Bryan R G Williams

Publications of Bryan R G Williams

• Dynamiting viruses with MxA.

  Authors: Anthony J Sadler, Bryan R G Williams

  Immunity. 10/2011; 35(4):491-3.

  The crystal structure of the interferon-induced member of the dynamin family MxA presented by Gao et al. (2011) in this issue of Immunity reveals the molecule's higher-order structure, therebyThe crystal structure of the interferon-induced member of the dynamin family MxA presented by Gao et al. (2011) in this issue of Immunity reveals the molecule's higher-order structure, thereby providing insight into the protein's antiviral action as a molecular machine.

• Analysis of microRNA turnover in mammalian cells following Dicer1 ablation.

  Authors: Michael P Gantier, Claire E McCoy, Irina Rusinova, Damien Saulep, Die Wang, Dakang Xu, Aaron T Irving, Mark A Behlke, Paul J Hertzog, Fabienne Mackay, Bryan R G Williams

  Nucleic acids research. 03/2011; 39(13):5692-703.

  Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to theAlthough microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function.

• Genetic modulation of TLR8 response following bacterial phagocytosis.

  Authors: Michael P Gantier, Aaron T Irving, Maria Kaparakis-Liaskos, Dakang Xu, Vanessa A Evans, Paul U Cameron, James A Bourne, Richard L Ferrero, Matthias John, Mark A Behlke, Bryan R G Williams

  Human mutation. 09/2010; 31(9):1069-79.

  Human Toll-like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 inHuman Toll-like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 in the detection of intracellular pathogenic bacteria, characterization of such a role for TLR8 is currently lacking. A recent genetic study has correlated the presence of a TLR8 single nucleotide polymorphism (SNP) (rs3764880:A>G; p.Met1Val) with the development of active tuberculosis, suggesting a role for TLR8 in the detection of phagosomal bacteria. Here we provide the first direct evidence that TLR8 sensing is activated in human monocytic cells following Helicobacter pylori phagocytosis. In addition, we show that rs3764880 fine tunes translation of the two TLR8 main isoforms, without affecting protein function. Although we show that TLR8 variant 2 (TLR8v2) is the prevalent form of TLR8 contributing to TLR8 function, we also uncover a role for the TLR8 long isoform (TLR8v1) in the positive regulation of TLR8 function in CD16(+)CD14(+) differentiated monocytes. Thus, TLR8 sensing can be activated following bacterial phagocytosis, and rs3764880 may play a role in the modulation of TLR8-dependent microbicidal response of infected macrophages.

• Viral apoptosis is induced by IRF-3-mediated activation of Bax.

  Authors: Saurabh Chattopadhyay, Joao T Marques, Michifumi Yamashita, Kristi L Peters, Kevin Smith, Avanti Desai, Bryan R G Williams, Ganes C Sen

  The EMBO journal. 04/2010; 29(10):1762-73.

  Upon infection with many RNA viruses, the cytoplasmic retinoic acid inducible gene-I (RIG-I) pathway activates the latent transcription factor IRF-3, causing its nuclear translocation and theUpon infection with many RNA viruses, the cytoplasmic retinoic acid inducible gene-I (RIG-I) pathway activates the latent transcription factor IRF-3, causing its nuclear translocation and the induction of many antiviral genes, including those encoding interferons. Here, we report a novel and distinct activity of IRF-3, in virus-infected cells, that induces apoptosis. Using genetically defective mouse and human cell lines, we demonstrated that, although both pathways required the presence of RIG-I, IPS1, TRAF3 and TBK1, only the apoptotic pathway required the presence of TRAF2 and TRAF6 in addition. More importantly, transcriptionally inactive IRF-3 mutants, such as the one missing its DNA-binding domain, could efficiently mediate apoptosis. Apoptosis was triggered by the direct interaction of IRF-3, through a newly identified BH3 domain, with the pro-apoptotic protein Bax, their co-translocation to the mitochondria and the resulting activation of the mitochondrial apoptotic pathway. Thus, IRF-3 is a dual-action cytoplasmic protein that, upon activation, translocates to the nucleus or to the mitochondrion and triggers two complementary antiviral responses of the infected cell.

• X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

  Authors: Kate L Jones, Michael Roche, Michael P Gantier, Nasim A Begum, Tasuku Honjo, Salvatore Caradonna, Bryan R G Williams, Johnson Mak

  The Journal of biological chemistry. 04/2010; 285(24):18603-14.

  It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinctIt has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations.

• Rational design of immunostimulatory siRNAs.

  Authors: Michael P Gantier, Stephen Tong, Mark A Behlke, Aaron T Irving, Martha Lappas, Ulrika W Nilsson, Eicke Latz, Nigel A J McMillan, Bryan R G Williams

  Molecular therapy : the journal of the American Society of Gene Therapy. 04/2010; 18(4):785-95.

  Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on theirShort-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies.

• Monitoring innate immune recruitment by siRNAs in mammalian cells.

  Authors: Michael P Gantier, Bryan R G Williams

  Methods in molecular biology (Clifton, N.J.). 01/2010; 623:21-33.

  The use of small interfering RNAs (siRNAs) in human therapy may be hindered by the recruitment of nonspecific effects such as the activation of innate immune responses. Recently, several innateThe use of small interfering RNAs (siRNAs) in human therapy may be hindered by the recruitment of nonspecific effects such as the activation of innate immune responses. Recently, several innate immune receptors have been implicated in the detection of siRNAs. This chapter provides a brief overview of the current knowledge of siRNA-induced innate immunity, as well as protocols for the rapid identification of siRNAs with innate immune stimulatory activity.

• Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication.

  Authors: Heather E Lynch, Caroline A Ray, Katrina L Oie, Justin J Pollara, Ian T D Petty, Anthony J Sadler, Bryan R G Williams, David J Pickup

  Virology. 08/2009;

  Modified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-kappaB transcription factors in cells of severalModified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-kappaB transcription factors in cells of several types. In human embryonic kidney (HEK 293T) cells, the MVA-induced depletion of IkappaBalpha required to activate NF-kappaB is inhibited by UV-inactivation of the virus, and begins before viral DNA replication. In HEK 293T, CHO, or RK13 cells, expression of the cowpox virus CP77 early gene, or the vaccinia virus K1L early gene suppresses MVA-induced IkappaBalpha depletion. In mouse embryonic fibroblasts (MEFs), MVA induction of IkappaBalpha depletion is dependent on the expression of mouse or human double-stranded RNA-activated protein kinase (PKR). These results demonstrate that events during the early phase of MVA replication can induce PKR-mediated processes contributing both to the activation of NF-kappaB signaling, and to processes that may restrict viral replication. This property may contribute to the efficacy of this vaccine virus.

• Promyelocytic leukemia zinc finger protein regulates interferon-mediated innate immunity.

  Authors: Dakang Xu, Michelle Holko, Anthony J Sadler, Bernadette Scott, Shigeki Higashiyama, Windy Berkofsky-Fessler, Melanie J McConnell, Pier Paolo Pandolfi, Jonathan D Licht, Bryan R G Williams

  Immunity. 07/2009; 30(6):802-16.

  Interferons (IFNs) direct innate and acquired immune responses and, accordingly, are used therapeutically to treat a number of diseases, yet the diverse effects they elicit are not fully understood.Interferons (IFNs) direct innate and acquired immune responses and, accordingly, are used therapeutically to treat a number of diseases, yet the diverse effects they elicit are not fully understood. Here, we identified the promyelocytic leukemia zinc finger (PLZF) protein as a previously unrecognized component of the IFN response. IFN stimulated an association of PLZF with promyelocytic leukemia protein (PML) and histone deacetylase 1 (HDAC1) to induce a decisive subset of IFN-stimulated genes (ISGs). Consequently, PLZF-deficient mice had a specific ISG expression defect and as a result were more susceptible to viral infection. This susceptibility correlated with a marked decrease in the expression of the key antiviral mediators and an impaired IFN-mediated induction of natural killer cell function. These results provide new insights into the regulatory mechanisms of IFN signaling and the induction of innate antiviral immunity.

• An antiviral response directed by PKR phosphorylation of the RNA helicase A.

  Authors: Anthony J Sadler, Olivier Latchoumanin, David Hawkes, Johnson Mak, Bryan R G Williams

  PLoS pathogens. 03/2009; 5(2):e1000311.

  The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the alpha subunitThe double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2alpha) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)-mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

• Regulation of CRABP-II expression by MycN in Wilms tumor.

  Authors: Anu Gupta, Patricia Kessler, Jawhar Rawwas, Bryan R G Williams

  Experimental cell research. 01/2009; 314(20):3663-8.

  Cellular retinoic acid binding protein II (CRABP-II) is overexpressed in a wide variety of cancers. Previously we have shown that CRABP-II expression levels are also elevated in neuroblastoma andCellular retinoic acid binding protein II (CRABP-II) is overexpressed in a wide variety of cancers. Previously we have shown that CRABP-II expression levels are also elevated in neuroblastoma and Wilms tumors. To elucidate the molecular mechanisms underlying the abnormal expression of CRABP-II in Wilms tumor, we studied the expression of MycN and CRABP-II in these tumor samples. Our data revealed that CRABP-II is overexpressed in Wilms tumor compared to normal adjacent non-neoplastic tissue and its levels are even higher in late stage tumors. Its expression correlates with MycN expression in tumors. The tumors that do not express MycN have no CRABP-II expression. The expression of CRABP-II is also regulated by methylation and its promoter is unmethylated in tumors. Knockdown of MycN by small interfering RNA leads to downregulation of CRABP-II. Thus our results suggest that both MycN and DNA methylation are responsible for CRABP-II expression in pediatric tumors and demethylation of CRABP-II may be an early event in tumor development.

• Differential Expression in Clear Cell Renal Cell Carcinoma Identified by Gene Expression Profiling.

  Authors: Brian R Lane, Jianbo Li, Ming Zhou, Denise Babineau, Pieter Faber, Andrew C Novick, Bryan R G Williams

  The Journal of urology. 01/2009;

  PURPOSE: Gene expression profiling has been shown to provide prognostic information on patients with solitary sporadic renal cell carcinoma. To our knowledge there is no reliable way to differentiatePURPOSE: Gene expression profiling has been shown to provide prognostic information on patients with solitary sporadic renal cell carcinoma. To our knowledge there is no reliable way to differentiate synchronous renal metastasis from bilateral primary tumors in patients with bilateral renal cell carcinoma. We present data using a custom kidney cancer cDNA array that can predict the outcome in patients with unilateral and bilateral renal cell carcinoma. MATERIALS AND METHODS: Fresh frozen tissue from 38 clear cell renal cell carcinomas was analyzed using a cancer cDNA array containing 3,966 genes relevant to cancer or kidney development. Median followup was 5.3 years. Cancer recurred in 12 patients (43%) and 11 (39%) had died by the last followup. RESULTS: Using a training data set of 8 tumors a 44 gene expression profile distinguishing aggressive and indolent clear cell renal cell carcinoma was identified. Of 29 single clear cell renal cell carcinomas 16 and 13 were predicted to be indolent and aggressive, respectively, by the gene expression profile. Recurrence-free survival at 5 years was 68% and 42% in these 2 groups, respectively (p = 0.032). Clear cell renal cell carcinoma classified as indolent or aggressive according to SSIGN (stage, size, grade and necrosis) score showed a 5-year recurrence-free survival rate of 78% and 42%, respectively (p = 0.021). On Cox proportional hazards analysis the gene expression profile was not an independent predictor of recurrence-free survival after accounting for SSIGN score. Gene expression profile classification correlated with cancer specific survival at 5 years in 4 of 4 patients with metachronous clear cell renal cell carcinoma but in only 2 of 4 with bilateral synchronous clear cell renal cell carcinoma. CONCLUSIONS: Gene expression profiling using a kidney cancer relevant cDNA array can differentiate between aggressive and indolent clear cell renal cell carcinomas. Gene expression profile results may be most useful for unilateral clear cell renal cell carcinoma when results are discordant with predictions of tumor behavior based on standard clinicopathological features. In addition, gene expression profiling can provide prognostic information that may help characterize tumors of unknown clinical stage, such as bilateral metachronous clear cell renal cell carcinoma.

• The p59 oligoadenylate synthetase-like protein possesses antiviral activity that requires the C-terminal ubiquitin-like domain.

  Authors: Joao Marques, Jangawar Anwar, Signe Eskildsen-Larsen, Dominique Rebouillat, Soren R Paludan, Ganes Sen, Bryan R G Williams, Rune Hartmann

  The Journal of general virology. 12/2008; 89(Pt 11):2767-72.

  Viral infection of mammalian cells prompts the innate immune system to initiate an antiviral response. The recognition of the virus triggers several antiviral signalling pathways, which among othersViral infection of mammalian cells prompts the innate immune system to initiate an antiviral response. The recognition of the virus triggers several antiviral signalling pathways, which among others include the family of 2'-5' oligoadenylate synthetase (OAS) proteins. The p59 protein encoded by the OAS-like (OASL) gene is an atypical member of the OAS family in the sense that it lacks the characteristic 2'-5' oligoadenylate synthetase activity. We decided to investigate the putative antiviral activity of p59 by ectopically expressing this protein in Vero cells and then infecting these cells with virus. We demonstrate that OASL has an antiviral effect against the single-stranded RNA virus picornavirus, encephalomyocarditis virus, but not against a large DNA virus, herpes simplex virus 1. Importantly, this antiviral activity was lost in a truncated version of p59 lacking the ubiquitin-like C-terminal domain of p59. Taken together our results indicate that p59 is indeed an antiviral protein that works through a novel mechanism distinct from other OAS proteins.

• The role of PACT in mediating gene induction, PKR activation, and apoptosis in response to diverse stimuli.

  Authors: Joao T Marques, Christine L White, Gregory A Peters, Bryan R G Williams, Ganes C Sen

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 09/2008; 28(8):469-76.

  PACT, the protein activator of the double-stranded (ds)RNA-activated protein kinase (PKR) has been shown to strongly interact with and activate PKR in cultured cells and in vitro. To further analyzePACT, the protein activator of the double-stranded (ds)RNA-activated protein kinase (PKR) has been shown to strongly interact with and activate PKR in cultured cells and in vitro. To further analyze the functions of PACT we have recently generated PACT knockout (KO) mice and described several developmental defects that are absent in PKR KO mice. Importantly, PACT has been previously suggested to be involved in different signaling pathways that include endoplasmic reticulum stress, serum deprivation, growth factor withdrawal, viral infection, and cytokine responses. In this study, we have analyzed the contribution of PACT to these pathways using cells derived from wildtype (WT) and PACT KO mice. Notably, we have been unable to detect any significant differences in the responses to stress stimuli comparing WT and PACT KO cells, although we have been able to validate the specific interaction between PACT and PKR. Taken together, our results reinforce the importance of genetic loss of function analysis to infer protein function.

• Protein kinase R-dependent regulation of interleukin-10 in response to double-stranded RNA.

  Authors: Arindam Chakrabarti, Anthony J Sadler, Niladri Kar, Howard A. Young, Robert H Silverman, Bryan R G Williams

  The Journal of biological chemistry. 08/2008;

  The double-stranded RNA-activated protein kinase R (PKR) is an important component of antiviral defense. PKR participates in different signaling pathways in response to various stimuli to regulateThe double-stranded RNA-activated protein kinase R (PKR) is an important component of antiviral defense. PKR participates in different signaling pathways in response to various stimuli to regulate translation via phosphorylation of the eukaryotic initiation factor 2alpha, and transcription via activating NF-kappaB and IRF-1, to induce pro-inflammatory cytokines. Here we show PKR regulates interleukin-10 induction in response to double-stranded RNA, bacterial lipopolysaccaride, and Sendai virus infection. Using chemical inhibitors, dominant negative constructs, and genetic knockouts, we demonstrate that the PKR-mediated interleukin-10 induction engages JNK and NF-kappaB. Together, our data demonstrate the role of PKR in regulating an anti-inflammatory cytokine. The findings have significance in antiviral, as well as broader innate immune responses.

• Interferon-inducible antiviral effectors.

  Authors: Anthony J Sadler, Bryan R G Williams

  Nature reviews. Immunology. 08/2008; 8(7):559-68.

  Since the discovery of interferons (IFNs), considerable progress has been made in describing the nature of the cytokines themselves, the signalling components that direct the cell response and theirSince the discovery of interferons (IFNs), considerable progress has been made in describing the nature of the cytokines themselves, the signalling components that direct the cell response and their antiviral activities. Gene targeting studies have distinguished four main effector pathways of the IFN-mediated antiviral response: the Mx GTPase pathway, the 2',5'-oligoadenylate-synthetase-directed ribonuclease L pathway, the protein kinase R pathway and the ISG15 ubiquitin-like pathway. As discussed in this Review, these effector pathways individually block viral transcription, degrade viral RNA, inhibit translation and modify protein function to control all steps of viral replication. Ongoing research continues to expose additional activities for these effector proteins and has revealed unanticipated functions of the antiviral response.

• Determinants of cytokine induction by small interfering RNA in human peripheral blood mononuclear cells.

  Authors: Maryam Zamanian-Daryoush, Joao T Marques, Michael P Gantier, Mark A Behlke, Matthias John, Patricia Rayman, James Finke, Bryan R G Williams

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 05/2008; 28(4):221-33.

  Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing.Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing. Chemically synthesized siRNAs, including traditional 19-mers with 2-nt 3' overhangs, longer duplexes with blunt or 3' overhangs, and asymmetric duplexes with a blunt end and a 2-nt 3' overhang, can evoke strong dose-dependent interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) release in human peripheral blood mononuclear cells (PBMCs). This response is independent of retinoic acid-inducible gene I but may involve endosomal toll-like receptors (TLRs). The immunostimulatory effect of the siRNAs is directly related to either or both of the strands of the duplex in a sequence-dependent manner. However, although some single-stranded RNAs and siRNAs potently evoked both IFN-alpha and TNF-alpha induction, these responses were not always coupled. In accordance with this, specific chemical modifications differentially altered cytokine production, suggesting recruitment of different TLRs in a sequence-dependent manner.

• TLR7 is involved in sequence-specific sensing of single-stranded RNAs in human macrophages.

  Authors: Michael P Gantier, Stephen Tong, Mark A Behlke, Dakang Xu, Simon Phipps, Paul S Foster, Bryan R G Williams

  Journal of immunology (Baltimore, Md. : 1950). 03/2008; 180(4):2117-24.

  Human TLR7 and 8 (hTLR7/8) have been implicated in the sequence-dependent detection of RNA oligonucleotides in immune cells. Although hTLR7 sequence-specific sensing of short RNAs has been inferredHuman TLR7 and 8 (hTLR7/8) have been implicated in the sequence-dependent detection of RNA oligonucleotides in immune cells. Although hTLR7 sequence-specific sensing of short RNAs has been inferred from studies of murine TLR7, this has yet to be established for hTLR7. We found that different short ssRNA sequences selectively induced either TNF-alpha or IFN-alpha in human PBMCs. The sequence-specific TNF-alpha response to ssRNAs observed in PBMCs could be replicated in activated human macrophage-like (THP-1) cells pretreated with IFN-gamma. Surprisingly, suppression of hTLR7 expression by RNA interference in this model reduced sensing of all immunostimulatory ssRNAs tested. Modulation of the relative expression ratio of hTLR7 to hTLR8 in THP-1 cells correlated with differential sensing of immunostimulatory sequences. Furthermore, the sequence-specific IFN-alpha induction profile in human PBMCs was accurately modeled by a sequence-specific activation of murine TLR7 in mouse macrophages. Thus, we demonstrate for the first time that hTLR7 is involved in sequence-specific sensing of ssRNAs. We establish a novel cell model for the prediction of TNF-alpha induction by short RNAs in human macrophages. Our results suggest that differential sequence-specific sensing of RNA oligonucleotides between human and mouse macrophages is due to the modulation of TLR7 sensing by human TLR8.

• Human immunodeficiency virus-1/surface glycoprotein 120 induces apoptosis through RNA-activated protein kinase signaling in neurons.

  Authors: Mehrdad Alirezaei, Debbie D Watry, Claudia F Flynn, William B Kiosses, Eliezer Masliah, Bryan R G Williams, Marcus Kaul, Stuart A Lipton, Howard S Fox

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 11/2007; 27(41):11047-55.

  Previous work has demonstrated that the surface glycoprotein (gp120) of human immunodeficiency virus-1 (HIV-1) can induce damage and apoptosis of neurons both in vitro and in vivo. In this report, wePrevious work has demonstrated that the surface glycoprotein (gp120) of human immunodeficiency virus-1 (HIV-1) can induce damage and apoptosis of neurons both in vitro and in vivo. In this report, we provide evidence that double-stranded RNA-activated protein kinase (PKR), a stress kinase, is involved in HIV/gp120-associated neurodegeneration. In cultures of mixed cortical cells, HIV/gp120 increased the protein level of PKR. Additionally, PKR was phosphorylated in neurons but not glia after exposure to gp120. The use of two independent pharmacological inhibitors of PKR activity abrogated neuronal cell death induced by gp120. Cortical neurons from PKR knock-out mice were significantly protected from neurotoxicity induced by gp120, further validating the pivotal proapoptotic function of PKR. gp120-induced phosphorylated PKR localized prominently to neuronal nuclei; PKR inhibition or the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] abrogated this effect. PKR inactivation also inhibited gp120-induced caspase-3 activation, consistent with its neuroprotective effect. Finally, brain tissue from individuals diagnosed with HIV-associated dementia (HAD), but not HIV infection alone, contained the activated form of PKR, which localized predominantly to neuronal nuclei. Together, these results identify PKR as a critical mediator of gp120 neurotoxicity, suggesting that activation of PKR contributes to the neuronal injury and cell death observed in HAD.

• Negative regulation of TLR-signaling pathways by activating transcription factor-3.

  Authors: Mark M Whitmore, Amaya Iparraguirre, Lindsey Kubelka, Wolfgang Weninger, Tsonwin Hai, Bryan R G Williams

  Journal of immunology (Baltimore, Md. : 1950). 10/2007; 179(6):3622-30.

  Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages andActivating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.