Charles Samuel - Academia.edu (original) (raw)

Papers by Charles Samuel

Biochemical and Biophysical Research Communications, 2007

Bacterial flagellin is recognized by Toll-like receptor (TLR5) and activates NF-jB which leads to... more Bacterial flagellin is recognized by Toll-like receptor (TLR5) and activates NF-jB which leads to the induction of proinflammatory gene expression. Salmonella expresses two flagellin proteins, FliC and FljB. We purified FliC and FljB and examined the ability of the Salmonella flagellins to activate the NF-jB transcription factor in human embryonic kidney cells. We found that FliC and FljB as purified proteins possessed a comparable specific activity for activation of NF-jB-dependent gene expression in HEK293 cells. We also determined the ability of UV-inactivated bacteria, both wild-type and fliC and fljB mutant strains, to activate NF-jB. Wild-type fliC + /fljB + Salmonella and the fliC + /fljB À mutant strain were robust activators, whereas the fliC À /fljB + and flhC À mutant strains were very poor activators. The NF-jB activation capacity of bacterial strains correlated with their flagellin expression level. Finally, Salmonella cell wall-associated polymeric flagellin displayed greatly reduced ability to activate NF-jB compared to purified monomeric flagellin.

Journal of Interferon Cytokine Research, 2002

The RNA-dependent protein kinase PKR promoter is interferon (IFN) inducible and possesses a novel... more The RNA-dependent protein kinase PKR promoter is interferon (IFN) inducible and possesses a novel 15-base pair (bp) constitutive activator element, designated kinase conserved sequence (KCS), in addition to an IFN-stimulated response element (ISRE). Deletion of the KCS element or point mutations within the KCS element greatly reduce both basal and IFN-inducible PKR promoter activity. The IFN-inducible RNA-specific adenosine deaminase ADAR1 promoter possesses a KCS-like (KCS-l) element. The sequences of the KCS and KCS-l elements and their positions relative to the cognate ISRE element are similar between the PKR and ADAR1 promoters. However, substitution of the ADAR1 KCS-l element for the KCS element of the PKR promoter resulted in significantly reduced basal and IFN-inducible promoter activities comparable to either point mutation or entire deletion of the PKR KCS element. The PKR KCS element selectively bound nuclear proteins more efficiently than did the ADAR1 KCS-l element. Reversing the positions of the KCS and ISRE elements of the PKR promoter relative to one another or reversing the orientation of either element while conserving the naturally occurring 4-bp spacing between the two elements did not significantly reduce basal or IFN-inducible promoter activity. Taken together, these results are consistent with the notion that the KCS and ISRE elements of the PKR promoter function as a unit.

Biochem Biophys Res Commun, 2007

Bacterial flagellin is recognized by Toll-like receptor (TLR5) and activates NF-κB which leads to... more Bacterial flagellin is recognized by Toll-like receptor (TLR5) and activates NF-κB which leads to the induction of proinflammatory gene expression. Salmonella expresses two flagellin proteins, FliC and FljB. We purified FliC and FljB and examined the ability of the Salmonella flagellins to activate the NF-κB transcription factor in human embryonic kidney cells. We found that FliC and FljB as purified proteins possessed a comparable specific activity for activation of NF-κB-dependent gene expression in HEK293 cells. We also determined the ability of UV-inactivated bacteria, both wild-type and fliC and fljB mutant strains, to activate NF-κB. Wild-type fliC+/fljB+Salmonella and the fliC+/fljB− mutant strain were robust activators, whereas the fliC−/fljB+ and flhC− mutant strains were very poor activators. The NF-κB activation capacity of bacterial strains correlated with their flagellin expression level. Finally, Salmonella cell wall-associated polymeric flagellin displayed greatly reduced ability to activate NF-κB compared to purified monomeric flagellin.

Virology, 1978

... References. VIROLOGY 89, 240251 (1978) Mechanism of Interferon Action Properties of an Interf... more ... References. VIROLOGY 89, 240251 (1978) Mechanism of Interferon Action Properties of an Interferonmediated Ribonucleolytic Activity from Mouse 1929 Cells DEBORAH A. EPPSTEIN ... SEN, GC, GUPTA, SL, BROWN, GE, LEBLEU, B., REBELLO, A., and LENGYEL, P. (1976a). ...

Cell Growth Differentiation the Molecular Biology Journal of the American Association For Cancer Research, Oct 1, 2002

The D-group cyclins play a key role in the progression of cells through the G 1 phase of the cell... more The D-group cyclins play a key role in the progression of cells through the G 1 phase of the cell cycle. Treatment of MCF-7 breast cancer cells with the cyclopentenone prostaglandin 15-deoxy-⌬ 12,14 -PGJ 2 (15d-PGJ 2 ) results in rapid down-regulation of cyclin D1 protein expression and growth arrest in the G 0 /G 1 phase of the cell cycle. 15d-PGJ 2 also down-regulates the expression of cyclin D1 mRNA; however, this effect is delayed relative to the effect on cyclin D1 protein levels, suggesting that the regulation of cyclin D1 occurs at least partly at the level of translation or protein turnover. Treatment of MCF-7 cells with 15d-PGJ 2 leads to a rapid increase in the phosphorylation of protein synthesis initiation factor eukaryotic initiation factor 2␣ (eIF-2␣) and a shift of cyclin D1 mRNA from the polysome-associated to free mRNA fraction, indicating that 15d-PGJ 2 inhibits the initiation of cyclin D1 mRNA translation. The selective rapid decrease in cyclin D1 protein accumulation is facilitated by its rapid turnover (t 1/2 ‫؍‬ 34 min) after inhibition of cyclin D1 protein synthesis. The half-life of cyclin D1 protein is not significantly altered in cells treated with 15d-PGJ 2 . Treatment of cells with 15d-PGJ 2 results in strong induction of heat shock protein 70 (HSP70) gene expression, suggesting that 15d-PGJ 2 might activate protein kinase R (PKR), an eIF-2␣ kinase shown previously to be responsive to agents that induce stress. 15d-PGJ 2 strongly stimulates eIF-2␣ phosphorylation and down-regulates cyclin D1 expression in a cell line derived from wild-type mouse embryo fibroblasts but has an attenuated effect in PKR-null cells, providing evidence that PKR is involved in mediating the effect of 15d-PGJ 2 on eIF-2␣ phosphorylation and cyclin D1 expression. In summary, treatment of MCF-7 cells with 15d-PGJ 2 results in increased phosphorylation of eIF-2␣ and inhibition of cyclin D1 mRNA translation initiation. At later time points, repression of cyclin D1 mRNA expression may also contribute to the decrease in cyclin D1 protein. reticulum; HSP, heat shock protein; GRP, glucose-regulated protein; BCS, bovine calf serum.

Journal of virology, Jan 13, 2015

Defective-interfering viral genome RNAs (DI-RNAs) can form during infections of negative strand R... more Defective-interfering viral genome RNAs (DI-RNAs) can form during infections of negative strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document frequent de novo generation of copyback DI-RNAs from independent rescue events both for vaccine (vac2) and wild-type (IC323) measles viruses as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C protein-deficient (C(KO)) measles viruses generated about ten times more DI-RNAs than parental virus, suggesting that C enhances processivity of the viral polymerase. We obtained nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and re-initiation sites and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA 1 (ADAR1), which catalyzes in double-stranded RNA the C6 deam...

Virology, Jan 15, 2015

Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cu... more Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cultured human cells, which is manifested by premature death of HAdV-12-infected cells. Whereas HAdV-2 induction of IFN-β expression was transient, HAdV-12-infected cells maintained high levels of IFN-β expression, protein kinase R (PKR) activation and eIF-2α phosphorylation throughout the infectious cycle. The importance of the IFN-inducible PKR kinase in restriction of HAdV-12 was supported by the enhanced growth of the virus following PKR knockdown in HeLa cells. Ectopic expression of HAdV-2 VA RNAI increased HAdV-12 hexon protein expression, suggesting that insufficient VA RNA expression contributes to the restricted growth of HAdV-12. Although some adenovirus species are known to persist in human lymphoid tissues, HAdV12 has so far not been found. Thus, it is possible that the inability of HAdV12 to evade the INF response may have implications for the virus to establish long-lasting o...

Virology, 1979

The effect of the duration of interferon treatment of mouse L929 fibroblasts on the expression of... more The effect of the duration of interferon treatment of mouse L929 fibroblasts on the expression of ribosome-associated and cell-sap-localized translation inhibition, protein phosphorylation, and messenger RNA (mRNA) methylation and degradation was investigated. The following results were obtained: (1) Ribosomal salt-wash fractions prepared from L929 cells treated for 12 or 18 hr significantly inhibited the translation of methylated reovirus mRNA catalyzed by the cell-free system prepared from untreated ascites tumor cells. The translation of reovirus mRNA was slightly stimulated by the ribosomal salt-wash fractions prepared from untreated cells and cells treated for 2 hr and was slightly inhibited by the salt-wash fraction from cells treated for 6 hr. (2) Cell-sap fractions prepared from untreated cells and from cells treated for 2 or 6 hr did not appreciably affect the translation of reovirus mRNA in the absence of double-stranded RNA (dsRNA); cell-sap fractions prepared from cells treated for 12 or 18 hr were slightly inhibitory. (3) Neither reovirus genome dsRNA nor polyriboinosinic acid-polyribocytidylic acid inhibited the translation of reovirus mRNA by the untreated ascites system. The inhibitory activity of ribosomal salt-wash fractions from interferon-treated cells was dsRNA independent, whereas dsRNA enhanced the inhibitory activity of cell-sap fractions from interferon-treated cells. (4) Interferon treatment significantly enhanced a cell-sap-independent phosphorylation of at least three proteins present in the ribosomal salt-wash fractions. Phosphorylation of an approximately 50,000-dalton component was maximally enhanced after 12 hr of interferon treatment and was increased by dsRNA; phosphorylation of 30,000- and &amp;amp;amp;lt;20,000-dalton components was enhanced earlier and was dsRNA independent. (5) An increase in the nuclease-mediated degradation of [3H]uridine-labeled methylated reovirus mRNA catalyzed by ribosomal salt-wash fractions was detectable after 6 hr of interferon treatment and was maximally enhanced after 12 hr of interferon treatment to about threefold the level of the untreated ribosomal salt-wash fraction. The degradation was not enhanced by dsRNA. The levels of reovirus mRNA degradation catalyzed by cell-sap fractions from untreated cells and from cells treated with interferon for 2 to 18 hr were comparable. (6) No significant difference was observed in the ability of cell-sap fractions from untreated cells or from cells treated with interferon for 2 to 18 hr to catalyze the methylation of unmethylated reovirus mRNA. In addition, none of the ribosomal salt-wash fractions significantly inhibited reovirus mRNA methylation catalyzed by untreated ascites cell-free extracts. These results suggest that interferon treatment may mediate multiple biochemical changes in murine cells, including the specific phosphorylation of protein(s) associated with ribosomes that possess interferon-mediated inhibitor(s) of viral mRNA translation.

Journal of Virology, 2008

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resist... more The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (⌬E3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ⌬E3L virus was increased by nearly 2 log 10 in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ⌬E3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ⌬E3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the ␣ subunit of protein synthesis initiation factor 2 (eIF-2␣) was elevated severalfold in ⌬E3L-infected

Journal of Virology, 2007

The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of bio... more The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient

Journal of Virology, 2014

Measles virus (MV) lacking expression of C protein (C KO ) is a potent activator of the double-st... more Measles virus (MV) lacking expression of C protein (C KO ) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C KO mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product.

Journal of Interferon & Cytokine Research, 2008

Recognition of cytoplasmic bacterial flagellin by the Nod-like receptor ICE protease-activating f... more Recognition of cytoplasmic bacterial flagellin by the Nod-like receptor ICE protease-activating factor (Ipaf) in macrophages leads to activation of caspase-1 and secretion of interleukin-1beta (IL-1beta). Salmonella possess two genes, fliC and fljB, that encode flagellin proteins. We examined the ability of purified FliC and FljB proteins to activate IL-1beta secretion in the mouse macrophage-like J774 cell line and in mouse primary peritoneal cells. We found that purified FliC and FljB flagellins possessed a comparable ability to activate IL-1beta secretion following introduction into the cytoplasm of J774 or primary cells. We also examined the ability of an attenuated Salmonella mutant strain (dam) deficient in DNA adenine methylase to activate IL-1beta secretion. Compared to infection of primary cells with wild-type Salmonella, IL-1beta secretion was reduced in cells infected with the dam mutant even though the two strains expressed similar levels of flagellin. As a control, cells infected with a flagellin-deficient (flhC) Salmonella strain did not show enhanced IL-1beta secretion.

Journal of Interferon & Cytokine Research, 2007

Salmonella enterica serovar Typhimurium is highly virulent and mediates robust interferon (IFN)-s... more Salmonella enterica serovar Typhimurium is highly virulent and mediates robust interferon (IFN)-stimulated gene (ISG) induction, whereas bacterial mutants that lack the DNA adenine methylase (Dam) are attenuated, elicit a reduced ISG activation profile, and establish immunity to murine typhoid fever. We show here that in contrast to observations in mice, infection of macrophage cell cultures with either wild-type (WT) or dam(-) mutant Salmonella resulted in surprisingly similar kinetics and amplitudes of induction of IFN-beta, the type I IFN-alpha,beta beacon gene Mx, and the type II IFN-gamma beacon inducible nitric oxide synthase (iNOS). Likewise, activation of NF-kappaB-dependent gene expression in epithelial cells was comparable with WT and dam(-) mutant Salmonella. In contrast, the flagellin-deficient flhC(-) mutant did not activate NF-kappaB in epithelial cells but activated ISG expression comparable to that of WT Salmonella in macrophage cells. WT and dam(-) strains displayed a similar Toll-like receptor 5 (TLR5)-dependent NF-kappaB activation, whereas the flhC(-) mutant lacked this activity. UV-inactivated Salmonella elicited similar ISG induction to that of viable Salmonella in macrophages and mediated the establishment of a functional antiviral state but displayed decreased cytocidal activity. These results establish that the inherent IFN system-inducing capacities of dam(-) and WT Salmonella strains in cultured macrophage and epithelial cells, unlike the mouse, are indistinguishable.

Journal of Interferon & Cytokine Research, 2002

Journal of Biological Chemistry, 2003

Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including ... more Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including virus multiplication and cell growth, differentiation, and apoptosis. The promoter of the PKR gene possesses a novel 15-bp element designated KCS, positioned upstream of a consensus interferon (IFN)-stimulated response element, that is required for both basal and interferoninducible transcription. Protein binding to the KCS element is not dependent upon IFN treatment and correlates with transcriptional activity of the PKR promoter. The identity of KCS-binding proteins (KBP) that selectively bind at the KCS element is largely unknown, except for the transcription factor Sp1. We now have purified KBP from HeLa cell nuclear extracts by ionexchange and DNA-affinity chromatography steps and then identified four constituent proteins of the KBP complex by mass spectrometry and immunochemistry: KBP120 and KBP45 are the damaged DNA-binding protein subunits, p127 DDB1 and p48 DDB2, respectively; KBP100 is the transcription factor Sp1; and KBP35 is the heterogeneous nuclear ribonucleoprotein A1. The steady-state levels of these four KCS-binding proteins in human cells are not altered by IFN treatment. Components of the KBP complex bind selectively and constitutively to the KCS element in the absence of IFN treatment, both in vitro as measured by competition electrophoretic mobility shift assay (EMSA) and DNA pull-down assays and in vivo as measured by chromatin immunoprecipitation assays. Depletion of DDB2 by antisense strategy reduces KBP complex formation by EMSA. These results provide new insight into the biochemical identity and activity of proteins involved in PKR promoter function.

Infection and Immunity, 2007

Salmonella enterica serovar Typhimurium deficient in DNA adenine methylase (Dam) are attenuated f... more Salmonella enterica serovar Typhimurium deficient in DNA adenine methylase (Dam) are attenuated for virulence in mice and confer heightened immunity in vaccinated animals. In contrast, infection of mice with wild-type (WT) strains or flagellin-deficient mutants of Salmonella causes typhoid fever. Here we examined the bacterial load and spatiotemporal kinetics of expression of several classes of host genes in Peyer's patches, the liver, and the spleen following oral infection of mice with WT, dam mutant, or flagellin-deficient (flhC) Salmonella. The genes evaluated included inflammatory (interleukin-1␤ [IL-1␤], tumor necrosis factor alpha), chemokine (macrophage inflammatory protein 2), Th1/Th2 indicator (IL-12p40, IL-4), and interferon system (beta interferon [IFN-␤], IFN-␥, protein Mx1 GTPase, RNA-dependent protein kinase, inducible nitric oxide synthase, suppressor of cytokine signaling 1) beacons. We showed that maximal interferon system and proinflammatory gene induction occurred by 5 days after infection and that the levels were comparable for the WT and flhC strains but were significantly lower for the dam mutant. Additionally, host gene expression in systemic tissues of individual animals was dependent on the bacterial load in the Peyer's patches for mice infected with WT, dam mutant, or flhC mutant Salmonella as early as 8 h after infection. Moreover, a bacterial load threshold in the Peyer's patches was necessary to stimulate the host gene induction in the liver and spleen. Taken together, these results suggest that bacterial load and the accompanying strain-specific cytokine signature are important determinants of the host innate immune response and associated disease manifestations observed in dam mutant Salmonella-infected animals compared to the immune response and disease manifestations observed in WT and flhC mutant Salmonella-infected animals.

Gene, 2003

The ADAR1 gene encodes an RNA-specific adenosine deaminase that alters the functional activity of... more The ADAR1 gene encodes an RNA-specific adenosine deaminase that alters the functional activity of both viral and cellular RNAs by posttranscriptional adenosine-to-inosine RNA editing. The interferon (IFN) responsive PI promoter of the ADAR1 gene possesses an IFNstimulated response element (ISRE) responsible for IFN-inducibility, as well as an adjacent upstream sequence, designated kinase conserved sequence-like (KCS-') element. The KCS-' element is similar to the 15-bp KCS element so far unique to the human and mouse RNAdependent PKR kinase gene promoters. The KCS element of the PKR kinase (PKR) promoter is essential for both basal and IFN-inducible PKR promoter activity. We have now examined the functional properties of the KCS-' element of the ADAR1 PI promoter. Electrophoretic mobility shift assays (EMSAs) detected constitutively expressed nuclear proteins that bound selectively to the ADAR1 KCS-' element. Competition EMSA and antibody supershift assays indicated that ADAR1 KCS-'-binding proteins shared some properties with PKR KCSbinding proteins. However, transient transfection analyses performed with ADAR1 PI promoter constructs possessing deletion and substitution mutant forms of the KCS-' element revealed that the ADAR1 KCS-' element was not essential for either basal or IFN-inducible promoter activity. Substitution of the ADAR1 KCS-' element with the PKR KCS element increased both basal and inducible ADAR1 PI promoter activity. These results suggest that the KCS-' element of the ADAR1 PI promoter is not functionally equivalent to the KCS element of the PKR promoter. q

Cell, 1986

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficie... more The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection. The growth of a viral mutant that is unable to produce the RNA is inhibited by interferon, while wild-type virus is not affected. VAI RNA prevents activation of the interferon-induced P1/eIF-2 alpha kinase. This inhibition can be reproduced in extracts of interferon-treated cells where purified VAI RNA prevents activation of latent kinase by double-stranded RNA.

Biochemical and Biophysical Research Communications, 2001

Since IFN production is also induced by treatment with poly(I:C), viral double-stranded (ds) RNA ... more Since IFN production is also induced by treatment with poly(I:C), viral double-stranded (ds) RNA has been postulated to play a direct role in the process. In the present study, we investigated the effect of dsRNA binding proteins on virus-induced activation of the IFN-␤ gene. We found that PACT, originally identified as protein activator for dsRNA-dependent protein kinase (PKR) and implicated in the regulation of translation, augmented IFN-␤ gene activation induced by Newcastle disease virus. Concomitantly with the augmented activity of IFN-␤ enhancer, increased activity of NF-B and IRF-3 and IRF-7 was observed. For the observed effect, the dsRNA-binding activity of PACT was essential. We identified residues of PACT that interact with a presumptive target molecule to exert its function. Furthermore, PACT colocalized with viral replication complex in the infected cells. Thus the observed effect of PACT is novel and PACT is involved in the regulation of viral replication and results in a marked increase of cellular IFN-␤ gene expression.