N
-methyladenosine (m
A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m
A within discreet ...regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m
A methyltransferases decreases RSV replication and gene expression whereas knockdown of m
A demethylases has the opposite effect. The G gene transcript contains the most m
A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m
A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m
A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m
A methylation upregulates RSV replication and pathogenesis and identify viral m
A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.
Traditionally, whooping cough or pertussis caused by the obligate human pathogen Bordetella pertussis (Bp) is described as an acute disease with severe symptoms. However, many individuals who ...contract pertussis are either asymptomatic or show very mild symptoms and yet can serve as carriers and sources of bacterial transmission. Biofilms are an important survival mechanism for bacteria in human infections and disease. However, bacterial determinants that drive biofilm formation in humans are ill-defined. In the current study, we show that Bp infection of well-differentiated primary human bronchial epithelial cells leads to formation of bacterial aggregates, clusters, and highly structured biofilms which are colocalized with cilia. These findings mimic observations from pathological analyses of tissues from pertussis patients. Distinct arrangements (mono-, bi-, and tri-partite) of the polysaccharide Bps, extracellular DNA, and bacterial cells were visualized, suggesting complex heterogeneity in bacteria-matrix interactions. Analyses of mutant biofilms revealed positive roles in matrix production, cell cluster formation, and biofilm maturity for three critical Bp virulence factors: Bps, filamentous hemagglutinin, and adenylate cyclase toxin. Adherence assays identified Bps as a new Bp adhesin for primary human airway cells. Taken together, our results demonstrate the multi-factorial nature of the biofilm extracellular matrix and biofilm development process under conditions mimicking the human respiratory tract and highlight the importance of model systems resembling the natural host environment to investigate pathogenesis and potential therapeutic strategies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate ...at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR >> KE, and of doxorubicin was JR ∼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines.
Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon ...(IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N6-methyladenosine (m6A) and RSV grown in m6A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m6A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m6A methylation enhances innate immune responses which in turn promote adaptive immunity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
N
6
-methyladenosine (m
6
A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m
6
A ...within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m
6
A methyltransferases decreases RSV replication and gene expression whereas knockdown of m
6
A demethylases has the opposite effect. The G gene transcript contains the most m
6
A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m
6
A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m
6
A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m
6
A methylation upregulates RSV replication and pathogenesis and identify viral m
6
A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.
5-methylcytosine (m
C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m
C ...methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m
C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m
C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m
C methylation of several abundant noncoding RNAs (ncRNAs). However, m
C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m
C methylation controls antiviral innate immunity through modulating the m
C methylome of ncRNAs and their expression.
Abstract
Ependymoma account for 6-12% of pediatric brain tumors and have a mean age of diagnosis of 4 years, a 5-year survival rate ranging from 39%-64%, and a progression free survival rate of ...23-45%. General use of chemotherapeutics remains highly ineffective for this disease therefore standard of care consists of surgery followed by radiation with total gross resection (TGR) having the best outcome (60-89% 5-year overall survival for TGR vs. 21-46% 5-year overall survival for subtotal resection (STR)) 6. Patients having had STR have an extremely high incidence of disease reoccurrence (43% to 72%) requiring additional surgeries and radiation with a median 2-year overall survival after relapse ranging from 20% to 49% 7. These statistics highlight the need for better adjuvant therapies. In an effort to identify new targets for treating ependymoma we have developed a mouse model that relies on the intracranial implantation of mouse supratentorial (forebrain) embryonic neuronal stem cells (STeNSC) overexpressing the tyrosine kinase receptor ephrin B2 (EphB2). Tumors develop within 4-8 months post-implantation, require receptor activation and are consistently found to be histologically, ultrastructurally and molecularly similar to the human disease.
Given the requirement for EphB2 activation in tumor development, it became necessary to identify the EphB2 initiated signaling pathways involved in this process. Among the many pathways initiated by EphB2 activation are those of the small GTPases of the Ras family (H-Ras, K-Ras, N-Ras) that regulate cellular processes including cell proliferation and transformation. Receptor activation is a multistep process involving receptor autophosphorylation of two critical tyrosines positions 604 and 610 within the juxtamembrane (Y604, Y610), receptor/ligand complex dimerization, release of juxtamembrane mediated kinase domain inhibition and finally kinase domain phosphorylation and activation. While EphB2-mediated activation of Ras involves the kinase domain of the receptor, Ras inhibition (RasGTP conversion to RasGDP) is achieved by activating the Ras GTPase activating protein p120RasGAP via its juxtamembrane. We took advantage of a previously identified EphB2 juxtamembrane mutation that inhibits p120RasGAP activation by converting the tyrosines at positions 604 and 610 to glutamic acids (Y604/610E) thus rendering Ras constitutively active. Ligand-mediated activation of this mutant resulted in an absence of p120RasGAP activation and an increase in Ras signaling (elevated levels of phosphorylated C-Raf, MEK1/2 and Erk1/2) in vitro as predicted. More importantly, overexpression of the EphB2(Y604/610E) protein in our model system showed no change in tumor penetrance (100% penetrance); however, there was a dramatic shortening of tumor latency from 207 days with EphB2 to 40 days with the Y604/610E mutation, supporting the role for Ras signaling in EphB2 mediated transformation.
Citation Format: Robert A. Johnson, Phylip Chen, Robert Lyons, Samuel Priddy. Importance of Ras signaling in EphB2-mediated ependymoma development. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2015-3285
Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon ...(IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N.sup.6 -methyladenosine (m.sup.6 A) and RSV grown in m.sup.6 A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m.sup.6 A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m.sup.6 A methylation enhances innate immune responses which in turn promote adaptive immunity.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. Currently, there are no FDA-approved vaccines to combat this ...virus. The large (L) polymerase protein of RSV replicates the viral genome and transcribes viral mRNAs. The L protein is organized as a core ring-like domain containing the RNA-dependent RNA polymerase and an appendage of globular domains containing an mRNA capping region and a cap methyltransferase region, which are linked by a flexible hinge region. Here, we found that the flexible hinge region of RSV L protein is tolerant to amino acid deletion or insertion. Recombinant RSVs carrying a single or double deletion or a single alanine insertion were genetically stable, highly attenuated in immortalized cells, had defects in replication and spread, and had a delay in innate immune cytokine responses in primary, well-differentiated, human bronchial epithelial (HBE) cultures. The replication of these recombinant viruses was highly attenuated in the upper and lower respiratory tracts of cotton rats. Importantly, these recombinant viruses elicited high levels of neutralizing antibody and provided complete protection against RSV replication. Taken together, amino acid deletions or insertions in the hinge region of the L protein can serve as a novel approach to rationally design genetically stable, highly attenuated, and immunogenic live virus vaccine candidates for RSV.
Despite tremendous efforts, there are no FDA-approved vaccines for human respiratory syncytial virus (RSV). A live attenuated RSV vaccine is one of the most promising vaccine strategies for RSV. However, it has been a challenge to identify an RSV vaccine strain that has an optimal balance between attenuation and immunogenicity. In this study, we generated a panel of recombinant RSVs carrying a single and double deletion or a single alanine insertion in the large (L) polymerase protein that are genetically stable, sufficiently attenuated, and grow to high titer in cultured cells, while retaining high immunogenicity. Thus, these recombinant viruses may be promising vaccine candidates for RSV.
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