Virology. Mutation rate and genotype variation of Ebola virus from Mali case sequences.

The occurrence of Ebola virus (EBOV) in West Africa during 2013-2015 is unprecedented. Early reports suggested that in this outbreak EBOV is mutating twice as fast as previously observed, which indicates the potential for changes in transmissibility and virulence and could render current molecular diagnostics and countermeasures ineffective. We have determined additional full-length sequences from two clusters of imported EBOV infections into Mali, and we show that the nucleotide substitution rate (9.6 × 10(-4) substitutions per site per year) is consistent with rates observed in Central African outbreaks. In addition, overall variation among all genotypes observed remains low. Thus, our data indicate that EBOV is not undergoing rapid evolution in humans during the current outbreak. This finding has important implications for outbreak response and public health decisions and should alleviate several previously raised concerns.

Identification of alpha interferon-induced genes associated with antiviral activity in Daudi cells and characterization of IFIT3 as a novel antiviral gene.

A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products.

Prolonged STAT1 activation related to the growth arrest of malignant lymphoma cells by interferon-alpha.

Multiple biologic effects of interferon-alpha (IFN-alpha), including cell growth inhibition and antiviral protection, are initiated by tyrosine phosphorylation of STAT proteins. Although this signal pathway has been intensively investigated, the relevance of STAT signal persistence has received scant attention. Using paired isogenic lymphoma cells (Daudi), which either are sensitive or resistant to growth inhibition by IFN-alpha, we found comparable initial tyrosine phosphorylation of multiple STAT proteins; however, the phosphorylation durations and associated DNA-binding activities diverged. Phosphorylation and DNA-binding capacity of STAT1 decreased after 4 to 8 hours in resistant cells, as compared with 24 to 32 hours in sensitive cells, whereas phosphorylation of STAT3 and STAT5b was briefer in both lines. Functional significance of the prolonged STAT1 signal, therefore, was explored by experimental interruption of tyrosine phosphorylation, either by premature withdrawal of the IFN-alpha or deferred addition of pharmacologically diverse antagonists: staurosporine (protein kinase inhibitor), phorbol 12-myristate 13-acetate (growth promoter), or aurintricarboxylic acid (ligand competitor). Results indicated that an approximately 18-hour period of continued STAT1 phosphorylation was associated with growth arrest, but that antiviral protection developed earlier. These differences provide novel evidence of a temporal dimension to IFN-alpha signal specificity and show that duration of STAT1 activation may be a critical variable in malignant cell responsiveness to antiproliferative therapy.

Priming of human monocytes for enhanced lipopolysaccharide responses: expression of alpha interferon, interferon regulatory factors, and tumor necrosis factor.

Culture of human monocytes with either granulocyte-macrophage colony-stimulating factor or gamma interferon (IFN-gamma) results in a primed state, during which these cells express heightened responses to bacterial lipopolysaccharide (LPS). The production of IFN-alpha in response to LPS by human monocytes has an absolute requirement for priming. Tumor necrosis factor (TNF) expression is also greatly enhanced in primed monocytes after LPS stimulation, but unlike IFN-alpha, TNF is readily expressed in unprimed monocytes as well. In an effort to determine the molecular events associated with IFN-alpha induction in this system, freshly isolated human monocytes were primed by culture with either IFN-gamma or granulocyte-macrophage colony-stimulating factor and then treated with LPS; expression of IFN-alpha subtype 2 (IFN-alpha 2), IFN regulatory factors (IRFs), and TNF was assessed by Northern (RNA blot) analysis. IRF-1 mRNA is expressed at high levels in monocytes and is regulated by both LPS and priming cytokines, but its expression alone does not correlate with the induction of IFN-alpha 2 expression. IRF-2 mRNA is expressed in a more gradual manner following LPS stimulation, implying a possible feedback mechanism for inhibiting IFN-alpha expression. However, nuclear run-on analysis indicates that IFN-alpha 2 is not transcriptionally modulated in this system, in striking contrast to TNF, which is clearly regulated at the transcriptional level. In addition, IFN-alpha 2 mRNA accumulation is superinduced when primed monocytes are treated with LPS plus cycloheximide, while TNF mRNA is relatively unaffected. The results demonstrate that priming can affect subsequent LPS-induced gene expression at different levels in human monocytes.

Evidence for multiple binding sites for several components of human lymphoblastoid interferon-alpha.

The antiproliferative and competitive binding activities of 20 purified components of human lymphoblastoid interferon (IFN)-alpha were compared with that of recombinant human IFN-alpha 2b on Daudi and AU937 cells. We observed broad ranges of antiproliferative activities of the IFN-alpha components on these cell lines. Daudi cells were more sensitive to the IFN-alpha components than AU937 cells; the concentrations of the components that resulted in 50% inhibition of cell growth ranged from 0.003 ng/ml to > 10 ng/ml on Daudi cells and 0.05 ng/ml to > 200 ng/ml on AU937 cells. Component o was the most active human IFN-alpha on both cell lines. Scatchard analysis demonstrated that the number of IFN-alpha 2b binding sites is greater on Daudi cells (12,700 binding sites/cell) than AU937 cells (3,300 binding sites/cell); however, their receptor affinities were similar. Component o, which exhibited high antiproliferative activity on both cell lines, had low affinity for the IFN-alpha 2b binding site on AU937 and Daudi cells. Several human IFN-alpha s (components b', b", and e) appeared to have high affinity binding sites but low antiproliferative activity on both Daudi and AU937 cells. These data suggest that there may be more than one binding site or receptor for human IFN-alpha, and/or there may be a multicomponent receptor involved in the biological actions of these molecules.

Purification and characterization of multiple components of human lymphoblastoid interferon-alpha.

Twenty-two components of human interferon-alpha (IFN-alpha) derived from Sendai virus-induced Namalwa cells were purified by sequential immunoadsorbent affinity chromatography using four monoclonal antibody affinity columns followed by ultrafiltration and reversed-phase high-performance liquid chromatography. The specific activity ranged from 0.2 to 2.6 x 10(8) IU/mg protein on Madin-Darby bovine kidney cells, 0.3 to 4.6 x 10(8) IU/mg protein on human WISH cells, and 10(4) to 7 x 10(5) units/mg protein on mouse L929 cells. The apparent molecular weights of the components ranged from 17,500 to 23,300 using nonreducing sodium dodecyl polyacrylamide-gel electrophoresis and 17,500 to 27,600 using reducing sodium dodecyl polyacrylamide-gel electrophoresis. The amino-terminal amino acid sequences were similar among the components as well as to those reported for the cloned human IFN-alpha genes (Pestka, S. (1986) Methods Enzymol. 119, 3-14). However, four components, f, i, l, and m, have amino-terminal amino acid sequences which appear to be unique when compared to those predicted from the cDNA clones. One component, pre-a, has a potential N-linked glycosylation site on the Asn of residues 2 through 4, Asn-Leu-Ser.

Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production.

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.

Aurin tricarboxylic acid, the anti-AIDS compound, prevents the binding of interferon-alpha to its receptor.

Binding of HIV to its receptor, the CD4 molecule of lymphocytes, can be prevented by chemical agents. These agents could be considered as potential anti-AIDS drugs. We have shown that aurin tricarboxylic acid (ATA, 3 microM) specifically blocks the binding of gp120, the HIV coat protein, to the CD4 molecule. We have also found that ATA prevents the binding of interferon-alpha to its receptor in a dose-dependent manner (12-50 microM range). Membrane potential shift, associated with binding of interferon-alpha to its receptor, was also blocked by ATA in a dose-dependent fashion. Our results indicate that potential anti-AIDS drugs should be screened for such undesired side effects.