branden; Langlois, Ryan A

2018-01-15

Influenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population, where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. MicroRNAs (miRNAs) have been used previously to generate attenuated influenza A viruses for use as a vaccine. Here, we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yields maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus, we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21-targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs. IMPORTANCE Influenza A virus circulates annually in both avian and human populations, causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. Micro

Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

PubMed

Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Li, Yao; Pang, Jing; Dong, Stephanie; Strauss-Soukup, Juliane K; Chen, Xian-Ming

2018-05-01

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes

PubMed Central

Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

2014-01-01

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. PMID:25071804

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib

PubMed Central

Bradford, James R.; Farren, Matthew; Powell, Steve J.; Runswick, Sarah; Weston, Susie L.; Brown, Helen; Delpuech, Oona; Wappett, Mark; Smith, Neil R.; Carr, T. Hedley; Dry, Jonathan R.; Gibson, Neil J.; Barry, Simon T.

2013-01-01

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers. PMID:23840389

An RNA pseudoknot is required for production of yellow fever virus subgenomic RNA by the host nuclease XRN1.

PubMed

Silva, Patrícia A G C; Pereira, Carina F; Dalebout, Tim J; Spaan, Willy J M; Bredenbeek, Peter J

2010-11-01

Cells and mice infected with arthropod-borne flaviviruses produce a small subgenomic RNA that is colinear with the distal part of the viral 3'-untranslated region (UTR). This small subgenomic flavivirus RNA (sfRNA) results from the incomplete degradation of the viral genome by the host 5'-3' exonuclease XRN1. Production of the sfRNA is important for the pathogenicity of the virus. This study not only presents a detailed description of the yellow fever virus (YFV) sfRNA but, more importantly, describes for the first time the molecular characteristics of the stalling site for XRN1 in the flavivirus genome. Similar to the case for West Nile virus, the YFV sfRNA was produced by XRN1. However, in contrast to the case for other arthropod-borne flaviviruses, not one but two sfRNAs were detected in YFV-infected mammalian cells. The smaller of these two sfRNAs was not observed in infected mosquito cells. The larger sfRNA could also be produced in vitro by incubation with purified XRN1. These two YFV sfRNAs formed a 5'-nested set. The 5' ends of the YFV sfRNAs were found to be just upstream of the previously predicted RNA pseudoknot PSK3. RNA structure probing and mutagenesis studies provided strong evidence that this pseudoknot structure was formed and served as the molecular signal to stall XRN1. The sequence involved in PSK3 formation was cloned into the Sinrep5 expression vector and shown to direct the production of an sfRNA-like RNA. These results underscore the importance of the RNA pseudoknot in stalling XRN1 and also demonstrate that it is the sole viral requirement for sfRNA production.

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA*

PubMed Central

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

2013-01-01

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 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 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

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

PubMed

Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

2016-10-01

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

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

MicroRNA-155 Modulates Acute Graft-versus-Host Disease by Impacting T Cell Expansion, Migration, and Effector Function.

PubMed

Zitzer, Nina C; Snyder, Katiri; Meng, Xiamoei; Taylor, Patricia A; Efebera, Yvonne A; Devine, Steven M; Blazar, Bruce R; Garzon, Ramiro; Ranganathan, Parvathi

2018-06-15

MicroRNA-155 (miR-155) is a small noncoding RNA critical for the regulation of inflammation as well as innate and adaptive immune responses. MiR-155 has been shown to be dysregulated in both donor and recipient immune cells during acute graft-versus-host disease (aGVHD). We previously reported that miR-155 is upregulated in donor T cells of mice and humans with aGVHD and that mice receiving miR-155-deficient (miR155 -/- ) splenocytes had markedly reduced aGVHD. However, molecular mechanisms by which miR-155 modulates T cell function in aGVHD have not been fully investigated. We identify that miR-155 expression in both donor CD8 + T cells and conventional CD4 + CD25 - T cells is pivotal for aGVHD pathogenesis. Using murine aGVHD transplant experiments, we show that miR-155 strongly impacts alloreactive T cell expansion through multiple distinct mechanisms, modulating proliferation in CD8 + donor T cells and promoting exhaustion in donor CD4 + T cells in both the spleen and colon. Additionally, miR-155 drives a proinflammatory Th1 phenotype in donor T cells in these two sites, and miR-155 -/- donor T cells are polarized toward an IL-4-producing Th2 phenotype. We further demonstrate that miR-155 expression in donor T cells regulates CCR5 and CXCR4 chemokine-dependent migration. Notably, we show that miR-155 expression is crucial for donor T cell infiltration into multiple target organs. These findings provide further understanding of the role of miR-155 in modulating aGVHD through T cell expansion, effector cytokine production, and migration. Copyright © 2018 by The American Association of Immunologists, Inc.

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

PubMed Central

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

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.

Rabies viruses leader RNA interacts with host Hsc70 and inhibits virus replication

PubMed Central

Zhang, Ran; Liu, Chuangang; Cao, Yunzi; Jamal, Muhammad; Chen, Xi; Zheng, Jinfang; Li, Liang; You, Jing; Zhu, Qi; Liu, Shiyong; Dai, Jinxia; Cui, Min; Fu, Zhen F.; Cao, Gang

2017-01-01

Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication. PMID:28388579

Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.

PubMed

Cui, Jiajia; Qin, Lingfeng; Zhang, Junwei; Abrahimi, Parwiz; Li, Hong; Li, Guangxin; Tietjen, Gregory T; Tellides, George; Pober, Jordan S; Mark Saltzman, W

2017-08-04

Human endothelial cells are initiators and targets of the rejection response. Pre-operative modification of endothelial cells by small interfering RNA transfection could shape the nature of the host response post-transplantation. Ablation of endothelial cell class II major histocompatibility complex molecules by small interfering RNA targeting of class II transactivator can reduce the capacity of human endothelial cells to recruit and activate alloreactive T cells. Here, we report the development of small interfering RNA-releasing poly(amine-co-ester) nanoparticles, distinguished by their high content of a hydrophobic lactone. We show that a single transfection of small interfering RNA targeting class II transactivator attenuates major histocompatibility complex class II expression on endothelial cells for at least 4 to 6 weeks after transplantation into immunodeficient mouse hosts. Furthermore, silencing of major histocompatibility complex class II reduces allogeneic T-cell responses in vitro and in vivo. These data suggest that poly(amine-co-ester) nanoparticles, potentially administered during ex vivo normothermic machine perfusion of human organs, could be used to modify endothelial cells with a sustained effect after transplantation.The use of gene silencing techniques in the treatment of post-transplantation host rejection is not long lasting and can have systemic effects. Here, the authors utilize a nanocarrier for siRNA for treatment of arteries ex vivo prior to implantation subsequently attenuating immune reaction in vivo.

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. © 2014 John Wiley & Sons Ltd.

Viral subversion of host functions for picornavirus translation and RNA replication

PubMed Central

Chase, Amanda J; Semler, Bert L

2012-01-01

Picornavirus infections lead to symptoms that can have serious health and economic implications. The viruses in this family (Picornaviridae) have a small genomic RNA and must rely on host proteins for efficient viral gene expression and RNA replication. To ensure their effectiveness as pathogens, picornaviruses have evolved to utilize and/or alter host proteins for the benefit of the virus life cycle. This review discusses the host proteins that are subverted during infection to aid in virus replication. It will also describe proteins and functions that are altered during infection for the benefit of the virus. PMID:23293659

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

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

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

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

PubMed

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

2016-03-01

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.

Alphavirus entry into host cells.

PubMed

Vancini, Ricardo; Hernandez, Raquel; Brown, Dennis

2015-01-01

Viruses have evolved to exploit the vast complexity of cellular processes for their success within the host cell. The entry mechanisms of enveloped viruses (viruses with a surrounding outer lipid bilayer membrane) are usually classified as being either endocytotic or fusogenic. Different mechanisms have been proposed for Alphavirus entry and genome delivery. Indirect observations led to a general belief that enveloped viruses can infect cells either by protein-assisted fusion with the plasma membrane in a pH-independent manner or by endocytosis and fusion with the endocytic vacuole in a low-pH environment. The mechanism of Alphavirus penetration has been recently revisited using direct observation of the processes by electron microscopy under conditions of different temperatures and time progression. Under conditions nonpermissive for endocytosis or any vesicular transport, events occur which allow the entry of the virus genome into the cells. When drug inhibitors of cellular functions are used to prevent entry, only ionophores are found to significantly inhibit RNA delivery. Arboviruses are agents of significant human and animal disease; therefore, strategies to control infections are needed and include development of compounds which will block critical steps in the early infection events. It appears that current evidence points to an entry mechanism, in which alphaviruses infect cells by direct penetration of cell plasma membranes through a pore structure formed by virus and, possibly, host proteins. © 2015 Elsevier Inc. All rights reserved.

Host Long Noncoding RNA lncRNA-PAAN Regulates the Replication of Influenza A Virus.

PubMed

Wang, Jing; Wang, Yujia; Zhou, Rui; Zhao, Jianyuan; Zhang, Yongxin; Yi, Dongrong; Li, Quanjie; Zhou, Jinming; Guo, Fei; Liang, Chen; Li, Xiaoyu; Cen, Shan

2018-06-16

The productive infection of influenza A virus (IAV) depends on host factors. However, the involvement of long non-coding RNAs (lncRNAs) in IAV infection remains largely uninvestigated. In this work, we have discovered a human lncRNA, named lncRNA-PAAN (PA-associated noncoding RNA) that enhances IAV replication. The level of lncRNA-PAAN increases upon infection of IAV, but not other viruses, nor interferon treatment, suggesting specific up-regulation of lncRNA-PAAN expression by IAV. Silencing lncRNA-PAAN significantly decreases IAV replication through impairing the activity of viral RNA-dependent RNA polymerase (RdRp). This function of lncRNA-PAAN is a result of its association with viral PA protein, a key component of IAV RNA polymerase complex. Consequently, depletion of lncRNA-PAAN prevents the formation of functional RdRp. Together, these results suggest that lncRNA-PAAN promotes the assembly of viral RNA polymerase, thus warranting efficient viral RNA synthesis. Elucidating the functions of lncRNAs in IAV infection is expected to advance our understanding of IAV pathogenesis and open new avenues to the development of novel anti-IAV therapeutics.

MicroRNA analysis in mouse neuro-2a cells after pseudorabies virus infection.

PubMed

Li, Yongtao; Zheng, Guanmin; Zhang, Yujuan; Yang, Xia; Liu, Hongying; Chang, Hongtao; Wang, Xinwei; Zhao, Jun; Wang, Chuanqing; Chen, Lu

2017-06-01

Pseudorabies virus (PRV), an alpha herpesvirus can enter the mammalian nervous system, causing Aujezsky's disease. Previous studies have reported an alteration of microRNA (miRNA) expression levels during PRV infections. However, knowledge regarding miRNA response in nervous cells to PRV infection is still unknown. To address this issue, small RNA libraries from infected and uninfected mouse neuroblastoma cells were assessed after Illumina deep sequencing. A total of eight viral miRNA were identified, and ten host miRNAs showed significantly different expression upon PRV infection. Among these, five were analyzed by stem-loop RT-qPCR, which confirmed the above data. Interestingly, these viral miRNAs were mainly found in the large latency transcript region of PRV, and predicted to target a variety of genes, forming a complicated regulatory network. Moreover, ten cellular miRNAs were expressed differently upon PRV infection, including nine upregulated and one downregulated miRNAs. Host targets of these miRNAs obtained by bioinformatics analysis belonged to large signaling networks, mainly encompassing calcium signaling pathway, cAMP signaling pathway, MAPK signaling pathway, and other nervous-associated pathways. These findings further highlighted miRNA features in nervous cells after PRV infection and contributed to unveil the underlying mechanisms of neurotropism as well as the neuropathogenesis of PRV.

Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells.

PubMed

Mann, Krin S; Johnson, Karyn N; Dietzgen, Ralf G

2015-02-01

RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

PubMed

Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

2015-09-01

Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cross-Species Translocation of mRNA from Host Plants into the Parasitic Plant Dodder1[OA

PubMed Central

Roney, Jeannine K.; Khatibi, Piyum A.; Westwood, James H.

2007-01-01

An intriguing new paradigm in plant biology is that systemically mobile mRNAs play a role in coordinating development. In this process, specific mRNAs are loaded into the phloem transport stream for translocation to distant tissues, where they may impact on developmental processes. However, despite its potential significance for plant growth regulation, mRNA trafficking remains poorly understood and challenging to study. Here, we show that phloem-mobile mRNAs can also traffic between widely divergent species from a host to the plant parasite lespedeza dodder (Cuscuta pentagona Engelm.). Reverse transcription-polymerase chain reaction and microarray analysis were used to detect specific tomato (Lycopersicon esculentum Mill.) transcripts in dodder grown on tomato that were not present in control dodder grown on other host species. Foreign transcripts included LeGAI, which has previously been shown to be translocated in the phloem, as well as nine other transcripts not reported to be mobile. Dodders are parasitic plants that obtain resources by drawing from the phloem of a host plant and have joint plasmodesmata with host cortical cells. Although viruses are known to move between dodder and its hosts, translocation of endogenous plant mRNA has not been reported. These results point to a potentially new level of interspecies communication, and raise questions about the ability of parasites to recognize, use, and respond to transcripts acquired from their hosts. PMID:17189329

Role of RNase MRP in viral RNA degradation and RNA recombination.

PubMed

Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

2011-01-01

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

RNA Viruses: ROS-Mediated Cell Death

PubMed Central

Reshi, Mohammad Latif; Su, Yi-Che; Hong, Jiann-Ruey

2014-01-01

Reactive oxygen species (ROS) are well known for being both beneficial and deleterious. The main thrust of this review is to investigate the role of ROS in ribonucleic acid (RNA) virus pathogenesis. Much evidences has accumulated over the past decade, suggesting that patients infected with RNA viruses are under chronic oxidative stress. Changes to the body's antioxidant defense system, in relation to SOD, ascorbic acid, selenium, carotenoids, and glutathione, have been reported in various tissues of RNA-virus infected patients. This review focuses on RNA viruses and retroviruses, giving particular attention to the human influenza virus, Hepatitis c virus (HCV), human immunodeficiency virus (HIV), and the aquatic Betanodavirus. Oxidative stress via RNA virus infections can contribute to several aspects of viral disease pathogenesis including apoptosis, loss of immune function, viral replication, inflammatory response, and loss of body weight. We focus on how ROS production is correlated with host cell death. Moreover, ROS may play an important role as a signal molecule in the regulation of viral replication and organelle function, potentially providing new insights in the prevention and treatment of RNA viruses and retrovirus infections. PMID:24899897

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector.

PubMed

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis -specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

PubMed Central

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria. PMID:28638803

Viral Evasion and Manipulation of Host RNA Quality Control Pathways

PubMed Central

2016-01-01

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

Orchestrating the Selection and Packaging of Genomic RNA by Retroviruses: An Ensemble of Viral and Host Factors

PubMed Central

Kaddis Maldonado, Rebecca J.; Parent, Leslie J.

2016-01-01

Infectious retrovirus particles contain two copies of unspliced viral RNA that serve as the viral genome. Unspliced retroviral RNA is transcribed in the nucleus by the host RNA polymerase II and has three potential fates: (1) it can be spliced into subgenomic messenger RNAs (mRNAs) for the translation of viral proteins; or it can remain unspliced to serve as either (2) the mRNA for the translation of Gag and Gag–Pol; or (3) the genomic RNA (gRNA) that is packaged into virions. The Gag structural protein recognizes and binds the unspliced viral RNA to select it as a genome, which is selected in preference to spliced viral RNAs and cellular RNAs. In this review, we summarize the current state of understanding about how retroviral packaging is orchestrated within the cell and explore potential new mechanisms based on recent discoveries in the field. We discuss the cis-acting elements in the unspliced viral RNA and the properties of the Gag protein that are required for their interaction. In addition, we discuss the role of host factors in influencing the fate of the newly transcribed viral RNA, current models for how retroviruses distinguish unspliced viral mRNA from viral genomic RNA, and the possible subcellular sites of genomic RNA dimerization and selection by Gag. Although this review centers primarily on the wealth of data available for the alpharetrovirus Rous sarcoma virus, in which a discrete RNA packaging sequence has been identified, we have also summarized the cis- and trans-acting factors as well as the mechanisms governing gRNA packaging of other retroviruses for comparison. PMID:27657110

Leishmania RNA virus: when the host pays the toll

PubMed Central

Hartley, Mary-Anne; Ronet, Catherine; Zangger, Haroun; Beverley, Stephen M.; Fasel, Nicolas

2012-01-01

The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic “metastatic factors” or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically

Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription.

PubMed

Bauer, David L V; Tellier, Michael; Martínez-Alonso, Mónica; Nojima, Takayuki; Proudfoot, Nick J; Murphy, Shona; Fodor, Ervin

2018-05-15

Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis.

PubMed

Martín-Hernández, Raquel; Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E; Meana, Aránzazu; Boonham, Neil

2017-01-01

Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host's cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite's survival within the cell.

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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia

PubMed Central

Silva, Ricardo Jorge; Cruz, Ana Rita; Mano, Miguel

2017-01-01

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells. PMID:28394930

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia.

PubMed

Sunkavalli, Ushasree; Aguilar, Carmen; Silva, Ricardo Jorge; Sharan, Malvika; Cruz, Ana Rita; Tawk, Caroline; Maudet, Claire; Mano, Miguel; Eulalio, Ana

2017-04-01

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.

RNA interference targets arbovirus replication in Culicoides cells.

PubMed

Schnettler, Esther; Ratinier, Maxime; Watson, Mick; Shaw, Andrew E; McFarlane, Melanie; Varela, Mariana; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

2013-03-01

Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

Transcriptional profiling of the host cell response to feline immunodeficiency virus infection.

PubMed

Ertl, Reinhard; Klein, Dieter

2014-03-19

Feline immunodeficiency virus (FIV) is a widespread pathogen of the domestic cat and an important animal model for human immunodeficiency virus (HIV) research. In contrast to HIV, only limited information is available on the transcriptional host cell response to FIV infections. This study aims to identify FIV-induced gene expression changes in feline T-cells during the early phase of the infection. Illumina RNA-sequencing (RNA-seq) was used identify differentially expressed genes (DEGs) at 24 h after FIV infection. After removal of low-quality reads, the remaining sequencing data were mapped against the cat genome and the numbers of mapping reads were counted for each gene. Regulated genes were identified through the comparison of FIV and mock-infected data sets. After statistical analysis and the removal of genes with insufficient coverage, we detected a total of 69 significantly DEGs (44 up- and 25 down-regulated genes) upon FIV infection. The results obtained by RNA-seq were validated by reverse transcription qPCR analysis for 10 genes. Out of the most distinct DEGs identified in this study, several genes are already known to interact with HIV in humans, indicating comparable effects of both viruses on the host cell gene expression and furthermore, highlighting the importance of FIV as a model system for HIV. In addition, a set of new genes not previously linked to virus infections could be identified. The provided list of virus-induced genes may represent useful information for future studies focusing on the molecular mechanisms of virus-host interactions in FIV pathogenesis.

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

Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus.

PubMed

Xin, Xiu; Wang, Hailong; Han, Lingling; Wang, Mingzhen; Fang, Hui; Hao, Yao; Li, Jiadai; Zhang, Hu; Zheng, Congyi; Shen, Chao

2018-05-01

Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G 2 /M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell. IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells. Copyright © 2018 American Society for Microbiology.

Perturbation of host-cell membrane is a primary mechanism of HIV cytopathology.

PubMed

Cloyd, M W; Lynn, W S

1991-04-01

Cytopathic viruses injure cells by a number of different mechanisms. The mechanism by which HIV-1 injures T cells was studied by temporally examining host-cell macromolecular syntheses, stages of the cell cycle, and membrane permeability following acute infection. T cells cytopathically infected at an m.o.i. of 1-5 grew normally for 24-72 hr, depending on the cell line, followed by the first manifestation of cell injury, slowing of cell division. At that time significant amounts of unintegrated HIV DNA and p24 core protein became detectable, and acridine orange flow cytometric cell cycle studies demonstrated the presence of fewer cells in the G2/M stage of the cell cycle. There was no change in the frequency of cells in the S-stage, and metabolic pulsing with radioactive precursors demonstrated that host-cell DNA, RNA, and protein syntheses were normal at that time and normal up to the time cells started to die (approximately 24 hr later), when all three decreased. Cellular lipid synthesis, however, was perturbed when cell multiplication slowed, with phospholipid synthesis reduced and neutral lipid synthesis enhanced. Permeability of the host-cell membrane to small molecules, such as Ca2+ and sucrose, was slightly enhanced early postinfection, and by the time of slowing of cell division, host membrane permeability was greatly increased to both Ca2+ and sucrose (Stokes radius 5.2 A) but not to inulin (Stokes radium 20 A). These changes in host-cell membrane permeability and phospholipid synthesis were not observed in acutely infected H9 cells, which are not susceptible to HIV cytopathology. Thus, HIV-1 appeared to predominantly injure T cells by perturbing host-cell membrane permeability and lipid synthesis, which is similar to the cytopathic mechanisms of paramyxoviruses.

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

Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

PubMed Central

Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

2017-01-01

Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

Ebola virus host cell entry.

PubMed

Sakurai, Yasuteru

2015-01-01

Ebola virus is an enveloped virus with filamentous structure and causes a severe hemorrhagic fever in human and nonhuman primates. Host cell entry is the first essential step in the viral life cycle, which has been extensively studied as one of the therapeutic targets. A virus factor of cell entry is a surface glycoprotein (GP), which is an only essential viral protein in the step, as well as the unique particle structure. The virus also interacts with a lot of host factors to successfully enter host cells. Ebola virus at first binds to cell surface proteins and internalizes into cells, followed by trafficking through endosomal vesicles to intracellular acidic compartments. There, host proteases process GPs, which can interact with an intracellular receptor. Then, under an appropriate circumstance, viral and endosomal membranes are fused, which is enhanced by major structural changes of GPs, to complete host cell entry. Recently the basic research of Ebola virus infection mechanism has markedly progressed, largely contributed by identification of host factors and detailed structural analyses of GPs. This article highlights the mechanism of Ebola virus host cell entry, including recent findings.

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

PubMed

Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

2017-07-01

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

When your cap matters: structural insights into self vs non-self recognition of 5' RNA by immunomodulatory host proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Daisy W.; Amarasinghe, Gaya K.

Cytosolic recognition of viral RNA is important for host innate immune responses. Differential recognition of self vs non-self RNA is a considerable challenge as the inability to differentiate may trigger aberrant immune responses. Recent work identified the composition of the RNA 5', including the 5' cap and its methylation state, as an important determinant of recognition by the host. Recent studies have advanced our understanding of the modified 5' RNA recognition and viral antagonism of RNA receptors. Here, we will discuss RIG-I and IFIT proteins as examples of host proteins that detect dsRNA and ssRNA, respectively.

Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication

PubMed Central

Slonchak, Andrii; Shannon, Rory P.; Pali, Gabor

2015-01-01

ABSTRACT West Nile virus (WNV) is a mosquito-transmitted flavivirus that naturally circulates between mosquitos and birds but can also infect humans, causing severe neurological disease. The early host response to WNV infection in vertebrates primarily relies on the type I interferon pathway; however, recent studies suggest that microRNAs (miRNAs) may also play a notable role. In this study, we assessed the role of host miRNAs in response to WNV infection in human cells. We employed small RNA sequencing (RNA-seq) analysis to determine changes in the expression of host miRNAs in HEK293 cells infected with an Australian strain of WNV, Kunjin (WNVKUN), and identified a number of host miRNAs differentially expressed in response to infection. Three of these miRNAs were confirmed to be significantly upregulated in infected cells by quantitative reverse transcription (qRT)-PCR and Northern blot analyses, and one of them, miR-532-5p, exhibited a significant antiviral effect against WNVKUN infection. We have demonstrated that miR-532-5p targets and downregulates expression of the host genes SESTD1 and TAB3 in human cells. Small interfering RNA (siRNA) depletion studies showed that both SESTD1 and TAB3 were required for efficient WNVKUN replication. We also demonstrated upregulation of mir-532-5p expression and a corresponding decrease in the expression of its targets, SESTD1 and TAB3, in the brains of WNVKUN -infected mice. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent a host antiviral response aimed at limiting WNVKUN infection and highlight the important role of miRNAs in controlling RNA virus infections in mammalian hosts. IMPORTANCE West Nile virus (WNV) is a significant viral pathogen that poses a considerable threat to human health across the globe. There is no specific treatment or licensed vaccine available for WNV, and deeper insight into how the virus interacts with the host is required to

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

PubMed

Hempstead, Andrew D; Isberg, Ralph R

2015-12-08

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.

Upregulation of Long Noncoding RNA Small Nucleolar RNA Host Gene 18 Promotes Radioresistance of Glioma by Repressing Semaphorin 5A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Rong; Department of Radiation Oncology, Fujian Medical University Union Hospital, Fuzhou, Fujian; Yao, Qiwei

Purpose: Although increasing evidence has shown that long noncoding RNAs play an important regulatory role in carcinogenesis and tumor progression, little is known about the role of small nucleolar RNA host gene 18 (SNHG18) in cancer. The goal of this study was to investigate the expression of SNHG18 and its clinical significance in glioma. Methods and Materials: Differences in the lncRNA expression profile between M059K and M059J cells were assessed by lncRNA expression microarray analysis. The expression and localization of SNHG18 in glioma cells or tissues was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH),more » respectively. the clinical associations of SNHG18 in glioma was evaluated by qRT-PCR, ISH and immunohistochemistry. The role of SNHG18 in glioma radiosensitivity was evaluated by colony formation assays, immunofluorescence, Western blot and tumor growth inhibition study. Results: The present study investigated the clinical associations of SNHG18 and its role in glioma. Our results showed that the expression of SNHG18 was remarkably upregulated in clinical glioma tissues compared with normal brain tissues. SNHG18 expression was associated with the clinical tumor grade and correlated negatively with isocitrate dehydrogenase 1 mutation. In addition, knockdown of SNHG18 with short hairpin RNA suppressed the radioresistance of glioma cells, and transgenic expression of SNHG18 had the opposite effect. Furthermore, xenograft tumors grown from cells with SNHG18 deletion were more radiosensitive than tumors grown from control cells. Further studies revealed that SNHG18 promotes radioresistance by inhibiting semaphorin 5A and that inhibition of semaphorin 5A expression abrogated the radiosensitizing effect caused by SNHG18 deletion. Conclusions: Our findings provide new insights into the role of SNHG18 in glioma and suggest its potential as a target for glioma therapy.« less

Host Genetic Background Strongly Affects Pulmonary microRNA Expression before and during Influenza A Virus Infection.

PubMed

Preusse, Matthias; Schughart, Klaus; Pessler, Frank

2017-01-01

Expression of host microRNAs (miRNAs) changes markedly during influenza A virus (IAV) infection of natural and adaptive hosts, but their role in genetically determined host susceptibility to IAV infection has not been explored. We, therefore, compared pulmonary miRNA expression during IAV infection in two inbred mouse strains with differential susceptibility to IAV infection. miRNA expression profiles were determined in lungs of the more susceptible strain DBA/2J and the less susceptible strain C57BL/6J within 120 h post infection (hpi) with IAV (H1N1) PR8. Even the miRNomes of uninfected lungs differed substantially between the two strains. After a period of relative quiescence, major miRNome reprogramming was detected in both strains by 48 hpi and increased through 120 hpi. Distinct groups of miRNAs regulated by IAV infection could be defined: (1) miRNAs ( n  = 39) whose expression correlated with hemagglutinin (HA) mRNA expression and represented the general response to IAV infection independent of host genetic background; (2) miRNAs ( n  = 20) whose expression correlated with HA mRNA expression but differed between the two strains; and (3) remarkably, miR-147-3p, miR-208b-3p, miR-3096a-5p, miR-3069b-3p, and the miR-467 family, whose abundance even in uninfected lungs differentiated nearly perfectly (area under the ROC curve > 0.99) between the two strains throughout the time course, suggesting a particularly strong association with the differential susceptibility of the two mouse strains. Expression of subsets of miRNAs correlated significantly with peripheral blood granulocyte and monocyte numbers, particularly in DBA/2J mice; miR-223-3p, miR-142-3p, and miR-20b-5p correlated most positively with these cell types in both mouse strains. Higher abundance of antiapoptotic (e.g., miR-467 family) and lower abundance of proapoptotic miRNAs (e.g., miR-34 family) and those regulating the PI3K-Akt pathway (e.g., miR-31-5p) were associated with the

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

PubMed

Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

PubMed

Nicholson, Linda J; Philippe, Marie; Paine, Alan J; Mann, Derek A; Dolphin, Colin T

2005-04-01

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.

Oncogenes and RNA splicing of human tumor viruses

PubMed Central

Ajiro, Masahiko; Zheng, Zhi-Ming

2014-01-01

Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein–Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis. PMID:26038756

Dengue Virus Selectively Annexes Endoplasmic Reticulum-Associated Translation Machinery as a Strategy for Co-opting Host Cell Protein Synthesis.

PubMed

Reid, David W; Campos, Rafael K; Child, Jessica R; Zheng, Tianli; Chan, Kitti Wing Ki; Bradrick, Shelton S; Vasudevan, Subhash G; Garcia-Blanco, Mariano A; Nicchitta, Christopher V

2018-04-01

A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translation capacity was subverted from host transcripts to DENV plus-strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV plus-strand RNA and host mRNAs indicated that DENV plus-strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely, the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication while minimizing the activation of antiviral and proteostatic stress response pathways. IMPORTANCE DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

PubMed

Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

2017-02-14

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 mRNAsmore » 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.« less

Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR.

PubMed

Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

2009-09-01

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

Dual Functions of Rift Valley Fever Virus NSs Protein: Inhibition of Host mRNA Transcription and Post-transcriptional Downregulation of Protein Kinase PKR

PubMed Central

Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C. J.; Makino, Shinji

2011-01-01

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-β mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase, PKR, to prevent phosphorylation of eIF2α and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts. PMID:19751406

A Molecular Approach Designed to Limit the Replication of Mature DENV2 in Host Cells.

PubMed

Raheel, Ummar; Jamal, Muhsin; Zaidi, Najam Us Sahar Sadaf

2015-09-01

Dengue virus (DENV) is an arthropod-borne virus, which belongs to the Flaviviridae family, and completes its life cycle in two hosts: humans and mosquitoes. For DENV maturation, the surface pre-membrane (prM) protein is cleaved to form a mature membrane protein (M) by furin, which is a cellular enzyme subsequently releasing the mature virus from the host dendritic cell. The objective of the current study was to inhibit mature DENV isotype 2 (DENV2) by RNA-interference in a Vero-81 cell line. Mature DENV2 was propagated in and isolated from U937 cells expressing dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin. Maturation of DENV2 was confirmed by Western blot analysis, where virus stock lacking prM was considered mature. Inhibition studies were carried out by transfection of Vero-81 cells with six synthetic siRNAs along with a control siRNA. Reduction in cellular DENV2 was observed also by focus-reduction assay, immunofluorescence assay (IFA), and real-time quantitative polymerase chain reaction (RT-qPCR). Cells transfected with DENV2SsiRNA2, which was targeting the structural region M of mature DENV2, was able to reduce DENV2 titer by up to 85% in focus reduction assays. A significant reduction in mature DENV2 RNA load was observed by RT-qPCR, confirming the previous findings. IFA also revealed reduced levels of cellular DENV2. These results demonstrated that mature DENV2 can be effectively inhibited by synthetic siRNA targeting the structural region of the genome. Mature DENV2 can be successfully inhibited by siRNAs, and specifically high knock-down efficiency is observed by siRNAs against M region of mature DENV2. This study shows that M represents a potential target for RNAi based inhibitory approaches.

Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome

PubMed Central

Douam, Florian; Hrebikova, Gabriela; Albrecht, Yentli E. Soto; Sellau, Julie; Sharon, Yael; Ding, Qiang; Ploss, Alexander

2017-01-01

Positive-sense RNA viruses pose increasing health and economic concerns worldwide. Our limited understanding of how these viruses interact with their host and how these processes lead to virulence and disease seriously hampers the development of anti-viral strategies. Here, we demonstrate the tracking of (+) and (−) sense viral RNA at single-cell resolution within complex subsets of the human and murine immune system in different mouse models. Our results provide insights into how a prototypic flavivirus, yellow fever virus (YFV-17D), differentially interacts with murine and human hematopoietic cells in these mouse models and how these dynamics influence distinct outcomes of infection. We detect (−) YFV-17D RNA in specific secondary lymphoid compartments and cell subsets not previously recognized as permissive for YFV replication, and we highlight potential virus–host interaction events that could be pivotal in regulating flavivirus virulence and attenuation. PMID:28290449

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA

PubMed Central

Chapman, Erich G; Moon, Stephanie L; Wilusz, Jeffrey; Kieft, Jeffrey S

2014-01-01

Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell’s RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5′ end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3′ end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction’s fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production. DOI: http://dx.doi.org/10.7554/eLife.01892.001 PMID:24692447

Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9 Virus Reveals an Inhibitory Role of NS1 in Host mRNA Maturation.

PubMed

Kuo, Rei-Lin; Chen, Chi-Jene; Tam, Ee-Hong; Huang, Chung-Guei; Li, Li-Hsin; Li, Zong-Hua; Su, Pei-Chia; Liu, Hao-Ping; Wu, Chih-Ching

2018-04-06

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.

Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

PubMed Central

Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

2012-01-01

Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. PMID:23063407

Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

PubMed

Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

2013-01-20

Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination

PubMed Central

Chao, Mei; Wang, Tzu-Chi; Lin, Chia-Chi; Yung-Liang Wang, Robert; Lin, Wen-Bin; Lee, Shang-En; Cheng, Ying-Yu; Yeh, Chau-Ting; Iang, Shan-Bei

2017-01-01

The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens (the only viral-encoded protein) and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription. PMID:28977829

Identification and analysis of host proteins that interact with the 3'-untranslated region of tick-borne encephalitis virus genomic RNA.

PubMed

Muto, Memi; Kamitani, Wataru; Sakai, Mizuki; Hirano, Minato; Kobayashi, Shintaro; Kariwa, Hiroaki; Yoshii, Kentaro

2018-04-02

Tick-borne encephalitis virus (TBEV) causes severe neurological disease, but the pathogenetic mechanism is unclear. The conformational structure of the 3'-untranslated region (UTR) of TBEV is associated with its virulence. We tried to identify host proteins interacting with the 3'-UTR of TBEV. Cellular proteins of HEK293T cells were co-precipitated with biotinylated RNAs of the 3'-UTR of low- and high-virulence TBEV strains and subjected to mass spectrometry analysis. Fifteen host proteins were found to bind to the 3'-UTR of TBEV, four of which-cold shock domain containing-E1 (CSDE1), spermatid perinuclear RNA binding protein (STRBP), fragile X mental retardation protein (FMRP), and interleukin enhancer binding factor 3 (ILF3)-bound specifically to that of the low-virulence strain. An RNA immunoprecipitation and pull-down assay confirmed the interactions of the complete 3'-UTRs of TBEV genomic RNA with CSDE1, FMRP, and ILF3. Partial deletion of the stem loop (SL) 3 to SL 5 structure of the variable region of the 3'-UTR did not affect interactions with the host proteins, but the interactions were markedly suppressed by deletion of the complete SL 3, 4, and 5 structures, as in the high-virulence TBEV strain. Further analysis of the roles of host proteins in the neurologic pathogenicity of TBEV is warranted. Copyright © 2018 Elsevier B.V. All rights reserved.

Zika Virus Alters the Expression Profile of microRNA-Related Genes in Liver, Lung, and Kidney Cell Lineages.

PubMed

Ferreira, Rafaella Nascimento; Holanda, Gustavo Moraes; Pinto Silva, Eliana Vieira; Casseb, Samir Mansour Moraes; Melo, Karla Fabiane Lopes; Carvalho, Carlos Alberto Marques; Lima, Juliana Abreu; Vasconcelos, Pedro Fernando Costa; Cruz, Ana Cecília Ribeiro

2018-06-07

Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (Flaviviridae). ZIKV infection is associated with alterations in various organs, including the liver, lungs, and kidneys. Studies on the influence of posttranscriptional control on viral infections have demonstrated that microRNAs (miRNAs) interfere with different stages of the replicative cycle of several viruses and may influence the disease outcome. To shed light on ZIKV-induced regulation of host miRNA-processing machinery in the above organs, we analyzed the expression of genes encoding key proteins of the miRNA pathway in different ZIKV-infected continuous primate cell lineages (HepG2, A549, and MA104) by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Expression of the genes encoding the miRNA-related proteins DGCR8, Ago1, and Ago3 in HepG2 cells and Drosha, Dicer, Ago2, and Ago3 in A549 and MA104 cells was significantly altered in the presence of ZIKV. Our results suggest that ZIKV modulates miRNA levels during infection in liver, lung, and kidney cells, which may be an additional mechanism of host cell subversion in these organs.

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

PubMed Central

Hyodo, Kiwamu; Taniguchi, Takako; Manabe, Yuki; Kaido, Masanori; Mise, Kazuyuki; Sugawara, Tatsuya; Taniguchi, Hisaaki; Okuno, Tetsuro

2015-01-01

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate. PMID:26020241

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing.

PubMed

Steuerman, Yael; Cohen, Merav; Peshes-Yaloz, Naama; Valadarsky, Liran; Cohn, Ofir; David, Eyal; Frishberg, Amit; Mayo, Lior; Bacharach, Eran; Amit, Ido; Gat-Viks, Irit

2018-06-01

The influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Ebola Virus Enters Host Cells by Macropinocytosis and Clathrin-Mediated Endocytosis

PubMed Central

Aleksandrowicz, Paulina; Marzi, Andrea; Biedenkopf, Nadine; Beimforde, Nadine; Becker, Stephan; Hoenen, Thomas; Feldmann, Heinz

2011-01-01

Virus entry into host cells is the first step of infection and a crucial determinant of pathogenicity. Here we show that Ebola virus-like particles (EBOV-VLPs) composed of the glycoprotein GP1,2 and the matrix protein VP40 use macropinocytosis and clathrin-mediated endocytosis to enter cells. EBOV-VLPs applied to host cells induced actin-driven ruffling and enhanced FITC-dextran uptake, which indicated macropinocytosis as the main entry mechanism. This was further supported by inhibition of entry through inhibitors of actin polymerization (latrunculin A), Na+/H+-exchanger (EIPA), and PI3-kinase (wortmannin). A fraction of EBOV-VLPs, however, colocalized with clathrin heavy chain (CHC), and VLP uptake was reduced by CHC small interfering RNA transfection and expression of the dominant negative dynamin II–K44A mutant. In contrast, we found no evidence that EBOV-VLPs enter cells via caveolae. This work identifies macropinocytosis as the major, and clathrin-dependent endocytosis as an alternative, entry route for EBOV particles. Therefore, EBOV seems to utilize different entry pathways depending on both cell type and virus particle size. PMID:21987776

Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing.

PubMed

Suresh, Bharathi; Ramakrishna, Suresh; Kim, Hyongbum

2017-01-01

The clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system represents an efficient tool for genome editing. It consists of two components: the Cas9 protein and a guide RNA. To date, delivery of these two components has been achieved using either plasmid or viral vectors or direct delivery of protein and RNA. Plasmid- and virus-free direct delivery of Cas9 protein and guide RNA has several advantages over the conventional plasmid-mediated approach. Direct delivery results in shorter exposure time at the cellular level, which in turn leads to lower toxicity and fewer off-target mutations with reduced host immune responses, whereas plasmid- or viral vector-mediated delivery can result in uncontrolled integration of the vector sequence into the host genome and unwanted immune responses. Cell-penetrating peptide (CPP), a peptide that has an intrinsic ability to translocate across cell membranes, has been adopted as a means of achieving efficient Cas9 protein and guide RNA delivery. We developed a method for treating human cell lines with CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs that leads to endogenous gene disruption. Here we describe a protocol for preparing an efficient CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs, as well as treatment methods to achieve safe genome editing in human cell lines.

Modified host cells with efflux pumps

DOEpatents

Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

2016-08-30

The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

PubMed Central

Veesenmeyer, Jeff L.; Andersen, Aaron W.; Lu, Xiaojun; Hussa, Elizabeth A.; Murfin, Kristen E.; Chaston, John M.; Dillman, Adler R.; Wassarman, Karen M.; Sternberg, Paul W.; Goodrich-Blair, Heidi

2014-01-01

Summary The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression. PMID:25041533

Single-cell RNA-sequencing: The future of genome biology is now

PubMed Central

Picelli, Simone

2017-01-01

ABSTRACT Genome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis. PMID:27442339

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes.

PubMed

Florentino, Pilar T V; Real, Fernando; Orikaza, Cristina M; da Cunha, Julia P C; Vitorino, Francisca N L; Cordero, Esteban M; Sobreira, Tiago J P; Mortara, Renato A

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas' disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo . Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas' disease.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

PubMed Central

Florentino, Pilar T. V.; Real, Fernando; Orikaza, Cristina M.; da Cunha, Julia P. C.; Vitorino, Francisca N. L.; Cordero, Esteban M.; Sobreira, Tiago J. P.; Mortara, Renato A.

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. PMID:29692765

A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure.

PubMed

Steckelberg, Anna-Lena; Akiyama, Benjamin M; Costantino, David A; Sit, Tim L; Nix, Jay C; Kieft, Jeffrey S

2018-06-19

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication

PubMed Central

Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H.; Züst, Roland; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; Silverman, Robert H.; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C.; Ziebuhr, John; Kalinke, Ulrich

2017-01-01

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses. PMID:28158275

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

PubMed

Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

2015-09-01

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN

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

The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

PubMed

Dauber, Bianca; Saffran, Holly A; Smiley, James R

2014-09-01

We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during

Ultrastructure of the replication sites of positive-strand RNA viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

2015-05-15

Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. Inmore » this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.« less

RNA trafficking in parasitic plant systems

PubMed Central

LeBlanc, Megan; Kim, Gunjune; Westwood, James H.

2012-01-01

RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host–parasite connections and the potential significance of host RNAs for the parasite. Additional research on host–parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking. PMID:22936942

Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nitschke, Matthias; Korte, Thomas; Tielesch, Claudia

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 suggestmore » that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.« less

Chlamydia Inhibit Host Cell Apoptosis by Degradation of Proapoptotic BH3-only Proteins

PubMed Central

Fischer, Silke F.; Vier, Juliane; Kirschnek, Susanne; Klos, Andreas; Hess, Simone; Ying, Songmin; Häcker, Georg

2004-01-01

Chlamydia are obligate intracellular bacteria that replicate in a vacuole inside a host cell. Chlamydial infection has been shown to protect the host cell against apoptotic stimuli. This is likely important for the ability of Chlamydia to reproduce in human cells. Here we show that resistance to apoptosis is conveyed by the destruction of the proapoptotic BH3-only proteins Bim/Bod, Puma, and Bad during infection. Apoptotic stimuli were blocked upstream of the mitochondrial activation of Bax/Bak. During infection with both species, Chlamydia trachomatis and Chlamydia pneumoniae, Bim protein gradually disappeared without noticeable changes in Bim mRNA. The disappearance was blocked by inhibitors of the proteasome. Infected cells retained sensitivity to Bim expressed by transfection, indicating functional relevance of the Bim disappearance. Fusion to Bim targeted the green fluorescent protein for destruction during infection. Analysis of truncation mutants showed that a short region of Bim containing the BH3 domain was sufficient for destruction during chlamydial infection. Like Bim, Puma and Bad proteins disappeared during infection. These results reveal a novel way by which microbes can interfere with the host cell's apoptotic machinery, and provide a molecular explanation of the cellular resistance to apoptosis during infection with Chlamydia. PMID:15452181

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

PubMed

Yang, Andrew C; du Bois, Haley; Olsson, Niclas; Gate, David; Lehallier, Benoit; Berdnik, Daniela; Brewer, Kyle D; Bertozzi, Carolyn R; Elias, Joshua E; Wyss-Coray, Tony

2018-06-13

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr Y43G ) and a phenylalanyl ( MmPhe T413G ) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr Y43G and MmPhe T413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr Y43G and MmPhe T413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Introduction to Single-Cell RNA Sequencing.

PubMed

Olsen, Thale Kristin; Baryawno, Ninib

2018-04-01

During the last decade, high-throughput sequencing methods have revolutionized the entire field of biology. The opportunity to study entire transcriptomes in great detail using RNA sequencing (RNA-seq) has fueled many important discoveries and is now a routine method in biomedical research. However, RNA-seq is typically performed in "bulk," and the data represent an average of gene expression patterns across thousands to millions of cells; this might obscure biologically relevant differences between cells. Single-cell RNA-seq (scRNA-seq) represents an approach to overcome this problem. By isolating single cells, capturing their transcripts, and generating sequencing libraries in which the transcripts are mapped to individual cells, scRNA-seq allows assessment of fundamental biological properties of cell populations and biological systems at unprecedented resolution. Here, we present the most common scRNA-seq protocols in use today and the basics of data analysis and discuss factors that are important to consider before planning and designing an scRNA-seq project. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Genomic segments RNA1 and RNA2 of Prunus necrotic ringspot virus codetermine viral pathogenicity to adapt to alternating natural Prunus hosts.

PubMed

Cui, Hongguang; Hong, Ni; Wang, Guoping; Wang, Aiming

2013-05-01

Prunus necrotic ringspot virus (PNRSV) affects Prunus fruit production worldwide. To date, numerous PNRSV isolates with diverse pathological properties have been documented. To study the pathogenicity of PNRSV, which directly or indirectly determines the economic losses of infected fruit trees, we have recently sequenced the complete genome of peach isolate Pch12 and cherry isolate Chr3, belonging to the pathogenically aggressive PV32 group and mild PV96 group, respectively. Here, we constructed the Chr3- and Pch12-derived full-length cDNA clones that were infectious in the experimental host cucumber and their respective natural Prunus hosts. Pch12-derived clones induced much more severe symptoms than Chr3 in cucumber, and the pathogenicity discrepancy between Chr3 and Pch12 was associated with virus accumulation. By reassortment of genomic segments, swapping of partial genomic segments, and site-directed mutagenesis, we identified the 3' terminal nucleotide sequence (1C region) in RNA1 and amino acid K at residue 279 in RNA2-encoded P2 as the severe virulence determinants in Pch12. Gain-of-function experiments demonstrated that both the 1C region and K279 of Pch12 were required for severe virulence and high levels of viral accumulation. Our results suggest that PNRSV RNA1 and RNA2 codetermine viral pathogenicity to adapt to alternating natural Prunus hosts, likely through mediating viral accumulation.

RNA-dependent RNA polymerases from flaviviruses and Picornaviridae.

PubMed

Lescar, Julien; Canard, Bruno

2009-12-01

Flaviviruses and picornaviruses are positive-strand RNA viruses that encode the RNA-dependent RNA polymerase (RdRp) required for replicating the viral genome in infected cells. Because of their specific and essential role in the virus life cycle, RdRps are prime targets for antiviral drugs. Recent structural data have shed light on the different strategies used by RdRps from flaviviruses and Picornaviridae to initiate RNA polymerization. New details about the catalytic mechanism, the role of metal ions, how these RdRps interact with other nonstructural (NS) viral and host-cell proteins as well as with the viral RNA genome have also been published. These advances contribute to give a more complete picture of the 3D structure and mechanism of a membrane-bound viral replication complex for these two classes of medically important human pathogens.

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

PubMed Central

Komoda, Keisuke; Naito, Satoshi; Ishikawa, Masayuki

2004-01-01

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses. PMID:14769932

Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

PubMed Central

Neupane, Biswas; Bai, Fengwei; Sherman, Michael B.; Choi, Kyung H.; Neelakanta, Girish

2018-01-01

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate

The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

PubMed Central

Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

2011-01-01

The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their ‘fresh’ host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its ‘fresh’ host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA. PMID:21729006

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

PubMed Central

Nakagawa, K.; Lokugamage, K.G.; Makino, S.

2017-01-01

Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

DNA forms of arboviral RNA genomes are generated following infection in mosquito cell cultures.

PubMed

Nag, Dilip K; Brecher, Matthew; Kramer, Laura D

2016-11-01

Although infections of vertebrate hosts by arthropod-borne viruses may lead to pathogenic outcomes, infections of vector mosquitoes result in persistent infections, where the virus replicates in the host without causing apparent pathological effects. It is unclear how persistent infections are established and maintained in mosquitoes. Several reports revealed the presence of flavivirus-like DNA sequences in the mosquito genome, and recent studies have shown that DNA forms of RNA viruses restrict virus replication in Drosophila, suggesting that DNA forms may have a role in developing persistent infections. Here, we sought to investigate whether arboviruses generate DNA forms following infection in mosquitoes. Our results with West Nile, Dengue, and La Crosse viruses demonstrate that DNA forms of the viral RNA genome are generated in mosquito cells; however, not the entire viral genome, but patches of viral RNA in DNA forms can be detected 24h post infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process.

PubMed

Forestier, Claire-Lise; Machu, Christophe; Loussert, Celine; Pescher, Pascale; Späth, Gerald F

2011-04-21

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Scaffold attachment factor B suppresses HIV-1 infection of CD4+ cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat.

PubMed

Ma, Li; Sun, Li; Jin, Xia; Xiong, Si-Dong; Wang, Jian-Hua

2018-06-10

The 5' end of HIV-1 long terminal repeat (LTR) promoter plays an essential role in driving viral transcription and productive infection. Multiple host and viral factors regulate LTR activity and modulate HIV-1 latency. Manipulation of the HIV-1 LTR provides a potential therapeutic strategy for combating HIV-1 persistence. In this study, we identified an RNA-/DNA-binding protein, Scaffold Attachment Factor B (SAFB1) as a host-cell factor that represses HIV-1 transcription. We found that SAFB1 bound to HIV-1 5`-LTR and significantly repressed 5`-LTR-driven-viral transcription and HIV-1 infection of CD4 + T cells. Mechanistically, SAFB1-mediated repression of HIV-1 transcription and infection was independent of its RNA- and DNA-binding capacities, instead, by binding to phosphorylated RNA polymerase II (RNA pol II), SAFB1 blocked its recruitment to the HIV-1 LTR. Of note, the SAFB1-mediated repression of HIV-1 transcription from proviral DNA maintained HIV-1 latency in CD4 + T cells. In summary, our findings reveal that SAFB1 binds to HIV-1-LTR and physically interacts with phosphorylated RNA pol II, repressing HIV-1 transcription initiation and elongation. Our findings improve the understanding of host modulation of HIV-1 transcription and latency and provide a new host-cell target for improved anti-HIV-1 therapies. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA.

PubMed

Vallery, Tenaya K; Withers, Johanna B; Andoh, Joana A; Steitz, Joan A

2018-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments. IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus. Copyright © 2018 American Society for Microbiology.

Movement of regulatory RNA between animal cells.

PubMed

Jose, Antony M

2015-07-01

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. © 2015 Wiley Periodicals, Inc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Hongxing, E-mail: Hongxing.Zhao@igp.uu.se; Chen, Maoshan; Lind, Sara Bergström

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 phasemore » 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. - Highlights: • The expression of 398 lncRNAs showed a distinct temporal pattern during Ad2 infection. • 80% of the deregulated lncRNAs were up-regulated during the late phase of infection. • The deregulated lncRNAs potentiallyinteract with 33 cellular RNA binding proteins. • These RBPs are involved in RNA splicing, nuclear export and translation. • Adenovirus exploits the cellular lncRNA network to optimize its replication.« less

Rare Cell Detection by Single-Cell RNA Sequencing as Guided by Single-Molecule RNA FISH.

PubMed

Torre, Eduardo; Dueck, Hannah; Shaffer, Sydney; Gospocic, Janko; Gupte, Rohit; Bonasio, Roberto; Kim, Junhyong; Murray, John; Raj, Arjun

2018-02-28

Although single-cell RNA sequencing can reliably detect large-scale transcriptional programs, it is unclear whether it accurately captures the behavior of individual genes, especially those that express only in rare cells. Here, we use single-molecule RNA fluorescence in situ hybridization as a gold standard to assess trade-offs in single-cell RNA-sequencing data for detecting rare cell expression variability. We quantified the gene expression distribution for 26 genes that range from ubiquitous to rarely expressed and found that the correspondence between estimates across platforms improved with both transcriptome coverage and increased number of cells analyzed. Further, by characterizing the trade-off between transcriptome coverage and number of cells analyzed, we show that when the number of genes required to answer a given biological question is small, then greater transcriptome coverage is more important than analyzing large numbers of cells. More generally, our report provides guidelines for selecting quality thresholds for single-cell RNA-sequencing experiments aimed at rare cell analyses. Copyright © 2018 Elsevier Inc. All rights reserved.

Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells.

PubMed

Li, Yang; Basavappa, Megha; Lu, Jinfeng; Dong, Shuwei; Cronkite, D Alexander; Prior, John T; Reinecker, Hans-Christian; Hertzog, Paul; Han, Yanhong; Li, Wan-Xiang; Cheloufi, Sihem; Karginov, Fedor V; Ding, Shou-Wei; Jeffrey, Kate L

2016-12-05

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens 1 . However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago 2 , remains unknown 3 . Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence 4-7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs) 8,9 . Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice 10,11 . However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells 12-21 . Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

T7 RNA polymerase-driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis.

PubMed

Juhas, Mario; Ajioka, James W

2017-11-01

The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome-encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase-driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

PubMed

Takeshita, Daijiro; Tomita, Kozo

2010-09-07

Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

Clinical significance of miRNA host gene promoter methylation in prostate cancer.

PubMed

Daniunaite, Kristina; Dubikaityte, Monika; Gibas, Povilas; Bakavicius, Arnas; Rimantas Lazutka, Juozas; Ulys, Albertas; Jankevicius, Feliksas; Jarmalaite, Sonata

2017-07-01

Only a part of prostate cancer (PCa) patients has aggressive malignancy requiring adjuvant treatment after radical prostatectomy (RP). Biomarkers capable to predict biochemical PCa recurrence (BCR) after RP would significantly improve preoperative risk stratification and treatment decisions. MicroRNA (miRNA) deregulation has recently emerged as an important phenomenon in tumor development and progression, however, the mechanisms remain largely unstudied. In the present study, based on microarray profiling of DNA methylation in 9 pairs of PCa and noncancerous prostate tissues (NPT), host genes of miR-155-5p, miR-152-3p, miR-137, miR-31-5p, and miR-642a, -b were analyzed for promoter methylation in 129 PCa, 35 NPT, and 17 benign prostatic hyperplasia samples (BPH) and compared to the expression of mature miRNAs and their selected targets (DNMT1, KDM1A, and KDM5B). The Cancer Genome Atlas dataset was utilized for validation. Methylation of mir-155, mir-152, and mir-137 host genes was PCa-specific, and downregulation of miR-155-5p significantly correlated with promoter methylation. Higher KDM5B expression was observed in samples with methylated mir-155 or mir-137 promoters, whereas upregulation of KDM1A and DNMT1 was associated with mir-155 and mir-152 methylation status, respectively. Promoter methylation of mir-155, mir-152, and mir-31 was predictive of BCR-free survival in various Cox models and increased the prognostic value of clinicopathologic factors. In conclusion, methylated mir-155, mir-152, mir-137, and mir-31 host genes are promising diagnostic and/or prognostic biomarkers of PCa. Methylation status of particular miRNA host genes as independent variables or in combinations might assist physicians in identifying poor prognosis PCa patients preoperatively. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

PubMed

Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.

PubMed

Nain, Minu; Mukherjee, Sriparna; Karmakar, Sonali Porey; Paton, Adrienne W; Paton, James C; Abdin, M Z; Basu, Anirban; Kalia, Manjula; Vrati, Sudhanshu

2017-03-15

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target. IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric

Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

DOE Office of Scientific and Technical Information (OSTI.GOV)

Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Riftmore » Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.« less

Human Cytomegalovirus Strategies to Maintain and Promote mRNA Translation

PubMed Central

Vincent, Heather A.; Ziehr, Benjamin; Moorman, Nathaniel J.

2016-01-01

mRNA translation requires the ordered assembly of translation initiation factors and ribosomal subunits on a transcript. Host signaling pathways regulate each step in this process to match levels of protein synthesis to environmental cues. In response to infection, cells activate multiple defenses that limit viral protein synthesis, which viruses must counteract to successfully replicate. Human cytomegalovirus (HCMV) inhibits host defenses that limit viral protein expression and manipulates host signaling pathways to promote the expression of both host and viral proteins necessary for virus replication. Here we review key regulatory steps in mRNA translation, and the strategies used by HCMV to maintain protein synthesis in infected cells. PMID:27089357

Role of host protein Ebp1 in influenza virus growth: intracellular localization of Ebp1 in virus-infected and uninfected cells.

PubMed

Honda, Ayae

2008-01-20

The cellular protein Ebp1 was identified to interact with PB1 protein of influenza virus RNA polymerase, and inhibit both RNA synthesis in vitro and influenza virus replication in vivo [Honda, A., Okamoto, T., Ishihama, A., 2007. Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes Cells 12, 133-142]. The intracellular localization of Ebp1 that is involved in cell proliferation control was analyzed by direct immunostaining of cells before and after influenza virus infection. Ebp1 was found to localize in the nuclear membrane of uninfected cells, and to form nuclear aggregates with viral P proteins in virus-infected cells.

Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA

PubMed Central

LeBlanc, Jason; Weil, Jason; Beemon, Karen

2013-01-01

After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases. PMID:23754689

Intracellular coordination of potyviral RNA functions in infection

PubMed Central

Mäkinen, Kristiina; Hafrén, Anders

2014-01-01

Establishment of an infection cycle requires mechanisms to allocate the genomes of (+)-stranded RNA viruses in a balanced ratio to translation, replication, encapsidation, and movement, as well as mechanisms to prevent translocation of viral RNA (vRNA) to cellular RNA degradation pathways. The ratio of vRNA allocated to various functions is likely balanced by the availability of regulatory proteins or competition of the interaction sites within regulatory ribonucleoprotein complexes. Due to the transient nature of viral processes and the interdependency between vRNA pathways, it is technically demanding to work out the exact molecular mechanisms underlying vRNA regulation. A substantial number of viral and host proteins have been identified that facilitate the steps that lead to the assembly of a functional potyviral RNA replication complex and their fusion with chloroplasts. Simultaneously with on-going viral replication, part of the replicated potyviral RNA enters movement pathways. Although not much is known about the processes of potyviral RNA release from viral replication complexes, the molecular interactions involved in these processes determine the fate of the replicated vRNA. Some viral and host cell proteins have been described that direct replicated potyviral RNA to translation to enable potyviral gene expression and productive infection. The antiviral defense of the cell causes vRNA degradation by RNA silencing. We hypothesize that also plant pathways involved in mRNA decay may have a role in the coordination of potyviral RNA expression. In this review, we discuss the roles of different potyviral and host proteins in the coordination of various potyviral RNA functions. PMID:24723931

Intracellular coordination of potyviral RNA functions in infection.

PubMed

Mäkinen, Kristiina; Hafrén, Anders

2014-01-01

Establishment of an infection cycle requires mechanisms to allocate the genomes of (+)-stranded RNA viruses in a balanced ratio to translation, replication, encapsidation, and movement, as well as mechanisms to prevent translocation of viral RNA (vRNA) to cellular RNA degradation pathways. The ratio of vRNA allocated to various functions is likely balanced by the availability of regulatory proteins or competition of the interaction sites within regulatory ribonucleoprotein complexes. Due to the transient nature of viral processes and the interdependency between vRNA pathways, it is technically demanding to work out the exact molecular mechanisms underlying vRNA regulation. A substantial number of viral and host proteins have been identified that facilitate the steps that lead to the assembly of a functional potyviral RNA replication complex and their fusion with chloroplasts. Simultaneously with on-going viral replication, part of the replicated potyviral RNA enters movement pathways. Although not much is known about the processes of potyviral RNA release from viral replication complexes, the molecular interactions involved in these processes determine the fate of the replicated vRNA. Some viral and host cell proteins have been described that direct replicated potyviral RNA to translation to enable potyviral gene expression and productive infection. The antiviral defense of the cell causes vRNA degradation by RNA silencing. We hypothesize that also plant pathways involved in mRNA decay may have a role in the coordination of potyviral RNA expression. In this review, we discuss the roles of different potyviral and host proteins in the coordination of various potyviral RNA functions.

Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

PubMed Central

VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

2014-01-01

The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

Innate immune restriction and antagonism of viral RNA lacking 2'-O methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyde, Jennifer L.; Diamond, Michael S., E-mail: diamond@borcim.wustl.edu; Molecular Microbiology, Washington University School of Medicine, St Louis., MO 63110

N-7 and 2′-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m{sup 7}GpppN; cap 1, m{sup 7}GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2′-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on theirmore » RNA through cap-snatching or virally-encoded 2′-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5′-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.« less

Leishmania cell surface prohibitin: role in host-parasite interaction.

PubMed

Jain, Rohit; Ghoshal, Angana; Mandal, Chitra; Shaha, Chandrima

2010-04-01

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95-100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host-parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild-type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI-link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti-prohibitin antibodies during macrophage-Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania-host interaction.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

PubMed

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Human Enterovirus 68 Interferes with the Host Cell Cycle to Facilitate Viral Production

PubMed Central

Wang, Zeng-yan; Zhong, Ting; Wang, Yue; Song, Feng-mei; Yu, Xiao-feng; Xing, Li-ping; Zhang, Wen-yan; Yu, Jing-hua; Hua, Shu-cheng; Yu, Xiao-fang

2017-01-01

Enterovirus D68 (EV-D68) is an emerging pathogen that recently caused a large outbreak of severe respiratory disease in the United States and other countries. Little is known about the relationship between EV-D68 virus and host cells. In this study, we assessed the effect of the host cell cycle on EV-D68 viral production, as well as the ability of EV-D68 to manipulate host cell cycle progression. The results suggest that synchronization in G0/G1 phase, but not S phase, promotes viral production, while synchronization in G2/M inhibits viral production. Both an early EV-D68 isolate and currently circulating strains of EV-D68 can manipulate the host cell cycle to arrest cells in the G0/G1 phase, thus providing favorable conditions for virus production. Cell cycle regulation by EV-D68 was associated with corresponding effects on the expression of cyclins and CDKs, which were observed at the level of the protein and/or mRNA. Furthermore, the viral non-structural protein 3D of EV-D68 prevents progression from G0/G1 to S. Interestingly, another member of the Picornaviridae family, EV-A71, differs from EV-D68 in that G0/G1 synchronization inhibits, rather than promotes, EV-A71 viral replication. However, these viruses are similar in that G2/M synchronization inhibits the production and activity of both viruses, which is suggestive of a common therapeutic target for both types of enterovirus. These results further clarify the pathogenic mechanisms of enteroviruses and provide a potential strategy for the treatment and prevention of EV-D68-related disease. PMID:28229049

Transcriptomic analysis reveals Toxoplasma gondii strain-specific differences in host cell response to dense granule protein GRA15.

PubMed

Liu, Qing; Gao, Wen-Wei; Elsheikha, Hany M; He, Jun-Jun; Li, Fa-Cai; Yang, Wen-Bin; Zhu, Xing-Quan

2018-06-19

Growth and replication of the protozoan parasite Toxoplasma gondii within host cell entail the production of several effector proteins, which the parasite exploits for counteracting the host's immune response. Despite considerable research to define the host signaling pathways manipulated by T. gondii and their effectors, there has been limited progress into understanding how individual members of the dense granule proteins (GRAs) modulate gene expression within host cells. The aim of this study was to evaluate whether T. gondii GRA15 protein plays any role in regulating host gene expression. Baby hamster kidney cells (BHK-21) were transfected with plasmids encoding GRA15 genes of either type I GT1 strain (GRA15 I ) or type II PRU strain (GRA15 II ). Gene expression patterns of transfected and nontransfected BHK-21 cells were investigated using RNA-sequencing analysis. GRA15 I and GRA15 II induced both known and novel transcriptional changes in the transfected BHK-21 cells compared with nontransfected cells. Pathway analysis revealed that GRA15 II was mainly involved in the regulation of tumor necrosis factor (TNF), NF-κB, HTLV-I infection, and NOD-like receptor signaling pathways. GRA15 I preferentially influenced the synthesis of unsaturated fatty acids in host cells. Our findings support the hypothesis that certain functions of GRA15 protein are strain dependent and that GRA15 modulates the expression of signaling pathways and genes with important roles in T. gondii pathophysiology. A greater understanding of host signaling pathways influenced by T. gondii effectors would allow the development of more efficient anti-T. gondii therapeutic schemes, capitalizing on disrupting parasite virulence factors to advance the treatment of toxoplasmosis.

Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection.

PubMed

Dapat, Clyde; Saito, Reiko; Suzuki, Hiroshi; Horigome, Tsuneyoshi

2014-01-22

The emergence of antiviral drug-resistant influenza viruses highlights the need for alternative therapeutic strategies. Elucidation of host factors required during virus infection provides information not only on the signaling pathways involved but also on the identification of novel drug targets. RNA interference screening method had been utilized by several studies to determine these host factors; however, proteomics data on influenza host factors are currently limited. In this study, quantitative phosphoproteomic analysis of human lung cell line (A549) infected with 2009 pandemic influenza virus A (H1N1) virus was performed. Phosphopeptides were enriched from tryptic digests of total protein of infected and mock-infected cells using a titania column on an automated purification system followed by iTRAQ labeling. Identification and quantitative analysis of iTRAQ-labeled phosphopeptides were performed using LC-MS/MS. We identified 366 phosphorylation sites on 283 proteins. Of these, we detected 43 upregulated and 35 downregulated proteins during influenza virus infection. Gene ontology enrichment analysis showed that majority of the identified proteins are phosphoproteins involved in RNA processing, immune system process and response to infection. Host-virus interaction network analysis had identified 23 densely connected subnetworks. Of which, 13 subnetworks contained proteins with altered phosphorylation levels during by influenza virus infection. Our results will help to identify potential drug targets that can be pursued for influenza antiviral drug development. Copyright © 2013 Elsevier B.V. All rights reserved.

Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45.

PubMed

Friedrich, Susann; Schmidt, Tobias; Schierhorn, Angelika; Lilie, Hauke; Szczepankiewicz, Grit; Bergs, Sandra; Liebert, Uwe G; Golbik, Ralph P; Behrens, Sven-Erik

2016-10-01

A prerequisite for the intracellular replication process of the Flavivirus West Nile virus (WNV) is the cyclization of the viral RNA genome, which enables the viral replicase to initiate RNA synthesis. Our earlier studies indicated that the p45 isoform of the cellular AU-rich element binding protein 1 (AUF1) has an RNA chaperone activity, which supports RNA cyclization and viral RNA synthesis by destabilizing a stem structure at the WNV RNA's 3'-end. Here we show that in mammalian cells, AUF1 p45 is consistently modified by arginine methylation of its C terminus. By a combination of different experimental approaches, we can demonstrate that the methyltransferase PRMT1 is necessary and sufficient for AUF1 p45 methylation and that PRMT1 is required for efficient WNV replication. Interestingly, in comparison to the nonmethylated AUF1 p45, the methylated AUF1 p45(aDMA) exhibits a significantly increased affinity to the WNV RNA termini. Further data also revealed that the RNA chaperone activity of AUF1 p45(aDMA) is improved and the methylated protein stimulates viral RNA synthesis considerably more efficiently than the nonmethylated AUF1 p45. In addition to its destabilizing RNA chaperone activity, we identified an RNA annealing activity of AUF1 p45, which is not affected by methylation. Arginine methylation of AUF1 p45 thus represents a specific determinant of its RNA chaperone activity while functioning as a WNV host factor. Our data suggest that the methylation modifies the conformation of AUF1 p45 and in this way affects its RNA binding and restructuring activities. © 2016 Friedrich et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

PubMed Central

Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

2013-01-01

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

A novel negative-stranded RNA virus mediates sex ratio in its parasitoid host

PubMed Central

Wang, Beibei; Yan, Zhichao; Hong, Jian; Werren, John H.; Song, Qisheng

2017-01-01

Parasitoid wasps are important natural enemies of arthropod hosts in natural and agricultural ecosystems and are often associated with viruses or virion-like particles. Here, we report a novel negative-stranded RNA virus from a parasitoid wasp (Pteromalus puparum). The complete viral genome is 12,230 nucleotides in length, containing five non-overlapping, linearly arranged open reading frames. Phylogenetically, the virus clusters with and is a novel member of the mononegaviral family Nyamiviridae, here designated as Pteromalus puparum negative-strand RNA virus 1 (PpNSRV-1). PpNSRV-1 is present in various tissues and life stages of the parasitoid wasp, and is transmitted vertically through infected females and males. Virus infections in field populations of P. puparum wasps ranged from 16.7 to 37.5%, without linearly correlating with temperature. PpNSRV-1 increased adult longevity and impaired several fitness parameters of the wasp, but had no influence on successful parasitism. Strikingly, PpNSRV-1 mediated the offspring sex ratio by decreasing female offspring numbers. RNA interference knockdown of virus open reading frame I eliminated these PpNSRV-1-induced effects. Thus, we infer that PpNSRV-1 has complex effects on its insect host including sex ratio distortion towards males, as well as possible mutualistic benefits through increasing wasp longevity. PMID:28278298

Global genomics and proteomics approaches to identify host factors as targets to induce resistance against Tomato bushy stunt virus.

PubMed

Nagy, Peter D; Pogany, Judit

2010-01-01

The success of RNA viruses as pathogens of plants, animals, and humans depends on their ability to reprogram the host cell metabolism to support the viral infection cycle and to suppress host defense mechanisms. Plus-strand (+)RNA viruses have limited coding potential necessitating that they co-opt an unknown number of host factors to facilitate their replication in host cells. Global genomics and proteomics approaches performed with Tomato bushy stunt virus (TBSV) and yeast (Saccharomyces cerevisiae) as a model host have led to the identification of 250 host factors affecting TBSV RNA replication and recombination or bound to the viral replicase, replication proteins, or the viral RNA. The roles of a dozen host factors involved in various steps of the replication process have been validated in yeast as well as a plant host. Altogether, the large number of host factors identified and the great variety of cellular functions performed by these factors indicate the existence of a truly complex interaction between TBSV and the host cell. This review summarizes the advantages of using a simple plant virus and yeast as a model host to advance our understanding of virus-host interactions at the molecular and cellular levels. The knowledge of host factors gained can potentially be used to inhibit virus replication via gene silencing, expression of dominant negative mutants, or design of specific chemical inhibitors leading to novel specific or broad-range resistance and antiviral tools against (+)RNA plant viruses. Copyright © 2010 Elsevier Inc. All rights reserved.

Myosin-Va-Dependent Cell-To-Cell Transfer of RNA from Schwann Cells to Axons

PubMed Central

Sotelo, José R.; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R.; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A.

2013-01-01

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration. PMID:23626749

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

PubMed

Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

2013-01-01

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

Viral tRNA Mimicry from a Biocommunicative Perspective

PubMed Central

Ariza-Mateos, Ascensión; Gómez, Jordi

2017-01-01

RNA viruses have very small genomes which limits the functions they can encode. One of the strategies employed by these viruses is to mimic key factors of the host cell so they can take advantage of the interactions and activities these factors typically participate in. The viral RNA genome itself was first observed to mimic cellular tRNA over 40 years ago. Since then researchers have confirmed that distinct families of RNA viruses are accessible to a battery of cellular factors involved in tRNA-related activities. Recently, potential tRNA-like structures have been detected within the sequences of a 100 mRNAs taken from human cells, one of these being the host defense interferon-alpha mRNA; these are then additional to the examples found in bacterial and yeast mRNAs. The mimetic relationship between tRNA, cellular mRNA, and viral RNA is the central focus of two considerations described below. These are subsequently used as a preface for a final hypothesis drawing on concepts relating to mimicry from the social sciences and humanities, such as power relations and creativity. Firstly, the presence of tRNA-like structures in mRNAs indicates that the viral tRNA-like signal could be mimicking tRNA-like elements that are contextualized by the specific carrier mRNAs, rather than, or in addition to, the tRNA itself, which would significantly increase the number of potential semiotic relations mediated by the viral signals. Secondly, and in particular, mimicking a host defense mRNA could be considered a potential new viral strategy for survival. Finally, we propose that mRNA’s mimicry of tRNA could be indicative of an ancestral intracellular conflict in which species of mRNAs invaded the cell, but from within. As the meaning of the mimetic signal depends on the context, in this case, the conflict that arises when the viral signal enters the cell can change the meaning of the mRNAs’ internal tRNA-like signals, from their current significance to that they had in the

Single-Cell RNA-Sequencing in Glioma.

PubMed

Johnson, Eli; Dickerson, Katherine L; Connolly, Ian D; Hayden Gephart, Melanie

2018-04-10

In this review, we seek to summarize the literature concerning the use of single-cell RNA-sequencing for CNS gliomas. Single-cell analysis has revealed complex tumor heterogeneity, subpopulations of proliferating stem-like cells and expanded our view of tumor microenvironment influence in the disease process. Although bulk RNA-sequencing has guided our initial understanding of glioma genetics, this method does not accurately define the heterogeneous subpopulations found within these tumors. Single-cell techniques have appealing applications in cancer research, as diverse cell types and the tumor microenvironment have important implications in therapy. High cost and difficult protocols prevent widespread use of single-cell RNA-sequencing; however, continued innovation will improve accessibility and expand our of knowledge gliomas.

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

2016-08-05

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

Reliance of Wolbachia on High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen of Drosophila Cells.

PubMed

White, Pamela M; Serbus, Laura R; Debec, Alain; Codina, Adan; Bray, Walter; Guichet, Antoine; Lokey, R Scott; Sullivan, William

2017-04-01

Wolbachia are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a Wolbachia -infected Drosophila cell line and genome-wide RNA interference (RNAi) screening to identify host factors that influence Wolbachia titer. By screening an RNAi library targeting 15,699 transcribed host genes, we identified 36 candidate genes that dramatically reduced Wolbachia titer and 41 that increased Wolbachia titer. Host gene knockdowns that reduced Wolbachia titer spanned a broad array of biological pathways including genes that influenced mitochondrial function and lipid metabolism. In addition, knockdown of seven genes in the host ubiquitin and proteolysis pathways significantly reduced Wolbachia titer. To test the in vivo relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of Wolbachia in the Drosophila oocyte. The presence of Wolbachia in either cell lines or oocytes dramatically alters the distribution and abundance of ubiquitinated proteins. Functional studies revealed that maintenance of Wolbachia titer relies on an intact host Endoplasmic Reticulum (ER)-associated protein degradation pathway (ERAD). Accordingly, electron microscopy studies demonstrated that Wolbachia is intimately associated with the host ER and dramatically alters the morphology of this organelle. Given Wolbachia lack essential amino acid biosynthetic pathways, the reliance of Wolbachia on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning Wolbachia with amino acids. In addition, the reliance of Wolbachia on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for Wolbachia ' s potent ability to prevent RNA virus replication. Copyright © 2017 by the Genetics Society of America.

Suppression of RNA Silencing by a Plant DNA Virus Satellite Requires a Host Calmodulin-Like Protein to Repress RDR6 Expression

PubMed Central

Li, Fangfang; Huang, Changjun; Li, Zhenghe; Zhou, Xueping

2014-01-01

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential

Single-cell transcriptional dynamics of flavivirus infection

PubMed Central

Bekerman, Elena

2018-01-01

Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level. PMID:29451494

Silencing VDAC1 Expression by siRNA Inhibits Cancer Cell Proliferation and Tumor Growth In Vivo

PubMed Central

Arif, Tasleem; Vasilkovsky, Lilia; Refaely, Yael; Konson, Alexander; Shoshan-Barmatz, Varda

2014-01-01

Alterations in cellular metabolism and bioenergetics are vital for cancer cell growth and motility. Here, the role of the mitochondrial protein voltage-dependent anion channel (VDAC1), a master gatekeeper regulating the flux of metabolites and ions between mitochondria and the cytoplasm, in regulating the growth of several cancer cell lines was investigated by silencing VDAC1 expression using small interfering RNA (siRNA). A single siRNA specific to the human VDAC1 sequence at nanomolar concentrations led to some 90% decrease in VDAC1 levels in the lung A549 and H358, prostate PC-3, colon HCT116, glioblastoma U87, liver HepG2, and pancreas Panc-1 cancer cell lines. VDAC1 silencing persisted 144 hours post-transfection and resulted in profound inhibition of cell growth in cancer but not in noncancerous cells, with up to 90% inhibition being observed over 5 days that was prolonged by a second transfection. Cells expressing low VDAC1 levels showed decreased mitochondrial membrane potential and adenoside triphosphate (ATP) levels, suggesting limited metabolite exchange between mitochondria and cytosol. Moreover, cells silenced for VDAC1 expression showed decreased migration, even in the presence of the wound healing accelerator basic fibroblast growth factor (bFGF). VDAC1-siRNA inhibited cancer cell growth in a Matrigel-based assay in host nude mice. Finally, in a xenograft lung cancer mouse model, chemically modified VDAC1-siRNA not only inhibited tumor growth but also resulted in tumor regression. This study thus shows that VDAC1 silencing by means of RNA interference (RNAi) dramatically inhibits cancer cell growth and tumor development by disabling the abnormal metabolic behavior of cancer cells, potentially paving the way for a more effective pipeline of anticancer drugs. PMID:24781191

RNA turnover and protein synthesis in fish cells.

PubMed

Smith, R W; Palmer, R M; Houlihan, D F

2000-03-01

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from 3H-phenylalanine and 3H-uridine incorporation, respectively, and expressed as % x day(-1) and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (approximately 175 microg RNA x mg(-1) cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 +/- 1.8)>RTP (25.1 +/- 1.7)>CHSE 214 (17.6 +/-1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised x microg(-1) RNA x day(-1), corresponded to the lowest RNA half-life, 75.4 +/- 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5-6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis.

Tobacco mosaic virus Movement Protein Enhances the Spread of RNA Silencing

PubMed Central

Vogler, Hannes; Kwon, Myoung-Ok; Dang, Vy; Sambade, Adrian; Fasler, Monika; Ashby, Jamie; Heinlein, Manfred

2008-01-01

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA. PMID:18389061

Insights into Host Cell Modulation and Induction of New Cells by the Corn Smut Ustilago maydis.

PubMed

Redkar, Amey; Matei, Alexandra; Doehlemann, Gunther

2017-01-01

Many filamentous fungal pathogens induce drastic modulation of host cells causing abnormal infectious structures such as galls, or tumors that arise as a result of re-programming in the original developmental cell fate of a colonized host cell. Developmental consequences occur predominantly with biotrophic phytopathogens. This suggests that these host structures result as an outcome of efficient defense suppression and intimate fungal-host interaction to suit the pathogen's needs for completion of its infection cycle. This mini-review mainly summarizes host cell re-programming that occurs in the Ustilago maydis - maize interaction, in which the pathogen deploys cell-type specific effector proteins with varying activities. The fungus senses the physiological status and identity of colonized host cells and re-directs the endogenous developmental program of its host. The disturbance of host cell physiology and cell fate leads to novel cell shapes, increased cell size, and/or the number of host cells. We particularly highlight the strategies of U. maydis to induce physiologically varied host organs to form the characteristic tumors in both vegetative and floral parts of maize.

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.

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

PubMed

Dilley, Kari A; Voorhies, Alexander A; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B; Lorenzi, Hernan; Basler, Christopher F; Shabman, Reed S

2017-01-01

Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing

PubMed Central

Voorhies, Alexander A.; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B.; Lorenzi, Hernan; Basler, Christopher F.; Shabman, Reed S.

2017-01-01

Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation. PMID:28636653

RNA viruses and microRNAs: challenging discoveries for the 21st century

PubMed Central

Swaminathan, Gokul; Martin-Garcia, Julio

2013-01-01

RNA viruses represent the predominant cause of many clinically relevant viral diseases in humans. Among several evolutionary advantages acquired by RNA viruses, the ability to usurp host cellular machinery and evade antiviral immune responses is imperative. During the past decade, RNA interference mechanisms, especially microRNA (miRNA)-mediated regulation of cellular protein expression, have revolutionized our understanding of host-viral interactions. Although it is well established that several DNA viruses express miRNAs that play crucial roles in their pathogenesis, expression of miRNAs by RNA viruses remains controversial. However, modulation of the miRNA machinery by RNA viruses may confer multiple benefits for enhanced viral replication and survival in host cells. In this review, we discuss the current literature on RNA viruses that may encode miRNAs and the varied advantages of engineering RNA viruses to express miRNAs as potential vectors for gene therapy. In addition, we review how different families of RNA viruses can alter miRNA machinery for productive replication, evasion of antiviral immune responses, and prolonged survival. We underscore the need to further explore the complex interactions of RNA viruses with host miRNAs to augment our understanding of host-virus interplay. PMID:24046280

Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

PubMed

Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

2017-10-20

Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

PubMed

Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

2013-04-17

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

PubMed Central

Moser, Joanna J.; Fritzler, Marvin J.

2010-01-01

Background GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. Methodology/Principal Findings RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. Conclusions/Significance The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

PubMed

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

A furoviral replicase recruits host HSP70 to membranes for viral RNA replication

PubMed Central

Yang, Jian; Zhang, Fen; Cai, Nian-Jun; Wu, Ne; Chen, Xuan; Li, Jing; Meng, Xiang-Feng; Zhu, Tong-Quan; Chen, Jian-Ping; Zhang, Heng-Mu

2017-01-01

Many host factors have been identified to be involved in viral infection. However, although furoviruses cause important diseases of cereals worldwide, no host factors have yet been identified that interact with furoviral genes or participate in the viral infection cycle. In this study, both TaHSP70 and NbHSP70 were up-regulated in Chinese wheat mosaic furovirus (CWMV)-infected plants. Their overexpression and inhibition were correlated with the accumulation of viral genomic RNAs, suggesting that the HSP70 genes could be necessary for CWMV infection. The subcellular distributions of TaHSP70 and NbHSP70 were significantly affected by CWMV infection or by infiltration of RNA1 alone. Further assays showed that the viral replicase encoded by CWMV RNA1 interacts with both TaHSP70 and NbHSP70 in vivo and vitro and that its region aa167–333 was responsible for the interaction. Subcellular assays showed that the viral replicase could recruit both TaHSP70 and NbHSP70 from the cytoplasm or nucleus to the granular aggregations or inclusion-like structures on the intracellular membrane system, suggesting that both HSP70s may be recruited into the viral replication complex (VRC) to promote furoviral replication. This is the first host factor identified to be involved in furoviral infection, which extends the list and functional scope of HSP70 chaperones. PMID:28367995

DUSP11 – An RNA phosphatase that regulates host and viral non-coding RNAs in mammalian cells

PubMed Central

Burke, James M.; Sullivan, Christopher S.

2017-01-01

ABSTRACT Dual-specificity phosphatase 11 (DUSP11) is a conserved protein tyrosine phosphatase (PTP) in metazoans. The cellular substrates and physiologic activities of DUSP11 remain largely unknown. In nematodes, DUSP11 is required for normal development and RNA interference against endogenous RNAs (endo-RNAi) via molecular mechanisms that are not well understood. However, mammals lack analogous endo-RNAi pathways and consequently, a role for DUSP11 in mammalian RNA silencing was unanticipated. Recent work from our laboratory demonstrated that DUSP11 activity alters the silencing potential of noncanonical viral miRNAs in mammalian cells. Our studies further uncovered direct cellular substrates of DUSP11 and suggest that DUSP11 is part of regulatory pathway that controls the abundance of select triphosphorylated noncoding RNAs. Here, we highlight recent findings and present new data that advance understanding of mammalian DUSP11 during gene silencing and discuss the emerging biological activities of DUSP11 in mammalian cells. PMID:28296624

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

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

Enforcing host cell polarity: an apicomplexan parasite strategy towards dissemination.

PubMed

Baumgartner, Martin

2011-08-01

The propagation of apicomplexan parasites through transmitting vectors is dependent on effective dissemination of parasites inside the mammalian host. Intracellular Toxoplasma and Theileria parasites face the challenge that their spread inside the host depends in part on the motile capacities of their host cells. In response, these parasites influence the efficiency of dissemination by altering adhesive and/or motile properties of their host cells. Theileria parasites do so by targeting signalling pathways that control host cell actin dynamics. The resulting enforced polar host cell morphology facilitates motility and invasiveness, by establishing focal adhesion and invasion structures at the leading edge of the infected cell. This parasite strategy highlights mechanisms of motility regulation that are also likely relevant for immune or cancer cell motility. Copyright © 2011 Elsevier Ltd. All rights reserved.

Chlamydia trachomatis Cellular Exit Alters Interactions with Host Dendritic Cells

PubMed Central

Sherrid, Ashley M.

2017-01-01

ABSTRACT The strategies utilized by pathogens to exit host cells are an area of pathogenesis which has received surprisingly little attention, considering the necessity of this step for infections to propagate. Even less is known about how exit through these pathways affects downstream host-pathogen interactions and the generation of an immune response. Chlamydia trachomatis exits host epithelial cells through two equally active mechanisms: lysis and extrusion. Studies have characterized the outcome of interactions between host innate immune cells, such as dendritic cells and macrophages, and free, extracellular Chlamydia bacteria, such as those resulting from lysis. Exit via extrusion generates a distinct, host-membrane-bound compartment of Chlamydia separate from the original infected cell. In this study, we assessed the effect of containment within extrusions upon the interaction between Chlamydia and host dendritic cells. Extrusion dramatically affected the outcome of Chlamydia-dendritic cell interactions for both the bacterium and the host cell. Dendritic cells rapidly underwent apoptosis in response to engulfment of an extrusion, while uptake of an equivalent dose of free Chlamydia had no such effect. Containment within an extrusion also prolonged bacterial survival within dendritic cells and altered the initial innate immune signaling by the dendritic cell. PMID:28223346

MicroRNA and Pathogenesis of Enterovirus Infection

PubMed Central

Ho, Bing-Ching; Yang, Pan-Chyr; Yu, Sung-Liang

2016-01-01

There are no currently available specific antiviral therapies for non-polio Enterovirus infections. Although several vaccines have entered clinical trials, the efficacy requires further evaluation, particularly for cross-strain protective activity. Curing patients with viral infections is a public health problem due to antigen alterations and drug resistance caused by the high genomic mutation rate. To conquer these limits in the development of anti-Enterovirus treatments, a comprehensive understanding of the interactions between Enterovirus and host cells is urgently needed. MicroRNA (miRNA) constitutes the biggest family of gene regulators in mammalian cells and regulates almost a half of all human genes. The roles of miRNAs in Enterovirus pathogenesis have recently begun to be noted. In this review, we shed light on recent advances in the understanding of Enterovirus infection-modulated miRNAs. The impacts of altered host miRNAs on cellular processes, including immune escape, apoptosis, signal transduction, shutdown of host protein synthesis and viral replication, are discussed. Finally, miRNA-based medication provides a promising strategy for the development of antiviral therapy. PMID:26751468

MicroRNA and Pathogenesis of Enterovirus Infection.

PubMed

Ho, Bing-Ching; Yang, Pan-Chyr; Yu, Sung-Liang

2016-01-06

There are no currently available specific antiviral therapies for non-polio Enterovirus infections. Although several vaccines have entered clinical trials, the efficacy requires further evaluation, particularly for cross-strain protective activity. Curing patients with viral infections is a public health problem due to antigen alterations and drug resistance caused by the high genomic mutation rate. To conquer these limits in the development of anti-Enterovirus treatments, a comprehensive understanding of the interactions between Enterovirus and host cells is urgently needed. MicroRNA (miRNA) constitutes the biggest family of gene regulators in mammalian cells and regulates almost a half of all human genes. The roles of miRNAs in Enterovirus pathogenesis have recently begun to be noted. In this review, we shed light on recent advances in the understanding of Enterovirus infection-modulated miRNAs. The impacts of altered host miRNAs on cellular processes, including immune escape, apoptosis, signal transduction, shutdown of host protein synthesis and viral replication, are discussed. Finally, miRNA-based medication provides a promising strategy for the development of antiviral therapy.

Bromovirus movement protein genes play a crucial role in host specificity.

PubMed Central

Mise, K; Allison, R F; Janda, M; Ahlquist, P

1993-01-01

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small. Images PMID:7682628

Bartonella entry mechanisms into mammalian host cells.

PubMed

Eicher, Simone C; Dehio, Christoph

2012-08-01

The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment. © 2012 Blackwell Publishing Ltd.

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication

PubMed Central

Fukuhara, Takeshi; Hosoya, Takamitsu; Shimizu, Saki; Sumi, Kengo; Oshiro, Takako; Yoshinaka, Yoshiyuki; Suzuki, Masaaki; Yamamoto, Naoki; Herzenberg, Leonore A.; Herzenberg, Leonard A.; Hagiwara, Masatoshi

2006-01-01

Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine–arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases. PMID:16840555

Dual RNA-seq of the plant pathogen phytophthora ramorum and its tanoak host

Treesearch

Katherine J. Hayden; Matteo Garbelotto; Brian J. Knaus; Richard C. Cronn; Hardeep Rai; Jessica W. Wright

2014-01-01

Emergent diseases are an ever-increasing threat to forests and forest ecosystems and necessitate the development of research tools for species that often may have few preexisting resources. We sequenced the mRNA expressed by the sudden oak death pathogen Phytophthora ramorum and its most susceptible forest host, tanoak, within the same tissue at two time points after...

Comparative Analysis of Single-Cell RNA Sequencing Methods.

PubMed

Ziegenhain, Christoph; Vieth, Beate; Parekh, Swati; Reinius, Björn; Guillaumet-Adkins, Amy; Smets, Martha; Leonhardt, Heinrich; Heyn, Holger; Hellmann, Ines; Enard, Wolfgang

2017-02-16

Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq, and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods, and it provides a framework for benchmarking further improvements of scRNA-seq protocols. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Nagy, Peter D; Pogany, Judit; Xu, Kai

2016-03-03

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.

Lipids in host-pathogen interactions: pathogens exploit the complexity of the host cell lipidome.

PubMed

van der Meer-Janssen, Ynske P M; van Galen, Josse; Batenburg, Joseph J; Helms, J Bernd

2010-01-01

Lipids were long believed to have a structural role in biomembranes and a role in energy storage utilizing cellular lipid droplets and plasma lipoproteins. Research over the last decades has identified an additional role of lipids in cellular signaling, membrane microdomain organization and dynamics, and membrane trafficking. These properties make lipids an attractive target for pathogens to modulate host cell processes in order to allow their survival and replication. In this review we will summarize the often ingenious strategies of pathogens to modify the lipid homeostasis of host cells, allowing them to divert cellular processes. To this end pathogens take full advantage of the complexity of the lipidome. The examples are categorized in generalized and emerging principles describing the involvement of lipids in host-pathogen interactions. Several pathogens are described that simultaneously induce multiple changes in the host cell signaling and trafficking mechanisms. Elucidation of these pathogen-induced changes may have important implications for drug development. The emergence of high-throughput lipidomic techniques will allow the description of changes of the host cell lipidome at the level of individual molecular lipid species and the identification of lipid biomarkers.

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...

Long non-coding RNA reprogramming (lncRNA-ROR) regulates cell apoptosis and autophagy in chondrocytes.

PubMed

Yang, Zhongmeng; Tang, Yuxing; Lu, Huading; Shi, Bo; Ye, Yongheng; Xu, Guoyong; Zhao, Qing

2018-06-12

Long Non-Coding RNA Reprogramming (lncRNA-ROR) plays an important role in regulating various biologic processes, whereas the effect of lncRNA-ROR in osteoarthritis (OA) is little studied. This study aimed to explore lncRNA-ROR expression in articular cartilage and identify the functional mechanism of lncRNA-ROR in OA. OA cartilage tissues were obtained from 15 OA patients, and 6 normal cartilage tissues were set as controls. Chondrocytes were isolated from the collected cartilage tissues. lncRNA-ROR was knockdown in normal cells and overexpressed in OA cells. Cell viability was determined with Cell Counting Kit-8 assay, and apoptosis was measured using flow cytometric analysis. Moreover, proteins and mRNAs involved in this study were also measured using Western blotting and quantitative real-time PCR (qPCR). Level of lncRNA-ROR was decreased in OA compared with normal chondrocytes, and overexpression of lncRNA-ROR dramatically promoted cell viability of OA chondrocytes. In addition, knockdown lncRNA-ROR inhibited apoptosis and promoted autophagy of normal chondrocytes. Moreover, lncRNA-ROR inhibited the expression of p53 in both mRNA and protein levels. Furthermore, we revealed that lncRNA-ROR regulated apoptosis and autophagy of chondrocytes via HIF1α and p53. The results indicated that lncRNA-ROR played a critical role in the pathogenesis of OA, suggesting that lncRNA-ROR could serve as a new potential therapeutic target for OA. © 2018 Wiley Periodicals, Inc.

Isolation of total RNA from yeast cell cultures.

PubMed

Ares, Manuel

2012-10-01

This article describes two procedures for isolating total RNA from yeast cell cultures. The first allows the convenient isolation of total RNA from early log-phase cultures (vegetative cells). RNA isolated in this way is intact and sufficiently pure for use in microarray experiments, primer extension, and RNase protection mapping. With additional treatment to remove contaminating genomic DNA, the preparation is suitable for reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), cDNA library construction, high-throughput sequencing of RNA, or other manipulations. However, compared to vegetative cells, the isolation of RNA from cells late in meiosis (asci and ascospores) requires additional effort. This is because a tough cell wall composed of heavily cross-linked polysaccharides and proteins is built around the four spores during meiosis and ascospore development. Therefore, an alternative protocol is presented for extracting RNA from cells late in meiosis. This alternative may also be preferable for cells from stationary cultures or from yeast strains and other fungal species isolated from the environment.

Viral microRNA effects on persistent infection of human lymphoid cells by polyomavirus SV40

PubMed Central

McNees, Adrienne L.; Harrigal, Lindsay J.; Kelly, Aoife; Minard, Charles G.; Wong, Connie

2018-01-01

Background Polyomaviruses, including simian virus 40 (SV40), display evidence of lymphotropic properties. This study analyzed the nature of SV40–human lymphocyte interactions in established cell lines and in primary lymphocytes. The effects of viral microRNA and the structure of the viral regulatory region on SV40 persistence were examined. Results SV40 DNA was maintained in infected B cell and myeloid cell lines during cell growth for at least 28 days. Limiting dilution analysis showed that low amounts of SV40 DNA (~2 copies per cell) were retained over time. Infected B cells remained viable and able to proliferate. Genome copies of the SV40 microRNA-null mutant persisted at higher levels than the DNA of wild-type viruses. Complex viral regulatory regions produced modestly higher DNA levels than simple regulatory regions. Viral large T-antigen protein was detected at low frequency and at low levels in infected B cells. Following infection of primary lymphocytes, SV40 DNA was detected in CD19+ B cells and CD14+ monocytes, but not in CD3+ T cells. Rescue attempts using either lysates of SV40-infected B lymphocytes, coculture of live cells, or infectious center assays all showed that replication-competent SV40 could be recovered on rare occasions. SV40 infections altered the expression of several B cell surface markers, with more pronounced changes following infections with the microRNA-null mutant. Conclusion These findings indicate that SV40 can establish persistent infections in human B lymphocytes. The cells retain low copy numbers of viral DNA; the infections are nonproductive and noncytolytic but can occasionally produce infectious virus. SV40 microRNA negatively regulates the degree of viral effects on B cells. Significance Lymphocytes may serve as viral reservoirs and may function to disseminate polyomaviruses to different tissues in a host. To our knowledge, this report is the first extensive analysis of viral microRNA effects on SV40 infection of human

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell.

PubMed

Hu, Zhilan; Guo, Donglin; Yip, Shirley S M; Zhan, Dejin; Misaghi, Shahram; Joly, John C; Snedecor, Bradley R; Shen, Amy Y

2013-01-01

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers.

MiRAR-miRNA Activity Reporter for Living Cells.

PubMed

Turk, Matthew A; Chung, Christina Z; Manni, Emad; Zukowski, Stephanie A; Engineer, Anish; Badakhshi, Yasaman; Bi, Yumin; Heinemann, Ilka U

2018-06-19

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.

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

USDA-ARS?s Scientific Manuscript database

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 ...

Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species

PubMed Central

Di Giallonardo, Francesca; Schlub, Timothy E.; Shi, Mang

2017-01-01

ABSTRACT Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide

Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species.

PubMed

Di Giallonardo, Francesca; Schlub, Timothy E; Shi, Mang; Holmes, Edward C

2017-04-15

Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies and

Human snoRNA-93 is processed into a microRNA-like RNA that promotes breast cancer cell invasion.

PubMed

Patterson, Dillon G; Roberts, Justin T; King, Valeria M; Houserova, Dominika; Barnhill, Emmaline C; Crucello, Aline; Polska, Caroline J; Brantley, Lucas W; Kaufman, Garrett C; Nguyen, Michael; Santana, Megann W; Schiller, Ian A; Spicciani, Julius S; Zapata, Anastasia K; Miller, Molly M; Sherman, Timothy D; Ma, Ruixia; Zhao, Hongyou; Arora, Ritu; Coley, Alexander B; Zeidan, Melody M; Tan, Ming; Xi, Yaguang; Borchert, Glen M

2017-01-01

Genetic searches for tumor suppressors have recently linked small nucleolar RNA misregulations with tumorigenesis. In addition to their classically defined functions, several small nucleolar RNAs are now known to be processed into short microRNA-like fragments called small nucleolar RNA-derived RNAs. To determine if any small nucleolar RNA-derived RNAs contribute to breast malignancy, we recently performed a RNA-seq-based comparison of the small nucleolar RNA-derived RNAs of two breast cancer cell lines (MCF-7 and MDA-MB-231) and identified small nucleolar RNA-derived RNAs derived from 13 small nucleolar RNAs overexpressed in MDA-MB-231s. Importantly, we find that inhibiting the most differentially expressed of these small nucleolar RNA-derived RNAs (sdRNA-93) in MDA-MB-231 cells results primarily in a loss of invasiveness, whereas increased sdRNA-93 expression in either cell line conversely results in strikingly enhanced invasion. Excitingly, we recently determined sdRNA-93 expressions in small RNA-seq data corresponding to 116 patient tumors and normal breast controls, and while we find little sdRNA-93 expression in any of the controls and only sporadic expression in most subtypes, we find robust expression of sdRNA-93 in 92.8% of Luminal B Her2+tumors. Of note, our analyses also indicate that at least one of sdRNA-93's endogenous roles is to regulate the expression of Pipox, a sarcosine metabolism-related protein whose expression significantly correlates with distinct molecular subtypes of breast cancer. We find sdRNA-93 can regulate the Pipox 3'UTR via standard reporter assays and that manipulating endogenous sdRNA-93 levels inversely correlates with altered Pipox expression. In summary, our results strongly indicate that sdRNA-93 expression actively contributes to the malignant phenotype of breast cancer through participating in microRNA-like regulation.

RNA-programmed genome editing in human cells

PubMed Central

Jinek, Martin; East, Alexandra; Cheng, Aaron; Lin, Steven; Ma, Enbo; Doudna, Jennifer

2013-01-01

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells. DOI: http://dx.doi.org/10.7554/eLife.00471.001 PMID:23386978

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.

tRNA modification profiles of the fast-proliferating cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Chao; Niu, Leilei; Song, Wei

Despite the recent progress in RNA modification study, a comprehensive modification profile is still lacking for mammalian cells. Using a quantitative HPLC/MS/MS assay, we present here a study where RNA modifications are examined in term of the major RNA species. With paired slow- and fast-proliferating cell lines, distinct RNA modification profiles are first revealed for diverse RNA species. Compared to mRNAs, increased ribose and nucleobase modifications are shown for the highly-structured tRNAs and rRNAs, lending support to their contribution to the formation of high-order structures. This study also reveals a dynamic tRNA modification profile in the fast-proliferating cells. In additionmore » to cultured cells, this unique tRNA profile has been further confirmed with endometrial cancers and their adjacent normal tissues. Taken together, the results indicate that tRNA is a actively regulated RNA species in the fast-proliferating cancer cells, and suggest that they may play a more active role in biological process than expected. -- Highlights: •RNA modifications were first examined in term of the major RNA species. •A dynamic tRNA modifications was characterized for the fast-proliferating cells. •The unique tRNA profile was confirmed with endometrial cancers and their adjacent normal tissues. •tRNA was predicted as an actively regulated RNA species in the fast-proliferating cancer cells.« less

MicroRNA-196b promotes cell proliferation and suppress cell differentiation in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Donglin, E-mail: caodlgz@sina.com; Hu, Liangshan; Lei, Da

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 partiallymore » 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.« less

Host-Derived CD70 Suppresses Murine Graft-versus-Host Disease by Limiting Donor T Cell Expansion and Effector Function.

PubMed

Leigh, Nicholas D; O'Neill, Rachel E; Du, Wei; Chen, Chuan; Qiu, Jingxin; Ashwell, Jonathan D; McCarthy, Philip L; Chen, George L; Cao, Xuefang

2017-07-01

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for hematologic and immunologic diseases. However, graft-versus-host disease (GVHD) may develop when donor-derived T cells recognize and damage genetically distinct normal host tissues. In addition to TCR signaling, costimulatory pathways are involved in T cell activation. CD27 is a TNFR family member expressed on T cells, and its ligand, CD70, is expressed on APCs. The CD27/CD70 costimulatory pathway was shown to be critical for T cell function and survival in viral infection models. However, the role of this pathway in allo-HCT is previously unknown. In this study, we have examined its contribution in GVHD pathogenesis. Surprisingly, Ab blockade of CD70 after allo-HCT significantly increases GVHD. Interestingly, whereas donor T cell- or bone marrow-derived CD70 plays no role in GVHD, host-derived CD70 inhibits GVHD as CD70 -/- hosts show significantly increased GVHD. This is evidenced by reduced survival, more severe weight loss, and increased histopathologic damage compared with wild-type hosts. In addition, CD70 -/- hosts have higher levels of proinflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-17. Moreover, accumulation of donor CD4 + and CD8 + effector T cells is increased in CD70 -/- versus wild-type hosts. Mechanistic analyses suggest that CD70 expressed by host hematopoietic cells is involved in the control of alloreactive T cell apoptosis and expansion. Together, our findings demonstrate that host CD70 serves as a unique negative regulator of allogeneic T cell response by contributing to donor T cell apoptosis and inhibiting expansion of donor effector T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

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

2016-09-13

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.

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

Comparison of Dengue Virus Type 2-Specific Small RNAs from RNA Interference-Competent and –Incompetent Mosquito Cells

PubMed Central

Scott, Jaclyn C.; Brackney, Doug E.; Campbell, Corey L.; Bondu-Hawkins, Virginie; Hjelle, Brian; Ebel, Greg D.; Olson, Ken E.; Blair, Carol D.

2010-01-01

The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts. PMID:21049014

Balance of power in host-virus arms races.

PubMed

Kok, Kin-Hang; Jin, Dong-Yan

2013-07-17

The sensing of viral RNA by the host innate immune system is mediated by RIG-I and its partner PACT. In this issue of Cell Host & Microbe, Luthra et al. (2013) show that the Ebola virus VP35 protein counteracts the action of PACT at the cost of compromising its own function in viral replication. Copyright © 2013 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vieira da Silva, Claudio; Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sao Paulo, Rua Botucatu, 862, 6o andar, 04023-062 Sao Paulo, SP; Alves da Silva, Erika

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 RHmore » 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.« less

Predicting bacteriophage proteins located in host cell with feature selection technique.

PubMed

Ding, Hui; Liang, Zhi-Yong; Guo, Feng-Biao; Huang, Jian; Chen, Wei; Lin, Hao

2016-04-01

A bacteriophage is a virus that can infect a bacterium. The fate of an infected bacterium is determined by the bacteriophage proteins located in the host cell. Thus, reliably identifying bacteriophage proteins located in the host cell is extremely important to understand their functions and discover potential anti-bacterial drugs. Thus, in this paper, a computational method was developed to recognize bacteriophage proteins located in host cells based only on their amino acid sequences. The analysis of variance (ANOVA) combined with incremental feature selection (IFS) was proposed to optimize the feature set. Using a jackknife cross-validation, our method can discriminate between bacteriophage proteins located in a host cell and the bacteriophage proteins not located in a host cell with a maximum overall accuracy of 84.2%, and can further classify bacteriophage proteins located in host cell cytoplasm and in host cell membranes with a maximum overall accuracy of 92.4%. To enhance the value of the practical applications of the method, we built a web server called PHPred (〈http://lin.uestc.edu.cn/server/PHPred〉). We believe that the PHPred will become a powerful tool to study bacteriophage proteins located in host cells and to guide related drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

PubMed

Kamachi, Yasuharu; Omasa, Takeshi

2018-04-01

Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™.

PubMed

Luck, Ashley N; Slatko, Barton E; Foster, Jeremy M

2017-01-01

Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues.

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

Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

PubMed

Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J; Jopling, Catherine L

2015-04-01

MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

PubMed Central

Collis, P S; O'Donnell, B J; Barton, D J; Rogers, J A; Flanegan, J B

1992-01-01

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis. Images PMID

Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity.

PubMed

Singh, Ajeet; Permar, Vipin; Jain, R K; Goswami, Suneha; Kumar, Ranjeet Ranjan; Canto, Tomas; Palukaitis, Peter; Praveen, Shelly

2017-08-01

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H 2 O 2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H 2 O 2 -responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSs S189R ) and the other in a non-Trp/GH3 motif (NSs L172R ). Tobacco rattle virus (TRV) expressing NSs S189R enhanced the PCD response, but not TRV-NSs L172R , while RNA silencing suppression activity was lost in TRV-NSs L172R , but not in TRV-NSs S189R . Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

Respiratory syncytial virus modifies microRNAs regulating host genes that affect virus replication

PubMed Central

Bakre, Abhijeet; Mitchell, Patricia; Coleman, Jonathan K.; Jones, Les P.; Saavedra, Geraldine; Teng, Michael; Tompkins, S. Mark

2012-01-01

Respiratory syncytial virus (RSV) causes substantial morbidity and life-threatening lower respiratory tract disease in infants, young children and the elderly. Understanding the host response to RSV infection is critical for developing disease-intervention approaches. The role of microRNAs (miRNAs) in post-transcriptional regulation of host genes responding to RSV infection is not well understood. In this study, it was shown that RSV infection of a human alveolar epithelial cell line (A549) induced five miRNAs (let-7f, miR-24, miR-337-3p, miR-26b and miR-520a-5p) and repressed two miRNAs (miR-198 and miR-595), and showed that RSV G protein triggered let-7f expression. Luciferase–untranslated region reporters and miRNA mimics and inhibitors validated the predicted targets, which included cell-cycle genes (CCND1, DYRK2 and ELF4), a chemokine gene (CCL7) and the suppressor of cytokine signalling 3 gene (SOCS3). Modulating let-7 family miRNA levels with miRNA mimics and inhibitors affected RSV replication, indicating that RSV modulates host miRNA expression to affect the outcome of the antiviral host response, and this was mediated in part through RSV G protein expression. PMID:22894925

Modulation of host cell function by Legionella pneumophila type IV effectors.

PubMed

Hubber, Andree; Roy, Craig R

2010-01-01

Macrophages and protozoa ingest bacteria by phagocytosis and destroy these microbes using a conserved pathway that mediates fusion of the phagosome with lysosomes. To survive within phagocytic host cells, bacterial pathogens have evolved a variety of strategies to avoid fusion with lysosomes. A virulence strategy used by the intracellular pathogen Legionella pneumophila is to manipulate host cellular processes using bacterial proteins that are delivered into the cytosolic compartment of the host cell by a specialized secretion system called Dot/Icm. The proteins delivered by the Dot/Icm system target host factors that play evolutionarily conserved roles in controlling membrane transport in eukaryotic cells, which enables L. pneumophila to create an endoplasmic reticulum-like vacuole that supports intracellular replication in both protozoan and mammalian host cells. This review focuses on intracellular trafficking of L. pneumophila and describes how bacterial proteins contribute to modulation of host processes required for survival within host cells.

Kupffer cell complement receptor clearance function and host defense.

PubMed

Loegering, D J

1986-01-01

Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.

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.

The RNA-binding protein repertoire of embryonic stem cells.

PubMed

Kwon, S Chul; Yi, Hyerim; Eichelbaum, Katrin; Föhr, Sophia; Fischer, Bernd; You, Kwon Tae; Castello, Alfredo; Krijgsveld, Jeroen; Hentze, Matthias W; Kim, V Narry

2013-09-01

RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells.

Analysis of Host-Takeover During SPO1 Infection of Bacillus subtilis.

PubMed

Stewart, Charles R

2018-01-01

When Bacillus subtilis is infected by bacteriophage SPO1, the phage directs the remodeling of the host cell, converting it into a factory for phage reproduction. Much synthesis of host DNA, RNA, and protein is shut off, and cell division is prevented. Here I describe the protocols by which we have demonstrated those processes, and identified the roles played by specific SPO1 gene products in causing those processes.

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; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

2017-10-17

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.

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.

MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

PubMed Central

Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

2016-01-01

MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.

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 cellularmore » 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.« less

Interferon-α Silencing by Small Interference RNA Increases Adenovirus Transduction and Transgene Expression in Huh7 Cells.

PubMed

Sobrevilla-Navarro, Ana Alondra; Sandoval-Rodríguez, Ana; García-Bañuelos, Jesús Javier; Armendariz-Borunda, Juan; Salazar-Montes, Adriana María

2018-04-01

Adenoviruses are the most common vectors used in clinical trials of gene therapy. In 2017, 21.2% of clinical trials used rAds as vectors. Systemic administration of rAds results in high tropism in the liver. Interferon types α and β are the major antiviral cytokines which orchestrate the host's immune response against rAd, limiting therapeutic gene expression and preventing subsequent vector administration. siRNA is small double-strand RNAs that temporally inhibit the expression of a specific gene. The aim is to evaluate the effect of IFN-α blocking by a specific siRNA on Ad-GFP transduction and on transgene expression in Huh7 cells in culture. Huh7 cells were cultured in DMEM and transfected with 70 nM of siRNA-IFN-α. Six hours later, the cells were exposed to 1 × 10 9  vp/ml of rAd-GFP for 24 h. Expression of IFN-α, TNF-α and the PKR gene was determined by RT-qPCR. Percentage of transduction was analyzed by flow cytometry and by qPCR. GFP expression was determined by western blot. 70 nM of siRNA-IFN-α inhibited 96% of IFN-α and 65% of TNF-α gene expression compared to an irrelevant siRNA. Percentage of transduction and transgene expression increased in these cells compared to an irrelevant siRNA. Inhibition of IFN-α expression by siRNA-IFN-α enabled a higher level of transduction and transgene expression GFP, highlighting the role of IFN-α in the elimination of adenovirus in transduced cells and thus suggesting that its inhibition could be an important strategy for gene therapy in clinical trials using adenovirus as a vector directed to liver diseases.

Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania.

PubMed

Padmanabhan, P K; Samant, M; Cloutier, S; Simard, M J; Papadopoulou, B

2012-12-01

Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved.

Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania

PubMed Central

Padmanabhan, P K; Samant, M; Cloutier, S; Simard, M J; Papadopoulou, B

2012-01-01

Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved. PMID:22767185

Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry.

PubMed

Taylor, Ethan Will; Ruzicka, Jan A; Premadasa, Lakmini; Zhao, Lijun

2016-01-01

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3' end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation.

Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

PubMed Central

Taylor, Ethan Will; Ruzicka, Jan A.; Premadasa, Lakmini; Zhao, Lijun

2016-01-01

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

Allocation of Interferon Gamma mRNA Positive Cells in Caecum Hallmarks a Protective Trait Against Histomonosis.

PubMed

Kidane, Fana Alem; Mitra, Taniya; Wernsdorf, Patricia; Hess, Michael; Liebhart, Dieter

2018-01-01

Histomonosis is a parasitic disease of gallinaceous birds characterized by necrotic lesions in cacum and liver that usually turns fatal in turkeys while it is less severe in chickens. Vaccination using in vitro attenuated Histomonas meleagridis has been experimentally shown to confer protection against histomonosis. The protective mechanisms that underpin the vaccine-induced immune response are not resolved so far. Therefore, the actual study aimed to evaluate the location and quantitative distribution patterns of signature cytokines of type 1 [interferon gamma (IFN-γ)] or type 2 [interleukin (IL)-13] immune responses in vaccinated or infected hosts. An intergroup and interspecies difference in the spatial and temporal distribution patterns of cytokine mRNA positive cells was evident. Quantification of cells showed a significantly decreased percentage of IFN-γ mRNA positive cells at 4 days post-inoculation (DPI) in caeca of turkeys inoculated exclusively with the attenuated or the virulent inocula, compared to control birds. The decrement was followed by a surge of cells expressing mRNA for IFN-γ or IL-13, reaching a peak of increment at 10 DPI. By contrast, turkeys challenged following vaccination showed a slight increment of cecal IFN-γ mRNA positive cells at 4 DPI after which positive cell counts became comparable to control birds. The increase in infected birds was accompanied by an extensive distribution of positively stained cells up to the muscularis layer of cecal tissue whereas the vaccine group maintained an intact mucosal structure. In chickens, the level of changes of positive cells was generally lower compared to turkeys. However, control chickens were found with a higher percentage of IFN-γ mRNA positive cells in cecum compared to their turkey counterparts indicating a higher resistance to histomonosis, similar to the observation in immunized turkeys. In chickens, it could be shown that the changes of cytokine-positive cells were related to

Influenza A Virus Infection of Human Respiratory Cells Induces Primary MicroRNA Expression*

PubMed Central

Buggele, William A.; Johnson, Karen E.; Horvath, Curt M.

2012-01-01

The cellular response to virus infection is initiated by recognition of the invading pathogen and subsequent changes in gene expression mediated by both transcriptional and translational mechanisms. In addition to well established means of regulating antiviral gene expression, it has been demonstrated that RNA interference (RNAi) can play an important role in antiviral responses. Virus-derived small interfering RNA (siRNA) is a primary antiviral response exploited by plants and invertebrate animals, and host-encoded microRNA (miRNA) species have been clearly implicated in the regulation of innate and adaptive immune responses in mammals and other vertebrates. Examination of miRNA abundance in human lung cell lines revealed endogenous miRNAs, including miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275, to specifically accumulate in response to infection with two influenza A virus strains, A/Udorn/72 and A/WSN/33. Known antiviral response pathways, including Toll-like receptor, RIG-I-like receptor, and direct interferon or cytokine stimulation did not alter the abundance of the tested miRNAs to the extent of influenza A virus infection, which initiates primary miRNA transcription via a secondary response pathway. Gene expression profiling identified 26 cellular mRNAs targeted by these miRNAs, including IRAK1, MAPK3, and other components of innate immune signaling systems. PMID:22822053

Genetic reprogramming of host cells by bacterial pathogens.

PubMed

Tran Van Nhieu, Guy; Arbibe, Laurence

2009-10-29

During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

RNA-seq in kinetoplastids: A powerful tool for the understanding of the biology and host-pathogen interactions.

PubMed

Patino, Luz Helena; Ramírez, Juan David

2017-04-01

The kinetoplastids include a large number of parasites responsible for serious diseases in humans and animals (Leishmania and Trypanosoma brucei) considered endemic in several regions of the world. These parasites are characterized by digenetic life cycles that undergo morphological and genetic changes that allow them to adapt to different microenvironments on their vertebrates and invertebrates hosts. Recent advances in ´omics´ technology, specifically transcriptomics have allowed to reveal aspects associated with such molecular changes. So far, different techniques have been used to evaluate the gene expression profile during the various stages of the life cycle of these parasites and during the host-parasite interactions. However, some of them have serious drawbacks that limit the precise study and full understanding of their transcriptomes. Therefore, recently has been implemented the latest technology (RNA-seq), which overcomes the drawbacks of traditional methods. In this review, studies that so far have used RNA-seq are presented and allowed to expand our knowledge regarding the biology of these parasites and their interactions with their hosts. Copyright © 2017 Elsevier B.V. All rights reserved.

Host cell processes that influence the intracellular survival of Legionella pneumophila.

PubMed

Shin, Sunny; Roy, Craig R

2008-06-01

Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.

Long non-coding RNA PlncRNA-1 promotes cell proliferation and hepatic metastasis in colorectal cancer.

PubMed

Jia, Gui-Qing; Zhang, Ming-Ming; Wang, Kang; Zhao, Gao-Ping; Pang, Ming-Hui; Chen, Zhe-Yu

2018-05-08

Emerging evidence has identified that long non-coding RNAs (lncRNAs) may play an important role in the pathogenesis of many cancer types, including colorectal cancer (CRC). However, the role of PlncRNA-1 in CRC remains unclear. The aim of our present study was to investigate the potential functions of PlncRNA-1 in CRC and to identify the underlying mechanisms of action. We demonstrated that up-regulated PlncRNA-1 in CRC tissues and cells promoted cell proliferation by accelerating cell cycle process and inhibiting cell apoptosis in vitro, enhanced tumor growth and matastasis in vivo and was associated with cell migration and invasion, EMT process of CRC cells. In addition, PlncRNA-1 was a target of miR-204 and enhanced the expression of an endogenous miR-204 target, MMP9 in CRC cells. Furthermore, we found that PlncRNA-1 activates Wnt/β-catenin pathway through the miR-204 in CRC cells. These results suggest that the PlncRNA-1/miR-204/ Wnt/β-catenin regulatory network may shed light on tumorigenesis in CRC. © 2018 Wiley Periodicals, Inc.

How pathogens use linear motifs to perturb host cell networks.

PubMed

Via, Allegra; Uyar, Bora; Brun, Christine; Zanzoni, Andreas

2015-01-01

Molecular mimicry is one of the powerful stratagems that pathogens employ to colonise their hosts and take advantage of host cell functions to guarantee their replication and dissemination. In particular, several viruses have evolved the ability to interact with host cell components through protein short linear motifs (SLiMs) that mimic host SLiMs, thus facilitating their internalisation and the manipulation of a wide range of cellular networks. Here we present convincing evidence from the literature that motif mimicry also represents an effective, widespread hijacking strategy in prokaryotic and eukaryotic parasites. Further insights into host motif mimicry would be of great help in the elucidation of the molecular mechanisms behind host cell invasion and the development of anti-infective therapeutic strategies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takeda, N.; Kuhn, R.J.; Yang, C.F.

1986-10-01

An in vitro poliovirus RNA-synthesizing system derived from a crude membrance fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing mucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T/sub 1/-resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized /sup 32/P-RNA. Incubation ofmore » preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. The data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.« less

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

PubMed

Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

PubMed

Khamina, Kseniya; Lercher, Alexander; Caldera, Michael; Schliehe, Christopher; Vilagos, Bojan; Sahin, Mehmet; Kosack, Lindsay; Bhattacharya, Anannya; Májek, Peter; Stukalov, Alexey; Sacco, Roberto; James, Leo C; Pinschewer, Daniel D; Bennett, Keiryn L; Menche, Jörg; Bergthaler, Andreas

2017-12-01

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

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. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

PubMed

Juergens, Hannes; Varela, Javier A; Gorter de Vries, Arthur R; Perli, Thomas; Gast, Veronica J M; Gyurchev, Nikola Y; Rajkumar, Arun S; Mans, Robert; Pronk, Jack T; Morrissey, John P; Daran, Jean-Marc G

2018-05-01

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

Select Host Restriction Factors Are Associated with HIV Persistence During Antiretroviral Therapy

PubMed Central

ABDEL-MOHSEN, Mohamed; WANG, Charlene; STRAIN, Matthew C.; LADA, Steven M.; DENG, Xutao; COCKERHAM, Leslie R.; PILCHER, Christopher D.; HECHT, Frederick M.; LIEGLER, Teri; RICHMAN, Douglas D.; DEEKS, Steven G.; PILLAI, Satish K.

2015-01-01

Objective The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir. Design We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals. Methods We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-LTR circle HIV-1 DNA, and immunophenotypes of CD4+ T cells in 72 HIV-1-infected subjects on suppressive ART (23 subjects initiated ART <1 year post-infection, and 49 subjects initiated ART >1 year post-infection). Correlations were analyzed using non-parametric tests. Results The enhanced expression of a few select host restriction factors, p21, schlafen 11, and PAF1, was strongly associated with reduced CD4+ T cell-associated HIV RNA during ART (p<0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4+ T cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in subjects who initiated ART during early versus chronic infection, and may contribute to the reduced reservoir size observed in these individuals. Conclusions Intrinsic immune responses modulate HIV persistence during suppressive ART, and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo. PMID:25602681

Pulse labeling of RNA of mammalian cells.

PubMed

Rovera, G; Berman, S; Baserga, R

1970-04-01

When cells from a hypotetraploid strain of Ehrlich ascites tumor are exposed to uridine-(3)H either in vivo or in vitro, the amount of radioactivity incorporated into RNA reaches a maximum within ten minutes, after which any further incorporation stops. (3)H-uridine triphosphate disappears from the acid soluble pool within 30 minutes and the findings indicate that the RNA of these cells can be pulse labeled without the use of any antibiotic or the need of a "chase." The stability of the pulse labeled RNA in the presence of pentobarbital (an inhibitor of RNA synthesis) indicates the virtual absence of RNA breakdown. However, actinomycin D, at a dosage of 250 mug/mouse in vivo and 10 mug/ml in vitro produces breakdown of labeled RNA, thus confirming earlier observations that the drug is not a suitable tool for RNA kinetics determinations. The pulse-labeled RNA leaves the nucleus slowly and some radioactive RNA is still present in the nuclear fraction after 24 hours. Radioactivity begins to appear in cytoplasmic ribosomal RNA after 20 minutes and continues to increase up to six hours.

Herpesvirus papio 2 encodes a virion host shutoff function.

PubMed

Bigger, John E; Martin, David W

2002-12-05

Infection of baboons with herpesvirus papio 2 (HVP-2) produces a disease that is similar to human infection with herpes simplex viruses (HSV). Molecular characterization of HVP-2 has demonstrated that the virion contains a factor which rapidly shuts off host cell protein synthesis after infection. Reduction of host cell protein synthesis occurs in parallel with the degradation of mRNA species. A homolog of the HSV virion host shutoff (vhs) gene was identified by Southern and DNA sequence analysis. The sequence of the HVP-2 vhs gene homolog had greater than 70% identity with the vhs genes of HSV 1 and 2. Disruption of the HVP-2 vhs open reading frame diminished the ability of the virus to shut off protein synthesis and degrade cellular mRNA, indicating that this gene was responsible for the vhs activity. The HVP-2 model system provides the opportunity to study the biological role of vhs in the context of a natural primate host. Further development of this system will provide a platform for proof-of-concept studies that will test the efficacy of vaccines that utilize vhs-deficient viruses.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

PubMed

O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine

PubMed Central

O’Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O’Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-01-01

AIM To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward’s clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells.

PubMed

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.

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

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Herpesviruses and Their Host Cells: A Successful Liaison.

PubMed

Adler, Barbara; Sattler, Christine; Adler, Heiko

2017-03-01

During a long history of coevolution, herpesviruses have reached a fine-tuned balance with their hosts, allowing them to successfully persist and spread to new hosts without causing too much damage. Only under certain circumstances, as in neonates or immunocompromised individuals, they may cause serious diseases. The delicate balance between herpesviruses and their hosts results from interactions of a great variety of viral and cellular factors which together shape the tropism for a particular host, tissue, or cell. Understanding these interactions will provide insight into the viral life cycle and cell biology in general. Moreover, it will also facilitate comprehension of herpesvirus pathogenesis, enabling the development of new strategies to combat herpesviruses in cases where they cause disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ikaros-Notch axis in host hematopoietic cells regulates experimental graft-versus-host disease

PubMed Central

Toubai, Tomomi; Sun, Yaping; Tawara, Isao; Friedman, Ann; Liu, Chen; Evers, Rebecca; Nieves, Evelyn; Malter, Chelsea; Chockley, Peter; Maillard, Ivan; Winandy, Susan

2011-01-01

Host hematopoietically derived APCs play a vital role in the initiation of GVH responses. However, the APC autonomous molecular mechanisms that are critical for the induction of GVHD are not known. We report here that the Ikaros-Notch axis in host hematopoietically derived APCs regulates the severity of acute GVHD across multiple clinically relevant murine models of experimental bone marrow transplantation. In the present study, Ikaros deficiency (Ik−/−) limited to host hematopoietically derived APCs enhanced donor T-cell expansion and intensified acute GVHD, as determined by survival and other GVHD-specific parameters. The Ik−/− conventional CD8+ and CD8−CD11c+ dendritic cells (DCs), the most potent APCs, showed no increase in the expression of activation markers or in response to TLR stimulation compared with wild-type controls. However, Ik−/− DCs demonstrated an enhanced stimulation of allogeneic T cells. Deficiency of Ikaros in the conventional CD8+ and CD8−CD11c+ DCs was associated with an increase in Notch signaling, the blockade of which mitigated the enhanced in vitro and in vivo allostimulatory capacity. Therefore, the Ikaros-Notch axis is a novel pathway that modulates DC biology in general, and targeting this pathway in host hematopoietically derived APCs may reduce GVHD. PMID:21471527

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

PubMed

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

2016-09-07

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

PubMed

Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

2014-03-01

Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression. © 2013 John Wiley & Sons Ltd.

High-throughput full-length single-cell mRNA-seq of rare cells.

PubMed

Ooi, Chin Chun; Mantalas, Gary L; Koh, Winston; Neff, Norma F; Fuchigami, Teruaki; Wong, Dawson J; Wilson, Robert J; Park, Seung-Min; Gambhir, Sanjiv S; Quake, Stephen R; Wang, Shan X

2017-01-01

Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial

Generation of Human Induced Pluripotent Stem Cells Using RNA-Based Sendai Virus System and Pluripotency Validation of the Resulting Cell Population.

PubMed

Chichagova, Valeria; Sanchez-Vera, Irene; Armstrong, Lyle; Steel, David; Lako, Majlinda

2016-01-01

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.

RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions

PubMed Central

Bolinger, Cheryl; Sharma, Amit; Singh, Deepali; Yu, Lianbo; Boris-Lawrie, Kathleen

2010-01-01

Retroviruses rely on host RNA-binding proteins to modulate various steps in their replication. Previously several animal retroviruses were determined to mediate Dhx9/RNA helicase A (RHA) interaction with a 5′ terminal post-transcriptional control element (PCE) for efficient translation. Herein PCE reporter assays determined HTLV-1 and HIV-1 RU5 confer orientation-dependent PCE activity. The effect of Dhx9/RHA down-regulation and rescue with siRNA-resistant RHA on expression of HIV-1NL4–3 provirus determined that RHA is necessary for efficient HIV-1 RNA translation and requires ATPase-dependent helicase function. Quantitative analysis determined HIV-1 RNA steady-state and cytoplasmic accumulation were not reduced; rather the translational activity of viral RNA was reduced. Western blotting determined that RHA-deficient virions assemble with Lys-tRNA synthetase, exhibit processed reverse transcriptase and contain similar level of viral RNA, but they are poorly infectious on primary lymphocytes and HeLa cells. The results demonstrate RHA is an important host factor within the virus-producer cell and within the viral particle. The identification of RHA-dependent PCE activity in cellular junD RNA and in six of seven genera of Retroviridae suggests conservation of this translational control mechanism among vertebrates, and convergent evolution of Retroviridae to utilize this host mechanism. PMID:20007598

Assessment of myoblast circular RNA dynamics and its correlation with miRNA during myogenic differentiation.

PubMed

Zhang, Pengpeng; Xu, Haixia; Li, Rui; Wu, Wei; Chao, Zhe; Li, Cencen; Xia, Wei; Wang, Lei; Yang, Jinzeng; Xu, Yongjie

2018-06-01

Myoblast differentiation is a highly complex process that is regulated by proteins as well as by non-coding RNAs. Circular RNAs have been identified as an emerging new class of non-coding RNA in the modulation of skeletal muscle development, whereas their expression profiles and functional regulation in myoblast differentiation remain unknown. In the present study, we performed deep RNA-sequencing of C2C12 myoblasts during cell differentiation and uncovered 37,751 unique circular RNAs derived from 6943 hosting genes. The ensuing qRT-PCR and RNA fluorescence in situ hybridization verification were carried out to confirm the RNA-sequencing results. An unbiased analysis demonstrated dynamic circular RNA expression changes in the process of myoblast differentiation, and the circular RNA abundances were independent from their cognate linear RNAs. Gene ontology analysis showed that many down-regulated circular RNAs were exclusive to cell division and the cell cycle, whereas up-regulated circular RNAs were related to the cell development process. Furthermore, interaction networks of circular RNA-microRNA were constructed. Several microRNAs well-known for myoblast regulation, such as miR-133, miR-24 and miR-23a, were in this network. In summary, this study showed that circular RNA expression dynamics changed during myoblast differentiation. Circular RNAs play a role in regulating the myoblast cell cycle and development by acting as microRNA binding sites to facilitate their regulation of gene expression during myoblast differentiation. These findings open a new avenue for future investigation of this emerging RNA class in skeletal muscle growth and development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

PubMed

Blomström, Anne-Lie; Gu, Quan; Barry, Gerald; Wilkie, Gavin; Skelton, Jessica K; Baird, Margaret; McFarlane, Melanie; Schnettler, Esther; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

2015-04-19

Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

CLL Cells Respond to B-Cell Receptor Stimulation with a MicroRNA/mRNA Signature Associated with MYC Activation and Cell Cycle Progression

PubMed Central

Pede, Valerie; Rombout, Ans; Vermeire, Jolien; Naessens, Evelien; Mestdagh, Pieter; Robberecht, Nore; Vanderstraeten, Hanne; Van Roy, Nadine; Vandesompele, Jo; Speleman, Frank; Philippé, Jan; Verhasselt, Bruno

2013-01-01

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation. PMID:23560086

Circular RNA HIPK3 promotes gallbladder cancer cell growth by sponging microRNA-124.

PubMed

Kai, Ding; Yannian, Liao; Yitian, Chen; Dinghao, Gong; Xin, Zhao; Wu, Ji

2018-06-18

Recent studies have implied that circHIPK3, an abundant circular RNA (circRNA), participates in tumorigenesis and cancer progression. Its expression and potential functions in human gallbladder cancer were examined in this study. We show that circHIPK3 is upregulated in human gallbladder cancer cells. But its level is low in gallbladder epithelial cells. circHIPK3 silencing by targeted siRNA potently inhibited survival and proliferation of established and primary human gallbladder cancer cells, while inducing cell apoptosis. Conversely, ectopic over-expression of circHIPK3 can further promote cancer cell proliferation. In gallbladder cancer cells, circHIPK3 sponged the tumor-suppressive microRNA-124 (miR-124) to sequester and inhibit its activity, thereby leading to increased expression of miR-124 targets, including ROCK1 (rho-associated protein kinase 1) and CDK6 (rho-associated protein kinase). Ectopic over-expression of miR-124 b y a lentiviral vector mimicked and abolished actions by circHIPK3 siRNA in gallbladder cancer cells. At last, we show that circHIPK3 is upregulated in human gallbladder cancer tissues, which is correlated with miR-124 downregulation and ROCK1-CDK6 upregulation. Together, we conclude that circHIPK3 promotes gallbladder cancer cell growth possibly by sponging miR-124. The over-expressed circHIPK3 could be a novel therapeutic target and diagnosis marker of human gallbladder cancer. Copyright © 2018. Published by Elsevier Inc.

Dynamics of Viral and Host Immune Cell MicroRNA Expression during Acute Infectious Mononucleosis

PubMed Central

Kaul, Vandana; Weinberg, Kenneth I.; Boyd, Scott D.; Bernstein, Daniel; Esquivel, Carlos O.; Martinez, Olivia M.; Krams, Sheri M.

2018-01-01

Epstein–Barr virus (EBV) is the etiological agent of acute infectious mononucleosis (IM). Since acute IM is a self-resolving disease with most patients regaining health in 1–3 weeks there have been few studies examining molecular signatures in early acute stages of the disease. MicroRNAs (miRNAs) have been shown, however, to influence immune cell function and consequently the generation of antibody responses in IM. In this study, we performed a comprehensive analysis of differentially expressed miRNAs in early stage uncomplicated acute IM. miRNAs were profiled from patient peripheral blood obtained at the time of IM diagnosis and at subsequent time points, and pathway analysis performed to identify important immune and cell signaling pathways. We identified 215 differentially regulated miRNAs at the most acute stage of infection when the patients initially sought medical help. The number of differentially expressed miRNAs decreased to 148 and 68 at 1 and 2 months post-primary infection, with no significantly changed miRNAs identified at 7 months post-infection. Interferon signaling, T and B cell signaling and antigen presentation were the top pathways influenced by the miRNAs associated with IM. Thus, a dynamic and regulated expression profile of miRNA accompanies the early acute immune response, and resolution of infection, in IM. PMID:29379474

Nuclear RNA quantification in protoplast cell-cycle phases.

PubMed

Bergounioux, C; Perennes, C; Brown, S C; Gadal, P

1988-01-01

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.

Influenza A Virus Dysregulates Host Histone Deacetylase 1 That Inhibits Viral Infection in Lung Epithelial Cells.

PubMed

Nagesh, Prashanth Thevkar; Husain, Matloob

2016-05-01

Viruses dysregulate the host factors that inhibit virus infection. Here, we demonstrate that human enzyme, histone deacetylase 1 (HDAC1) is a new class of host factor that inhibits influenza A virus (IAV) infection, and IAV dysregulates HDAC1 to efficiently replicate in epithelial cells. A time-dependent decrease in HDAC1 polypeptide level was observed in IAV-infected cells, reducing to <50% by 24 h of infection. A further depletion (97%) of HDAC1 expression by RNA interference increased the IAV growth kinetics, increasing it by >3-fold by 24 h and by >6-fold by 48 h of infection. Conversely, overexpression of HDAC1 decreased the IAV infection by >2-fold. Likewise, a time-dependent decrease in HDAC1 activity, albeit with slightly different kinetics to HDAC1 polypeptide reduction, was observed in infected cells. Nevertheless, a further inhibition of deacetylase activity increased IAV infection in a dose-dependent manner. HDAC1 is an important host deacetylase and, in addition to its role as a transcription repressor, HDAC1 has been lately described as a coactivator of type I interferon response. Consistent with this property, we found that inhibition of deacetylase activity either decreased or abolished the phosphorylation of signal transducer and activator of transcription I (STAT1) and expression of interferon-stimulated genes, IFITM3, ISG15, and viperin in IAV-infected cells. Furthermore, the knockdown of HDAC1 expression in infected cells decreased viperin expression by 58% and, conversely, the overexpression of HDAC1 increased it by 55%, indicating that HDAC1 is a component of IAV-induced host type I interferon antiviral response. Influenza A virus (IAV) continues to significantly impact global public health by causing regular seasonal epidemics, occasional pandemics, and zoonotic outbreaks. IAV is among the successful human viral pathogens that has evolved various strategies to evade host defenses, prevent the development of a universal vaccine, and acquire

Influenza A Virus Dysregulates Host Histone Deacetylase 1 That Inhibits Viral Infection in Lung Epithelial Cells

PubMed Central

Nagesh, Prashanth Thevkar

2016-01-01

ABSTRACT Viruses dysregulate the host factors that inhibit virus infection. Here, we demonstrate that human enzyme, histone deacetylase 1 (HDAC1) is a new class of host factor that inhibits influenza A virus (IAV) infection, and IAV dysregulates HDAC1 to efficiently replicate in epithelial cells. A time-dependent decrease in HDAC1 polypeptide level was observed in IAV-infected cells, reducing to <50% by 24 h of infection. A further depletion (97%) of HDAC1 expression by RNA interference increased the IAV growth kinetics, increasing it by >3-fold by 24 h and by >6-fold by 48 h of infection. Conversely, overexpression of HDAC1 decreased the IAV infection by >2-fold. Likewise, a time-dependent decrease in HDAC1 activity, albeit with slightly different kinetics to HDAC1 polypeptide reduction, was observed in infected cells. Nevertheless, a further inhibition of deacetylase activity increased IAV infection in a dose-dependent manner. HDAC1 is an important host deacetylase and, in addition to its role as a transcription repressor, HDAC1 has been lately described as a coactivator of type I interferon response. Consistent with this property, we found that inhibition of deacetylase activity either decreased or abolished the phosphorylation of signal transducer and activator of transcription I (STAT1) and expression of interferon-stimulated genes, IFITM3, ISG15, and viperin in IAV-infected cells. Furthermore, the knockdown of HDAC1 expression in infected cells decreased viperin expression by 58% and, conversely, the overexpression of HDAC1 increased it by 55%, indicating that HDAC1 is a component of IAV-induced host type I interferon antiviral response. IMPORTANCE Influenza A virus (IAV) continues to significantly impact global public health by causing regular seasonal epidemics, occasional pandemics, and zoonotic outbreaks. IAV is among the successful human viral pathogens that has evolved various strategies to evade host defenses, prevent the development of a universal

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma.

PubMed

Suzuki, Shigeki; Hoshino, Hiroaki; Yoshida, Kazuma; Nakanishi, Jun; Tsuchiya-Hirata, Shizu; Kobuke, Seiji; Haruyama, Naoto; Nishimura, Fusanori; Shiba, Hideki

2018-01-15

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC

Ectromelia virus accumulates less double-stranded RNA compared to vaccinia virus in BS-C-1 cells.

PubMed

Frey, Tiffany R; Lehmann, Michael H; Ryan, Colton M; Pizzorno, Marie C; Sutter, Gerd; Hersperger, Adam R

2017-09-01

Most orthopoxviruses, including vaccinia virus (VACV), contain genes in the E3L and K3L families. The protein products of these genes have been shown to combat PKR, a host defense pathway. Interestingly, ectromelia virus (ECTV) contains an E3L ortholog but does not possess an intact K3L gene. Here, we gained insight into how ECTV can still efficiently evade PKR despite lacking K3L. Relative to VACV, we found that ECTV-infected BS-C-1 cells accumulated considerably less double-stranded (ds) RNA, which was due to lower mRNA levels and less transcriptional read-through of some genes by ECTV. The abundance of dsRNA in VACV-infected cells, detected using a monoclonal antibody, was able to activate the RNase L pathway at late time points post-infection. Historically, the study of transcription by orthopoxviruses has largely focused on VACV as a model. Our data suggest that there could be more to learn by studying other members of this genus. Copyright © 2017 Elsevier Inc. All rights reserved.

Replication of tobacco mosaic virus RNA.

PubMed Central

Buck, K W

1999-01-01

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the

Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xiaofei; College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036; Deng, Ping

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 themore » 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. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.« less

MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii

PubMed Central

Sahni, Abha; Narra, Hema P.; Patel, Jignesh; Sahni, Sanjeev K.

2017-01-01

MicroRNAs (miRNAs) mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R. rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01) and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01). Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs). Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections. PMID:28698491

Fierce Competition between Toxoplasma and Chlamydia for Host Cell Structures in Dually Infected Cells

PubMed Central

Romano, Julia D.; de Beaumont, Catherine; Carrasco, Jose A.; Ehrenman, Karen; Bavoil, Patrik M.

2013-01-01

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients. PMID:23243063

Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

PubMed

Romano, Julia D; de Beaumont, Catherine; Carrasco, Jose A; Ehrenman, Karen; Bavoil, Patrik M; Coppens, Isabelle

2013-02-01

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

Secretion of RNA-Containing Extracellular Vesicles by the Porcine Whipworm, Trichuris suis.

PubMed

Hansen, Eline Palm; Kringel, Helene; Williams, Andrew R; Nejsum, Peter

2015-06-01

Trichuris suis is a common parasitic helminth of pigs. As with many other parasites, T. suis ensures its own survival by evading host immune responses, but little is known about how this is achieved. MicroRNAs (miRNA) have been shown to be involved in various immunological processes by post-transcriptional regulation of specific genes, and the potential of using these molecules as biomarkers of disease is currently being examined. It has recently been shown that parasites may secrete extracellular structures such as exosomes and microvesicles, containing proteins and miRNA. The fusion of these structures with host cells has been demonstrated, and a role of exosome-derived miRNA in host gene regulation has been suggested. In the present study, we show that exosome- and microvesicular-like structures are secreted by T. suis L1 larvae and also demonstrate the presence of miRNA-sized RNA inside these structures. A potential role of these molecules in host-parasite interactions is suggested. In addition, an electron-dense layer covering the surface of the larvae was observed, which may play a function in host immune evasion.

Modeling RNA interference in mammalian cells

PubMed Central

2011-01-01

Background RNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers. Results We tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects. Conclusions Our model has a simple mathematical form, amenable to analytical investigations and a small set of

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.

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

An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

PubMed

Shiao, Yih-Horng; Lupascu, Sorin T; Gu, Yuhan D; Kasprzak, Wojciech; Hwang, Christopher J; Fields, Janet R; Leighty, Robert M; Quiñones, Octavio; Shapiro, Bruce A; Alvord, W Gregory; Anderson, Lucy M

2009-10-19

Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

miRNA-regulated cancer stem cells: understanding the property and the role of miRNA in carcinogenesis.

PubMed

Chakraborty, Chiranjib; Chin, Kok-Yong; Das, Srijit

2016-10-01

Over the last few years, microRNAs (miRNA)-controlled cancer stem cells have drawn enormous attention. Cancer stem cells are a small population of tumor cells that possess the stem cell property of self-renewal. Recent data shows that miRNA regulates this small population of stem cells. In the present review, we explained different characteristics of cancer stem cells as well as miRNA regulation of self-renewal and differentiation in cancer stem cells. We also described the migration and tumor formation. Finally, we described the different miRNAs that regulate various types of cancer stem cells, such as prostate cancer stem cells, head and neck cancer stem cells, breast cancer stem cells, colorectal cancer stem cells, lung cancer stem cells, gastric cancer stem cells, pancreatic cancer stem cells, etc. Extensive research is needed in order to employ miRNA-based therapeutics to control cancer stem cell population in various cancers in the future.

Gene expression distribution deconvolution in single-cell RNA sequencing.

PubMed

Wang, Jingshu; Huang, Mo; Torre, Eduardo; Dueck, Hannah; Shaffer, Sydney; Murray, John; Raj, Arjun; Li, Mingyao; Zhang, Nancy R

2018-06-26

Single-cell RNA sequencing (scRNA-seq) enables the quantification of each gene's expression distribution across cells, thus allowing the assessment of the dispersion, nonzero fraction, and other aspects of its distribution beyond the mean. These statistical characterizations of the gene expression distribution are critical for understanding expression variation and for selecting marker genes for population heterogeneity. However, scRNA-seq data are noisy, with each cell typically sequenced at low coverage, thus making it difficult to infer properties of the gene expression distribution from raw counts. Based on a reexamination of nine public datasets, we propose a simple technical noise model for scRNA-seq data with unique molecular identifiers (UMI). We develop deconvolution of single-cell expression distribution (DESCEND), a method that deconvolves the true cross-cell gene expression distribution from observed scRNA-seq counts, leading to improved estimates of properties of the distribution such as dispersion and nonzero fraction. DESCEND can adjust for cell-level covariates such as cell size, cell cycle, and batch effects. DESCEND's noise model and estimation accuracy are further evaluated through comparisons to RNA FISH data, through data splitting and simulations and through its effectiveness in removing known batch effects. We demonstrate how DESCEND can clarify and improve downstream analyses such as finding differentially expressed genes, identifying cell types, and selecting differentiation markers. Copyright © 2018 the Author(s). Published by PNAS.

The role of arginine and arginine-metabolizing enzymes during Giardia – host cell interactions in vitro

PubMed Central

2013-01-01

Background Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections. Results RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline. Conclusions Giardia affects the host’s arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy. PMID:24228819

Stress and death of cnidarian host cells play a role in cnidarian bleaching.

PubMed

Paxton, Camille W; Davy, Simon K; Weis, Virginia M

2013-08-01

Coral bleaching occurs when there is a breakdown of the symbiosis between cnidarian hosts and resident Symbiodinium spp. Multiple mechanisms for the bleaching process have been identified, including apoptosis and autophagy, and most previous work has focused on the Symbiodinium cell as the initiator of the bleaching cascade. In this work we show that it is possible for host cells to initiate apoptosis that can contribute to death of the Symbiodinium cell. First we found that colchicine, which results in apoptosis in other animals, causes cell death in the model anemone Aiptasia sp. but not in cultured Symbiodinium CCMP-830 cells or in cells freshly isolated from host Aiptasia (at least within the time frame of our study). In contrast, when symbiotic Aiptasia were incubated in colchicine, cell death in the resident Symbiodinium cells was observed, suggesting a host effect on symbiont mortality. Using live-cell confocal imaging of macerated symbiotic host cell isolates, we identified a pattern where the initiation of host cell death was followed by mortality of the resident Symbiodinium cells. This same pattern was observed in symbiotic host cells that were subjected to temperature stress. This research suggests that mortality of symbionts during temperature-induced bleaching can be initiated in part by host cell apoptosis.

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

Probing Xist RNA Structure in Cells Using Targeted Structure-Seq

PubMed Central

Rutenberg-Schoenberg, Michael; Simon, Matthew D.

2015-01-01

The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5’-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function. PMID:26646615

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

PubMed Central

de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

2015-01-01

ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although

A Systematic Approach to Time-series Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture.

PubMed

Hsu, Han-Hsiu; Araki, Michihiro; Mochizuki, Masao; Hori, Yoshimi; Murata, Masahiro; Kahar, Prihardi; Yoshida, Takanobu; Hasunuma, Tomohisa; Kondo, Akihiko

2017-03-02

Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture.

Bacterial effectors target the plant cell nucleus to subvert host transcription.

PubMed

Canonne, Joanne; Rivas, Susana

2012-02-01

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) 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.

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

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1.

PubMed

Kuchipudi, Suresh V; Dunham, Stephen P; Nelli, Rahul; White, Gavin A; Coward, Vivien J; Slomka, Marek J; Brown, Ian H; Chang, Kin Chow

2012-01-01

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and 'classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host-pathogen relationships for highly pathogenic and zoonotic avian influenza.

RNA synthetic mechanisms employed by diverse families of RNA viruses.

PubMed

McDonald, Sarah M

2013-01-01

RNA viruses are ubiquitous in nature, infecting every known organism on the planet. These viruses can also be notorious human pathogens with significant medical and economic burdens. Central to the lifecycle of an RNA virus is the synthesis of new RNA molecules, a process that is mediated by specialized virally encoded enzymes called RNA-dependent RNA polymerases (RdRps). RdRps directly catalyze phosphodiester bond formation between nucleoside triphosphates in an RNA-templated manner. These enzymes are strikingly conserved in their structural and functional features, even among diverse RNA viruses belonging to different families. During host cell infection, the activities of viral RdRps are often regulated by viral cofactor proteins. Cofactors can modulate the type and timing of RNA synthesis by directly engaging the RdRp and/or by indirectly affecting its capacity to recognize template RNA. High-resolution structures of RdRps as apoenzymes, bound to RNA templates, in the midst of catalysis, and/or interacting with regulatory cofactor proteins, have dramatically increased our understanding of viral RNA synthetic mechanisms. Combined with elegant biochemical studies, such structures are providing a scientific platform for the rational design of antiviral agents aimed at preventing and treating RNA virus-induced diseases. Copyright © 2013 John Wiley & Sons, Ltd.

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

Trafficking arms: oomycete effectors enter host plant cells.

PubMed

Birch, Paul R J; Rehmany, Anne P; Pritchard, Leighton; Kamoun, Sophien; Beynon, Jim L

2006-01-01

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.

The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Lei; Zhang, Qing; Yang, Yu

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 formore » 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.« less

Exploring the single-cell RNA-seq analysis landscape with the scRNA-tools database.

PubMed

Zappia, Luke; Phipson, Belinda; Oshlack, Alicia

2018-06-25

As single-cell RNA-sequencing (scRNA-seq) datasets have become more widespread the number of tools designed to analyse these data has dramatically increased. Navigating the vast sea of tools now available is becoming increasingly challenging for researchers. In order to better facilitate selection of appropriate analysis tools we have created the scRNA-tools database (www.scRNA-tools.org) to catalogue and curate analysis tools as they become available. Our database collects a range of information on each scRNA-seq analysis tool and categorises them according to the analysis tasks they perform. Exploration of this database gives insights into the areas of rapid development of analysis methods for scRNA-seq data. We see that many tools perform tasks specific to scRNA-seq analysis, particularly clustering and ordering of cells. We also find that the scRNA-seq community embraces an open-source and open-science approach, with most tools available under open-source licenses and preprints being extensively used as a means to describe methods. The scRNA-tools database provides a valuable resource for researchers embarking on scRNA-seq analysis and records the growth of the field over time.

A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing

DOE PAGES

Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.; ...

2016-12-21

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich formore » pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.« less