Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. IMPORTANCE: Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

Simultaneous uncoupled expression and purification of the Dengue virus NS3 protease and NS2B co-factor domain.

Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B-NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B-NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B-NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies.

Riboflavin and ultraviolet light: impact on dengue virus infectivity.

BACKGROUND: Dengue viruses (DENV 1-4) are emerging across the world, and these viruses pose a risk to transfusion safety. Pathogen inactivation may be an alternative approach for managing the risk of DENV transfusion transmission. This study aimed to investigate the ability of riboflavin and UV light to inactivate DENV 1-4 in platelet concentrates. MATERIALS AND METHODS: DENV 1-4 were spiked into buffy coat-derived platelet concentrates in additive solution (SSP+) before being treated with riboflavin and UV light. Infectious virus was quantified pre- and posttreatment, and the reduction in viral infectivity was calculated. RESULTS: All four DENV serotypes were modestly reduced after treatment. The greatest amount of reduction in infectivity was observed for DENV-4 (1·81 log reduction) followed by DENV-3 (1·71 log reduction), DENV-2 (1·45 log reduction) and then DENV-1 (1·28 log reduction). CONCLUSION: Our study demonstrates that DENV 1-4 titres are modestly reduced following treatment with riboflavin and UV light. With the increasing number of transfusion-transmitted cases of DENV around the globe, and the increasing incidence and geographical distribution of DENV, additional approaches for maintaining blood safety may be required in the future.

Identification of residues in West Nile virus pre-membrane protein that influence viral particle secretion and virulence.

The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNV(NY99)) and a weakly virulent Australian subtype (WNV(KUN)). Five amino acid differences occur in WNV(NY99) compared with WNV(KUN) (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNV(NY99) prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNV(KUN) prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNV(KUN) prM to the same level as that of WNV(NY99), and enhanced the secretion of WNV(KUN) prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNV(KUN) infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

Complete remission with immunotherapy: Case report of a patient with metastatic bladder cancer to the humerus.

Bladder cancer is the sixth most common malignancy in the United States. Cisplatin combination regimens are first line therapy for patients with metastatic urothelial bladder cancer who are eligible candidates and no treatments have shown to improve outcome compared to chemotherapy for the past 20 years. Significant advances were made in past 2-3 years and the most significant was the introduction of checkpoints inhibitors in bladder cancer treatment. We present a patient diagnosed with metastatic urothelial carcinoma who progressed while on cisplatin/gemcitabine chemotherapy in the form of oligometastasis to the bone. He has achieved a durable complete response with atezolizumab.

Human interleukin 1 beta is not secreted from hamster fibroblasts when expressed constitutively from a transfected cDNA.

To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.

West Nile Virus NS2B/NS3 protease as an antiviral target.

West Nile Virus (WNV) has spread rapidly during the last decade across five continents causing disease and fatalities in humans and mammals. It highlights the serious threat to both our health and the economy posed by viruses crossing species, in this case from migratory birds via mosquitoes to mammals. There is no vaccine or antiviral drug for treating WNV infection. One attractive target for antiviral development is a viral trypsin-like serine protease, encoded by the N-terminal 184 amino acids of NS3, which is only active when tethered to its cofactor, NS2B. This protease, NS2B/NS3pro, cleaves the viral polyprotein to release structural and non-structural viral proteins that are essential in viral replication and assembly of new virus particles. Disruption of this protease activity is lethal for virus replication. The NS3 protein also has other enzymes within its sequence (helicase, nucleoside triphosphatase, RNA triphosphatase), all of which are tightly regulated through localisation within membranous compartments in the infected cell. This review describes the various roles of NS3, focussing on NS2B-NS3 protease and its function and regulation in WNV replication and infection. Current advances towards development of antiviral inhibitors of NS2B/NS3pro are examined along with obstacles to their development as an antiviral therapy.

Modified influenza virosomes: recent advances and potential in gene delivery.

Influenza virosomes have proven to be effective vehicles for the delivery of antigens in the vaccination of humans against a number of pathogens. However, their potential as a means for gene delivery has yet to be realized. Chemical modification of viruses is emerging as a new strategy for production of safe and efficient gene delivery systems. Influenza virosomes exhibit many of the features of the virus, such as for cell binding, uptake and endosomal escape, which can be easily engineered into designer delivery vehicles capable of safe, efficient and cell-specific cargo delivery. This review focuses on the next generation of influenza virosomes and highlights aspects of their modification that may lead to simple but effective gene delivery vehicles.

Induction of alpha-fetoprotein synthesis in differentiating F9 teratocarcinoma cells is accompanied by a genome-wide loss of DNA methylation.

F9 teratocarcinoma cells can be grown as monolayers or aggregates, and upon treatment with retinoic acid they will differentiate into parietal or visceral endoderm, respectively. Visceral endoderm specifically synthesizes alpha-fetoprotein and albumin mRNAs, which are not found in parietal endoderm. In contrast, both endoderms produce enhanced levels of the major histocompatibility antigen (H2) mRNA compared with F9 cells. F9 cells contain highly methylated DNA as judged by restriction enzyme digestion. However, upon differentiation into visceral endoderm, there is a genome-wide loss of methylation in induced, silent, and constitutively expressed genes. Experiments in which methylation loss is induced via the methyltransferase inhibitor 5-azacytidine result in no induction of alpha-fetoprotein mRNA and no morphological differentiation, suggesting that methylation loss alone is not sufficient to induce the visceral endoderm phenotype. Likewise, 5-azacytidine treatment of differentiated cells does not result in enhanced expression of alpha-fetoprotein mRNA. However, the patterns of loss of DNA methylation at all sites examined after differentiation or 5-azacytidine treatment were remarkably similar, suggesting that the two occur by a similar mechanism, the inhibition of DNA methyltransferase activity. These results argue that the specificity for methylation loss at a given site is an inherent property of aggregated F9 cell chromatin. This system provides a model for studying a tissue-specific change in DNA methylation upon differentiation.

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Pharmacological modulation of cytokine action and production through signaling pathways.

The action or production of cytokines is mediated through a number of signal transduction pathways which have been elucidated recently. These include pathways integrating the activation of extracellular receptors and subsequent intracellular events leading to alterations of gene expression, cytoskeletal organization, DNA synthesis and cell survival, and the direct activation of intracellular transcription factors via cell permeable hormones. Discovery and characterization of many of these pathways has been aided by the use of compounds which inhibit them. In turn the inhibitors, many of which are already in the clinic, have provided significant insight into the pharmacological importance of each pathway and its potential for providing more potent, selective and safer alternatives. This review summarizes the current state of knowledge about several of these pathways, how they regulate cytokine action or production, and their potential for pharmacological intervention.

Structural basis of inhibitor selectivity in MAP kinases.

BACKGROUND: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

Product release is rate-limiting for catalytic processing by the Dengue virus protease.

Dengue Virus (DENV) is the most prevalent global arbovirus, yet despite an increasing burden to health care there are currently no therapeutics available to treat infection. A potential target for antiviral drugs is the two-component viral protease NS2B-NS3pro, which is essential for viral replication. Interactions between the two components have been investigated here by probing the effect on the rate of enzyme catalysis of key mutations in a mobile loop within NS2B that is located at the interface of the two components. Steady-state kinetic assays indicated that the mutations greatly affect catalytic turnover. However, single turnover and fluorescence experiments have revealed that the mutations predominantly affect product release rather than substrate binding. Fluorescence analysis also indicated that the addition of substrate triggers a near-irreversible change in the enzyme conformation that activates the catalytic centre. Based on this mechanistic insight, we propose that residues within the mobile loop of NS2B control product release and present a new target for design of potent Dengue NS2B-NS3 protease inhibitors.

Solar Coronal Jets: Observations, Theory, and Modeling.

Chromospheric and coronal jets represent important manifestations of ubiquitous solar transients, which may be the source of significant mass and energy input to the upper solar atmosphere and the solar wind. While the energy involved in a jet-like event is smaller than that of "nominal" solar flares and Coronal Mass Ejections (CMEs), jets share many common properties with these major phenomena, in particular, the explosive magnetically driven dynamics. Studies of jets could, therefore, provide critical insight for understanding the larger, more complex drivers of the solar activity. On the other side of the size-spectrum, the study of jets could also supply important clues on the physics of transients close or at the limit of the current spatial resolution such as spicules. Furthermore, jet phenomena may hint to basic process for heating the corona and accelerating the solar wind; consequently their study gives us the opportunity to attack a broad range of solar-heliospheric problems.

Kinetics and mechanism of the glutathione-dependent reduction of dehydromethionine.

Dehydromethionine (S-methylisothiazolidine-3-carboxylic acid) is reduced by glutathione (aqueous solution, 25 degrees C) to give methionine and glutathione disulfide in a reaction that is dependent on pH and the nature and concentration of the buffer utilized. The data are consistent with proton-assisted, rate-limiting attack of glutathione thiolate anion on the sulfilimine sulfur with concomitant cleavage of the sulfur-nitrogen bond. The data predict a half-life for dehydromethionine ranging between 33 and 330 min in the presence of physiological concentrations of glutathione and phosphate buffer, suggesting that dehydromethionine and similar sulfilimines may have a sufficient lifetime in vivo to permit their use as carriers of oxidizing equivalents.

Substratum acidification and proteinase activation by murine B16F10 melanoma cultures.

Murine B16F10 melanoma cells, cultured within 0.7% agarose gels containing the fluorescent proteinase substrate acetamidofluorescein-BSA, catalyze the hydrolysis of the substrate in the region immediately surrounding the cell. Fluorescence ratio measurements on hydrolyzed substrate correlate with an average pH of 5.5 +/- 0.2 in the adjacent substratum region. Enzymatic activity within the gel is partially inhibited by leupeptin, pepstatin, phenylmethylsulfonyl fluoride. EDTA and by anti-human cathepsin B, suggesting potential roles for thiol-, aspartic- and metalloproteinases. The time-course of fluorescence intensity, correlated with substratum pH measurements, suggest that substrate hydrolysis is catalyzed by enzymes with pH optima of < 5.5. Invasion by these cells through thin barriers of reconstituted basement membrane gel (Matrigel) is totally blocked by the thiol proteinase inhibitor, leupeptin. It is suggested that secreted or cell-surface acid proteinase enzymes, activated by the cell-mediated local hyperacidity, are involved in substrate hydrolysis and that these enzymes may be important in invasiveness by this cell-line.

Substratum acidification by murine B16F10 melanoma cultures.

Murine B16F10 melanoma cells, adherent to thin films of crosslinked, fluorescein-labeled collagen I, and covered by a thin layer of 0.7% agarose, exhibit a decrease in fluorescence emission in the substratum region immediately beneath adherent cells. The relative diminution in fluorescence intensity is dependent on excitation wavelength and is observed following excitation at 490 nm, but is not observed following excitation at 452 nm. The decrease in fluorescence emission is not due to quenching or concentration effects and is attributed to the decrease in extracellular pH in the substratum region. Fluorescence measurements of (I490/I452) within these substratum regions, correlate with an average extracellular pH of 6.4 +/- 0.2 which drops to pH less than 5 after 5 h. It is suggested that this region is sufficiently acidic to activate secreted or cell-surface acid proteinase enzymes and that the activity of these enzymes may be important in invasiveness by this cell-line.

Binding of inhibitors to the major glutathione S-transferase from bovine brain. Competitive binding between bilirubin and glutathione.

The binding of non-substrate ligands to the glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) from bovine brain has been investigated kinetically by monitoring the inhibition of the enzyme-catalyzed reaction between glutathione and 1-chloro-2,4-dinitrobenzene. Bilirubin, thyroxine, lithocholic acid, retinoic acid and retinol are competitive inhibitors with respect to glutathione. Cooperative binding effects are observed with lithocholic acid, retinoic acid and retinol while cooperative binding is not observed with thyroxine or bilirubin. Bilirubin is the most potent inhibitor with constants of 0.1 and 110 microM. 50% of the total activity is lost upon binding to the high-affinity site and the remainder is lost at higher bilirubin concentrations. In spite of the apparently favorable binding for bilirubin, it is estimated that the high intracellular concentrations of reduced glutathione will saturate the enzyme and allow only a small fraction of the bilirubin in brain to bind to the enzyme. It is concluded that the binding of these ligands may be of minor importance in vivo.

Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages.

Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant. Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect. 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM. The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.

Kinetic analysis of the intracellular conjugation of monochlorobimane by IC-21 murine macrophage glutathione-S-transferase.

Monochlorobimane (MCB) reacts with glutathione (GSH) in a reaction catalyzed by the glutathione-S-transferase (GST) isozymes. The diffusion of MCB through cell membranes is rapid and the fluorescence conjugates are relatively insensitive to quenching and to pH effects, and are expelled slowly from the cell, allowing the rate of fluorescence increase to be used to probe the dynamics of the intracellular reaction. Using low-light microscopic cytometry to monitor the initial rates of fluorescence increase for the GST-catalyzed reaction within IC-21 macrophages yields Vmax = 8.4 x 10(-16) mol s-1 cell-1 and KMCBm = 65 microM. Combining these data with an integrated Michaelis analysis of the reaction course yields KIP approximately 1.5 x 10(-5) M, and KmGSH approximately 3.0 x 10(-4) M (at [MCB] = 50 microM). The values of Vmax and KMCBm for the cell-free (extracellular) GST-catalyzed conjugation reaction are 1.2 x 10(-18) mol s-1 cell-1 and 3.1 microM, respectively. The values of Vmax for the intra- and extracellular conjugation reactions differ by 700-fold, suggesting the presence of an intracellular activator for this enzyme system.