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Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC50 = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.

Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here a novel class of allosteric inhibitor of NS5B that shows potent affinity for the NS5B enzyme and effective inhibition of subgenomic HCV RNA replication in HUH-7 cells. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.

We investigated the effects of recombinant interleukin-3 (IL-3), granulocyte-macrophage and granulocyte colony-stimulating factors (GM- CSF and G-CSF), and erythropoietin (Ep) on the number of human hematopoietic progenitors after two to ten days of incubation in liquid cultures deprived of fetal bovine serum (FBS). The source of progenitor cells was normal human marrow depleted of T lymphocytes and/or adherent cells. When adherent cell-depleted marrow was cultured without growth factors, the number of progenitor cells was relatively constant for periods up to eight days. In contrast, a progressive decline in the number of progenitor cells was detected in cultures of nonadherent, T- cell-depleted marrow cells. In both cases, the addition of IL-3 increased by two- to fourfold over input the number of erythroid burst- forming cells (BFU-E) per culture. The number of BFU-E peaked either at day 4 or 8. G-CSF had no effect on the number of progenitor cells per culture. GM-CSF and Ep had no effect in cultures of nonadherent marrow cells but maintained the number of BFU-E in cultures of nonadherent, T- cell-depleted marrow cells. The addition of a neutralizing anti-GM-CSF monoclonal antibody, but not anti-IL-3 neutralizing antiserum, decreased the number of BFU-E in cultures of nonadherent marrow cells. None of the growth factors investigated enhanced the number of GM progenitors to the same degree as the number of BFU-E. However, in cultures of nonadherent, T-cell-depleted marrow cells, IL-3 and GM-CSF maintained the number of GM progenitors up to eight days. These results indicate that IL-3 alone is capable of increasing the number of BFU-E and of maintaining the number of GM progenitors in liquid culture, whereas GM-CSF and Ep are capable of maintaining, but not increasing, BFU-E in this system.

Gamma-globin chain synthesis has been evaluated in individual bursts and subcolonies that were generated by normal adult blood BFU-Es in methylcellulose cultures containing semipurified erythropoietin (Ep) and then analyzed via either isoelectric focusing (IEF) of globin chains or immunofluorescence techniques. At variance with previously reported results, based on plasma clot culture and immunofluorescence, all bursts and subcolonies analyzed synthesize gamma-globin chains. Identification of gamma-chains has been confirmed by preparative IEF of HbF, followed by either carboxymethylcellulose chromatography or IEF analysis of the resulting globin chains. In all bursts analyzed, the relative synthesis of the two types of gamma-globin chains (G gamma and A gamma) shows an adult ratio (i.e., approximately 1:1). The results obtained via IEF have been confirmed by immunofluorescence studies, which apparently showed presence of at least some HbF-positive cells within all scrutinized bursts or subcolonies. The significance of these studies is discussed in the light of current hypotheses on mechanism(s) underlying HbF synthesis in normal adults.

Addition of prostaglandins of the E series (PGE1, PGE2) in methylcellulose cultures of murine marrow results in a dose-dependent inhibition of the cloning efficiency of both BFU-E and CFU-C. However, CFU-E growth is unaffected. The inhibitory action of PGE is progressively overcome by increasing amounts of colony-stimulating factor (CSF), and with some limitations, also of erythropoietin (Ep). Addition of PGF2 alpha' associated or not with indomethacin, does not exert any significant effect on these hemopoietic precursors. In an attempt to unvail the mechanism(s) underlying these phenomena, dibutyryl-cyclic AMP (db-cAMP), theophylline (an inhibitor of phosphodiesterase), or theophylline + PGE were plated at various concentrations. Both db-cAMP and theophylline induce an inhibitory influence on both BFU-E and CFU-C growth, which mimicks that by PGEs; additionally, theophylline potentiates the inhibitory action of PGE1. In all these studies, the CFU-E number was not significantly modified. PGE action on BFU-E proliferation is clearly species-dependent, since PGE1 addition to human marrow methylcellulose cultures induces a significant enhancement of the number of both BFU-E and CFU-E derived colonies. This action was abolished upon removal of adherent cells, thus suggesting that PGE1 evokes a release of factor(s) enhancing human erythroid colony growth by adherent cells.

Haemoglobin switching in humans provides a unique model for investigating the mechanisms underlying expression of a develop-mentally regulated gene family. Numerous studies have focused on the switch from fetal to adult (that is, γ → β) globin1,2, but little is known about the embryonic → fetal (that is, ζ → α and ε → γ) switches, as well as the transition from ‘primitive’ yolk sac to ‘definitive’ liver erythropoiesis3–7. Here we have studied the embryonic→fetal haemoglobin switches in yolk sac, liver and circulating blood erythroblasts from 25 embryos and 6 fetuses. Globin synthesis was also evaluated in purified ‘primitive’ and ‘definitive’ erythroblasts. Primitive erythroblasts synthesize essentially ζ and ε chains at 5 weeks and α- and ε-globin with a minor aliquot of ζ and γ chains at 6–7 weeks, whereas definitive erythroblasts produce α and γ + β-globin at 6 weeks but only α and γ + β chains from 8 weeks onward. In both lineages the ζ → α and the ε → γ switches are asynchronous, the former preceding the latter. Furthermore, ζ- and β-globin synthesis is restricted to primitive and definitive erythroblasts respectively. These findings are discussed in terms of a monoclonal model for haemoglobin switching in early human ontogeny.

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.

Under serum-deprived and chemically defined culture conditions, the growth of Rauscher erythroleukemia cells is mediated by an autocrine mechanism. The growth-promoting activity is produced by fresh or irradiated cells and resembles the activity of interleukin-3 (IL-3) in its ability to sustain colony formation from three of four IL-3- dependent cell lines and to induce formation of granulocyte/macrophage (GM) colonies and, in the presence of erythropoietin (Ep), of erythroid bursts and mixed erythroid colonies. IL-3, IL-1, IL-4, IL-6, GM colony- stimulating factor (GM-CSF), G-CSF, M-CSF, Ep, and media conditioned by concanavalin A-stimulated mouse spleen cells or phytohemagglutinin- stimulated LBRM 33 cells were unable to induce proliferation of the Rauscher erythroleukemia cells. Northern analysis of total and polyA- selected RNA extracted from untreated Rauscher cells or from cells 24 hours after irradiation showed the presence of message for M-CSF but not for IL-3, IL-1, GM-CSF, or G-CSF. The production of IL-6 was excluded by a sensitive bioassay. These results indicate that the autocrine growth of the Rauscher cell line is mediated by a growth factor different from IL-3, but with similar biological activity. Activation of the expression of such a growth factor during viral infection may contribute to the generation of leukemic cells that have the property to grow in vitro and generate Rauscher erythroleukemia cell lines.

The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.

The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.

We report that bromodeoxyuridine (BrdU) addition in semi-solid cultures of normal adult erythroid progenitors causes a sharp rise of gamma- globin gene expression in erythroid colonies. Control studies were carefully carried out to exclude the possibility of toxic effects exerted by the drug in these experimental conditions. In particular, BrdU addition induces a sharp increase in the level of relative gamma- globin synthesis and content in pooled BFU-E-derived colonies: this rise is clearly observed in single bursts of the mature type (largely composed of late erythroblasts) but not in immature ones (essentially comprising early erythroblasts). Furthermore, it is associated with an increase of the G gamma/G gamma + A gamma synthetic ratio from adult up to fetal like values. Reactivation of gamma-synthesis was observed even if BrdU was added to colonies composed essentially of early erythroblasts, ie, when BrdU was added to either bursts at day 10 of culture or late CFU-E-derived clones at day 1. These in vitro observations indicate modulation of gamma-synthesis at the stage of erythroblasts from normal adults. At the molecular level we suggest that BrdU, by replacing thymidine in DNA, may inhibit the switch from a fetal-like biosynthetic program expressed in early erythroblastic differentiation to the adult program expressed in later stages of maturation.

Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold-conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane.

Gamma-globin chain synthesis has been evaluated in individual bursts and subcolonies that were generated by normal adult blood BFU-Es in methylcellulose cultures containing semipurified erythropoietin (Ep) and then analyzed via either isoelectric focusing (IEF) of globin chains or immunofluorescence techniques. At variance with previously reported results, based on plasma clot culture and immunofluorescence, all bursts and subcolonies analyzed synthesize γ-globin chains. Identification of γ-chains has been confirmed by preparative IEF of HbF, followed by either carboxymethylcellulose chromatography or IEF analysis of the resulting globin chains. In all bursts analyzed, the relative synthesis of the two types of γ-globin chains (Gγ and Aγ) shows an adult ratio (i.e., approximately 1:1). The results obtained via IEF have been confirmed by immunofluorescence studies, which apparently showed presence of at least some HbF-positive cells within all scrutinized bursts or subcolonies. The significance of these studies is discussed in the light of current hypotheses on mechanism(s) underlying HbF synthesis in normal adults.

Palmitylation of vesicular stomatitis virus G and Sindbis virus E1 glycoproteins has been studied in relation to the transport from the endoplasmic reticulum (ER) to the Golgi complex. Incubation of infected cells at 15 °C prevents the transport of newly synthesized membrane proteins from the ER to the Golgi (Saraste, J., and Kuismanen, E. (1984) Cell 38, 535–549). In these conditions, also palmitylation of G protein and of E1 glycoprotein is blocked. When the transport is restored by increasing the temperature, palmitylation occurs quickly and is followed by the complete trimming of peripheral mannose residues due to mannosidase I (a putative cis-Golgi function). Immunofluorescence analysis showed that the G glycoprotein accumulated at 15 °C in structures distinct from both ER and Golgi. These studies suggest that transport from the ER to the cis-Golgi involves intermediate compartments.

We obtained Sindbis defective-interfering particles by nine and undiluted passages of standard virus on chicken embryo fibroblasts. These particles contain a deleted 20S RNA molecule which has mRNA activity, as shown by translation in cell-free systems in vitro. In infected cells, this mRNA activity appeared to be totally inhibited except at very late times postinfection.

The influence of recombinant erythropoietin (Ep) and interleukin-3 (IL- 3) on the proliferation and differentiation of murine hematopoietic stem and progenitor cells was investigated in serum-deprived cultures. The differentiation of progenitor cells, purified by collecting blast cell colonies from spleen cell cultures of 5-fluorouracil-treated mice, was evaluated by scoring the number and type of colonies appearing after eight days in semisolid culture. IL-3 induced the formation of both erythroid and granulocyte-macrophage colonies in a concentration- dependent fashion, the plateau being reached at 300 U/mL. However, concentrations of IL-3 alone that had little or no effect (less than or equal to 10 U/mL) induced maximal numbers of erythroid bursts in the presence of Ep (1.5 IU/mL). In the presence of Ep alone, no colonies were seen. Proliferation of quiescent hematopoietic stem cells, purified by cell sorting and evaluated by spleen colony assay (CFU-S), was investigated by measuring the total cell number and CFU-S content and the DNA histogram at 20 and 48 hours of liquid culture. Almost no cells or CFU-S survived 20 hours of incubation without the addition of IL-3. The presence of either IL-3 (400 U/mL) or the combination of EP and IL-3 (10 U/mL), supported the maintenance of nearly 40% of sorted CFU-S for 48 hours. Approximately 10% of these cells were in the S phase of the cell cycle at 20 hours and an increase in the total cell number per culture, but not in the CFU-S content, was detected at 48 hours. These data indicate that IL-3 exerts a differentiative and proliferative effect on early stem and progenitor cells, which is concentration dependent. At IL-3 concentrations, which had little or no activity alone, Ep acted synergistically to induce both proliferation of stem cells and differentiation of erythroid progenitors.

Erythroid bursts from cord or adult blood were grown in methylcellulose cultures (3 international units of erythropoietin per plate). On day 13, single bursts were picked up and reincubated for 16-24 hr with [3H]leucine. Radioactive globin chains [alpha,beta,G gamma, and A gamma (Ala-136)] were analyzed by either isoelectric focusing on polyacrylamide gels and fluorography or carboxymethylcellulose chromatography. In all cases, alpha to non-alpha globin radioactivity ratios were close to 1. In single cord blood bursts, the values of both gamma-to-beta and G gamma-to-A gamma ratios were spread over a large spectrum and further characterized by a continuous rather than a bimodal distribution. Morever, the G gamma-to-A gamma ratios demonstrated in single bursts appeared to be directly correlated with the respective gamma-to-beta ratios. These data suggest that both the gamma leads to beta and the G gamma leads to A gamma switches are mediated via mechanisms modulating the relative activities of the different genes in the non-alpha globin gene cluster rather than via selection of clones committed to the preferential synthesis of beta and A gamma globins. In contrast with the results obtained with cord blood, individual adult blood bursts synthesize a lower and hence relatively more uniform amount of gamma globin chains.

The synthesis of embryonic (zeta, epsilon), fetal (alpha, gamma), and adult (beta) globin was evaluated in human yolk sacs (YS) and livers at different ontogenic stages (i.e., from 6 through 10-12 wk of age) by means of analytical isoelectric focusing. Globin production was comparatively evaluated in vivo (i.e., in directly labeled erythroblasts from YS and liver) and in vitro [i.e., in erythroid bursts generated in culture by erythroid burst-forming units (BFU-E) from the same erythropoietic tissues]. Erythroid bursts generated in vitro by BFU-E from 6-wk livers and YS show essentially a "fetal" globin synthetic pattern: this is in sharp contrast to the "embryonic" pattern in corresponding liver and YS erythroblasts directly labeled in vivo. The invitro phenomenon suggests that (i) 6-wk BFU-E constitute a new generation of progenitors, which have already switched from an embryonic to a fetal program, and/or (ii) expression of their fetal program is induced by unknown in vitro factor(s), which may underlie the in vivo switch at later ontogenic stages. It is emphasized that 6- to 7-wk BFU-E are endowed with the potential for in vitro synthesis of not only epsilon- and gamma-chains but also some beta-globin. In general, we observed an inverse correlation between the levels of epsilon- and beta-chain synthesis. These results, together with previous studies on fetal, perinatal, and adult BFU-E, are compatible with models suggesting that in ontogeny the chromatin configuration is gradually modified at the level of the non-alpha gene cluster, thus leading to a 5'----3' activation of globin genes in a balanced fashion.