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Robert T Sarisky

Publications and source records attributed to Robert T Sarisky.

At least 19 recordsLinked to original sources
PubMed2007

Biochemical and functional analyses of the human Toll-like receptor 3 ectodomain.

The structure of the human Toll-like receptor 3 (TLR3) ectodomain (ECD) was recently solved by x-ray crystallography, leading to a number of models concerning TLR3 function (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585; Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980) The structure revealed four pairs of cysteines that are putatively involved in disulfide bond formation, several residues that are predicted to be involved in dimerization between ECD subunits, and surfaces that could bind to poly(I:C). In addition, there are two loops that protrude from the central solenoid structure of the protein. We examined the recombinant TLR3 ECD for disulfide bond formation, poly(I:C) binding, and protein-protein interaction. We also made over 80 mutations in the residues that could affect these features in the full-length TLR3 and examined their effects in TLR3-mediated NF-kappaB activation. A number of mutations that affected TLR3 activity also affected the ability to act as dominant negative inhibitors of wild type TLR3. Loss of putative RNA binding did not necessarily affect dominant negative activity. All of the results support a model where a dimer of TLR3 is the form that binds RNA and activates signal transduction.

Amino Acid Sequence↗
PubMed2006

Factors that affect in vitro measurement of the susceptibility of herpes simplex virus to nucleoside analogues.

OBJECTIVES: To identify factors that contribute to variability of HSV antiviral susceptibility breakpoints. METHODS: Acyclovir and penciclovir IC(50)'s for 12 HSV clinical isolates were measured in two laboratories using plaque reduction assay (PRA), an enzyme immunoassay (EIA)-based antigen reduction, and DNA hybridization on Vero, A549, MRC-5, HEL299 and HELG monolayers. Pair-wise comparisons were performed to evaluate variables including testing laboratory, technique, monolayer, and antiviral. The proportion of false results was analyzed using a conventional susceptibility IC(50) breakpoint of 2 microg/ml. RESULTS: Acyclovir-resistant HSV isolates were correctly identified by all methods. In contrast, there were 6-67% of susceptible isolates incorrectly characterized as drug-resistant. Variables associated with these errors included testing site, assay method, cell line and antiviral. A549, DNA hybridization, and penciclovir were associated with the highest IC(50)'s, whereas the PRA, EIA, and human fibroblast-monolayers provided the best differentiation between susceptible and resistant HSV isolates. CONCLUSIONS: The current recommendations to use a single discriminating value to define HSV resistance to nucleoside analogues can be problematic. False results are influenced in various degrees by the laboratory method, tissue culture and antivirals.

Acyclovir↗
PubMed2006

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function.

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32 degrees C, 37 degrees C, or 39.5 degrees C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype.

Amino Acid Sequence↗
PubMed2006

A pathway analysis of poly(I:C)-induced global gene expression change in human peripheral blood mononuclear cells.

To gain global pathway perspective of ex vivo viral infection models using human peripheral blood mononuclear cells (PBMCs), we conducted expression analysis on PBMCs of healthy donors. RNA samples were collected at 3 and 24 h after PBMCs were challenged with the Toll-like receptor-3 (TLR3) agonist polyinosinic acid-polycytidylic acid [poly(I:C)] and analyzed by internally developed cDNA microarrays and TaqMan PCR. Our results demonstrate that poly(I:C) challenge can elicit certain gene expression changes, similar to acute viral infection. Hierarchical clustering revealed distinct immediate early, early-to-late, and late gene regulation patterns. The early responses were innate immune responses that involve TLR3, the NF-kappaB-dependent pathway, and the IFN-stimulated pathway, whereas the late responses were mostly cell-mediated immune response that involve activation of cell adhesion, cell mobility, and phagocytosis. Overall, our results expanded the utilities of this ex vivo model, which could be used to screen molecules that can modulate viral stress-induced inflammation, in particular those mediated via TLRs.

Cluster Analysis↗
PubMed2006

Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation.

Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function. Exogenous addition of purified extracellular domain of TLR3 (hTLR3 ECD) expressed in human embryonic kidney cells was found to inhibit TLR3-dependent signaling, thus providing a reagent for structural and functional characterization. Approximately 35% of the mass of the hTLR3 ECD was due to posttranslational modification, with N-linked glycosyl groups contributing substantially to the additional mass. Cells treated with tunicamycin, an inhibitor of glycosylation, prevented TLR3-induced NF-kappaB activation, confirming that N-linked glycosylation is required for bioactivity of this receptor. Further, mutations in two of these predicted glycosylation sites impaired TLR3 signaling without obviously affecting the expression of the protein. Single-particle structures reconstructed from electron microscopy images and two-dimensional crystallization revealed that hTLR3 ECD forms a horseshoe structure similar to the recently elucidated x-ray structure of the protein expressed in insect cells using baculovirus vectors (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585 and Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980). There are, however, notable differences between the human cell-derived and insect cell-derived structures, including features attributable to glycosylation.

Amino Acid Sequence↗
PubMed2006

3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones, potent inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

Recently, we disclosed a new class of HCV polymerase inhibitors discovered through high-throughput screening (HTS) of the GlaxoSmithKline proprietary compound collection. This interesting class of 3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones potently inhibits HCV polymerase enzymatic activity and inhibits the ability of the subgenomic HCV replicon to replicate in Huh-7 cells. This report will focus on the structure-activity relationships (SAR) of substituents on the quinolinone ring, culminating in the discovery of 1-(2-cyclopropylethyl)-3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-6-fluoro-4-hydroxy-2(1H)-quinolinone (130), an inhibitor with excellent potency in biochemical and cellular assays possessing attractive molecular properties for advancement as a clinical candidate. The potential for development and safety assessment profile of compound 130 will also be discussed.

Animals↗
PubMed2006

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids: potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids is reported. Starting from commercially available chiral Fmoc-protected alpha-amino acids loaded onto Wang resin, Fmoc removal, reductive amination followed by amide bond formation, and base-catalyzed cyclization with simultaneous cleavage from the resin provided the desired products. Compounds described are potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

Animals↗
PubMed2006

Not-so-sweet sixteen: the role of IL-16 in infectious and immune-mediated inflammatory diseases.

Over the past two decades, our understanding of interleukin-16 (IL-16) has increased substantially. Initial studies characterizing IL-16 as a chemotactic cytokine (but not a chemokine) just scratched the surface of the unique properties of this cytokine. Since then, scientists have determined that IL-16 has a wide range of effects on cells, including upregulation of CD25, induction of cells to progress to the G(1) phase, inhibition of antigen- specific proliferation yet with retained antigen nonspecific proliferative properties, and discovery of a novel neuronal form with unique properties. Recently, a plethora of studies have implicated IL-16 in exacerbation of infectious, immune-mediated, and autoimmune inflammatory disorders, including atopic dermatitis, irritable bowel syndrome, systemic lupus erythematosus, neurodegenerative disorders, and viral infections. Herein, we review the body of evidence supporting a role for IL-16 in infectious and immune-mediated inflammatory disorders and explore the known and possible mechanism of actions in the numerous diseases.

Animals↗
PubMed2006

The cytoplasmic domain of the F protein of Human respiratory syncytial virus is not required for cell fusion.

The cytoplasmic domains of the fusion proteins encoded by several viruses play a role in cell fusion and contain sites for palmitoylation associated with viral protein trafficking and virus assembly. The fusion (F) protein of Human respiratory syncytial virus (HRSV) has a predicted cytoplasmic domain of 26 residues containing a single palmitoylated cysteine residue that is conserved in bovine RSV F protein, but not in the F proteins of other pneumoviruses such as pneumonia virus of mice, human metapneumovirus and avian pneumovirus. The cytoplasmic domains in other paramyxovirus fusion proteins such as Newcastle disease virus F protein play a role in fusion. In this study, it was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue (C550S) of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion.

Cell Fusion↗
PubMed2005

Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein.

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus). That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP), to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.

Animals↗
PubMed2005

Modulating toll-like receptor signalling as a novel antiinfective approach.

The innate immune response against invading microorganisms results in the deployment of phagocytes, including macrophages and dendritic cells to recognize pathogen-associated molecular patterns. Activation of Toll-like receptors (TLRs) expressed on these cells is a critical step in the initiation of this response, triggering the production of pro- and antiinflammatory cytokines to dampen microbial pathogenesis. Importantly, TLR activation also mediates dendritic cell maturation, a critical step in bridging the innate and adaptive arms of the immune system. Balancing the role of TLRs as central mediators of overlapping signaling pathways, whether directly through ligand interactions or via secondary adaptor molecules, mandates exquisite specificity. Further, understanding the immunopharmacology of TLR cross-talk during infection may help to provide insight into innate immunity and the mechanisms of immune-response subversion by pathogens. The continual and rapid emergence of drug resistance to traditional antimicrobial agents highlights the medical need for new treatment approaches. Herein, the discovery and development of TLR agonist and antagonist therapies for infectious diseases as adjunct to, or in place of, conventional treatment paradigms is discussed.

Anti-Infective Agents↗
PubMed2004

Non-nucleoside inhibitors of the HCV polymerase.

Chronic hepatitis C virus (HCV) infection is the leading cause of chronic liver disease. Current therapy using pegylated interferon-alpha with ribavirin is poorly tolerated and confers an overall sustained virological response around 56%. Compounds exhibiting an improved safety profile with similar or enhanced antiviral properties may represent future treatment options. Several drug discovery programmes are ongoing to directly target the viral enzymes involved in HCV replication. Recent clinical success using a peptidomimetic inhibitor of the viral serine protease has demonstrated proof-of-concept for the use of direct antiviral agents in reducing viral load. The RNA-dependent RNA polymerase (RdRp) of HCV is also required for viral RNA replication and thus represents an attractive drug discovery target. Preclinical characterization of several non-nucleoside inhibitors (NNIs) of the HCV RdRp have been described, including a promising series of benzothiadiazine derivatives which have been shown to efficiently block viral RNA synthesis in HCV replicon cell systems. Herein, the antiviral activity, mode of action, resistance profiling and therapeutic potential of the benzothiadiazine class of compounds for clinical development are explored.

Drug Resistance, Viral↗
PubMed2004

Sequence requirements for the development of a chimeric HCV replicon system.

The hepatitis C virus (HCV) 3'nontranslated region (3'NTR) is important for virus infection and replicon replication. Here, we constructed a panel of chimera replicons containing non-structural (NS) and 3'NTR sequences from different HCV strains or types, and examined the requirements for stable replication. A subgenomic replicon chimera comprising the polymerase and 3'NTR from HCV strain Con1, and other non-structural genes from type 1a strain H77, supported stable colony formation and replication in Huh7 cells. However, extending the type 1a sequence to include 132 amino acids of NS5B resulted in a defective HCV replicon. In contrast, a similar chimera containing HCV strain J4 sequences linked in cis to Con1 NS5B and 3'NTR supported stable replication suggesting that the interaction between the NS proteins and the 3'NTR may represent a critical determinant. Lastly, the type 1a 3'NTR from pCV-J4L6S was unable to confer replication when paired with non-structural coding sequences from BB7 or J4 and the 3'NTR from Con1 was unable to confer replication when paired with J4 or H77 sequences. These results highlighted the importance of sequence specific interaction among 3'NTR and two distinct subdomains of the NS coding region as a determinant in supporting stable replication of subgenomic replicons. The results underscore the importance of directly cloning 3'NTR sequences from relevant clinical samples.

3' Untranslated Regions↗
PubMed2004

Mapping cooperative activity of the hepatitis C virus RNA-dependent RNA polymerase using genotype 1a-1b chimeras.

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (RdRp) which is essential for viral replication. NS5B expression in bacteria generated 20- to 50-fold lower yield and 100-fold less product per mol of enzyme for gentoype 1a RdRp than type 1b. Further, unlike type 1b RdRp, type 1a enzyme failed to exhibit cooperative properties in the assays described herein. Differences in thermal stability may partially account for the inability to efficiently oligomerize. Superose gel filtration analyses confirm differences between these RdRp preparations, although affinity for the column rather than size may account for the differences in migration. To further address this complexity, a panel of RdRp type 1a-type 1b chimeras were evaluated and implicate a role for the thumb subdomain of genotype 1b RdRp as critical for cooperative function.

Amino Acid Sequence↗
PubMed2004

What is disrupting IFN-alpha's antiviral activity?

Despite advances in treatment strategies for hepatitis C virus (HCV), a significant proportion of patients fail to achieve viral clearance following treatment with pegylated interferon (IFN)-alpha plus ribavirin. Many of these individuals show elevated levels of tumor necrosis factor (TNF)-alpha compared with normal controls, and recent data have implicated this cytokine in the negative regulation of IFN-alpha. Although a therapeutic opportunity for TNF-alpha antagonists might exist for reducing inflammation in chronic HCV disease, further exploration is required to identify the key mediators of responsiveness to IFN-alpha. In particular, the interplay should be clarified between host response factors [e.g. IFN-alpha, IFN-gamma, suppressor of cytokine signaling (SOCS), TNF-alpha and others] and pathogen-associated molecular patterns [PAMPs, e.g. lipopolysaccharide (LPS) and CpG DNA] in HCV disease; this information might guide future therapies aimed at improving IFN-alpha responsiveness.

Cytokines↗