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Ebola virus disease (EVD) represents a global health threat. The etiological agents of EVD are six species of Orthoebolaviruses, with Orthoebolavirus zairense (EBOV) having the greatest public health and medical significance. EVD pathogenesis occurs as a result of broad cellular tropism of the virus, robust viral replication and a potent and dysregulated production of cytokines. In vivo, tissue macrophages are some of the earliest cells infected and contribute significantly to virus load and cytokine production. While EBOV is known to infect macrophages and to generate high titer virus in the liver, EBOV infection of liver macrophages, Kupffer cells, has not previously been examined in tissue culture or experimentally manipulated in vivo. Here, we employed primary murine Kupffer cells (KC) and an immortalized murine Kupffer cell line (ImKC) to assess EBOV-eGFP replication in liver macrophages. KCs and ImKCs were highly permissive for EBOV infection and IFN-γ polarization of these cells suppressed their permissiveness to infection. The kinetics of IFN-γ-elicited antiviral responses were examined using a biologically contained model of EBOV infection termed EBOV ΔVP30. The antiviral activity of IFN-γ was transient, but a modest ~3-fold reduction of infection persisted for as long as 6 days post-treatment. To assess the interferon-stimulated gene products (ISGs) responsible for protection, the efficacy of secreted ISGs induced by IFN-γ was evaluated and secreted ISGs failed to block EBOV ΔVP30. Our studies define new cellular tools for the study of EBOV infection that can potentially aid the development of new antiviral therapies. Furthermore, our data underscore the importance of macrophages in EVD pathogenesis and those IFN-γ-elicited ISGs that help to control EBOV infection.

Plasmodium parasite-specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.

Filoviruses cause severe hemorrhagic fever in humans. The archetypal virus of this group, Ebola virus, is responsible for the current filovirus epidemic in West Africa. Filoviruses infect most mammalian cells, resulting in broad species tropism and likely contributing to rapid spread of virus throughout the body. A thorough understanding of filovirus entry events will facilitate the development of therapeutics against these critical steps in the viral life cycle. This review summarizes the current understanding of filovirus entry and discusses some of the recent advancements in therapeutic strategies that target entry.

Identification of host factors that are needed for Zaire Ebolavirus (EBOV) entry provides insights into the mechanism(s) of filovirus uptake, and these factors may serve as potential antiviral targets. In order to identify novel host genes and pathways involved in EBOV entry, gene array findings in the National Cancer Institute's NCI-60 panel of human tumor cell lines were correlated with permissivity for EBOV glycoprotein (GP)-mediated entry. We found that the gene encoding the γ2 subunit of AMP-activated protein kinase (AMPK) strongly correlated with EBOV transduction in the tumor panel. The AMPK inhibitor compound C inhibited infectious EBOV replication in Vero cells and diminished EBOV GP-dependent, but not Lassa fever virus GPC-dependent, entry into a variety of cell lines in a dose-dependent manner. Compound C also prevented EBOV GP-mediated infection of primary human macrophages, a major target of filoviral replication in vivo. Consistent with a role for AMPK in filovirus entry, time-of-addition studies demonstrated that compound C abrogated infection when it was added at early time points but became progressively less effective when added later. Compound C prevented EBOV pseudovirion internalization at 37°C as cell-bound particles remained susceptible to trypsin digestion in the presence of the inhibitor but not in its absence. Mouse embryonic fibroblasts lacking the AMPKα1 and AMPKα2 catalytic subunits were significantly less permissive to EBOV GP-mediated infection than their wild-type counterparts, likely due to decreased macropinocytic uptake. In total, these findings implicate AMPK in macropinocytic events needed for EBOV GP-dependent entry and identify a novel cellular target for new filoviral antivirals.

The Iowa Center for Research on Botanical Dietary Supplements seeks to optimize Echinacea, Hypericum, and Prunella botanical supplements for human-health benefit, emphasizing antiviral, anti-inflammatory and anti-pain activities. This mini-review reports on ongoing studies on Hypericum. The Center uses the genetically diverse, well-documented Hypericum populations collected and maintained at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS), and the strength of research in synthetic chemistry at Iowa State University to tap natural diversity, to help discover key constituents and interactions among constituents that impact bioactivity and toxicity. The NCRPIS has acquired more than 180 distinct populations of Hypericum, with a focus on Hypericum perforatum L. (Hypericaceae), representing about 13% of currently recognized taxa. Center chemists have developed novel synthetic pathways for key flavones, acyl phloroglucinols, hyperolactones and a tetralin that have been found in Hypericum, and these compounds are used as standards and for bioactivity studies. Both light-dependent and light-independent anti-viral activities have been identified by using bioactivity-guided fractionation of H. perforatum and a HIV-1 infection test system. Our Center has focused on light-independent activity, potentially due to novel chemicals, and polar fractions are undergoing further fractionation. Anti-inflammatory activity has been found to be light-independent, and fractionation of a flavonoid-rich extract revealed four compounds (amentoflavone, chlorogenic acid, pseudohypericin and quercetin) that interacted in the light to inhibit lipopolysaccharide-induced prostaglandin E(2) activity. The Center continues to explore novel populations of H. perforatum and related species to identify constituents and interactions of constituents that contribute to potential health benefits related to infection.

Ongoing studies have developed strategies for identifying key bioactive compounds and chemical profiles in Echinacea with the goal of improving its human health benefits. Antiviral and antiinflammatory-antipain assays have targeted various classes of chemicals responsible for these activities. Analysis of polar fractions of E. purpurea extracts showed the presence of antiviral activity, with evidence suggesting that polyphenolic compounds other than the known HIV inhibitor, cichoric acid, may be involved. Antiinflammatory activity differed by species, with E. sanguinea having the greatest activity and E. angustifolia, E. pallida, and E. simulata having somewhat less. Fractionation and studies with pure compounds indicate that this activity is explained, at least in part, by the alkamide constituents. Ethanol extracts from Echinacea roots had potent activity as novel agonists of TRPV1, a mammalian pain receptor reported as an integrator of inflammatory pain and hyperalgesia and a prime therapeutic target for analgesic and antiinflammatory drugs. One fraction from E. purpurea ethanol extract was bioactive in this system. Interestingly, the antiinflammatory compounds identified to inhibit prostaglandin E(2) production differed from those involved in TRPV1 receptor activation.

The rapid evolution of HIV drug resistance is a major cause of AIDS treatment failure. Superinfection, the infection of an already infected cell by additional virions, can be a major factor contributing to the evolution of drug resistance. However, the pattern and consequences of superinfection in HIV populations are far from fully understood. In this paper we study the implications of the fact that superinfection is regulated by HIV. We propose that superinfection is negatively associated with the success of the virus, so that more successful viruses are less likely to allow superinfection. We use computational models to investigate the effect that regulated superinfection would have on the evolution of drug resistance in HIV population. We find that regulated, fitness-associated superinfection can provide a distinct advantage to the virus in adapting to anti-HIV drugs in comparison with unregulated superinfection. Based on the results of the computational models and on current biological evidence, we suggest that the mechanism of fitness-associated regulation of coinfection in HIV is plausible, and that its investigation can lead to new ways to fight viral drug resistance.

Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb-HEXIM1-7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb-HEXIM1-7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

Lentiviruses vary in their dependence on a functional tat gene during their viral life cycle. To begin to understand the viral and cellular parameters controlling equine infectious anemia virus (EIAV) transactivation, we investigated Tat function and Tat and LTR structural requirements necessary for successful transactivation. EIAV Tat expression was required for detection of viral antigens from a full-length provirus. The level of transactivation by EIAV Tat as measured by LTR-CAT assays correlated well with viral antigen expression. Using horse/mouse somatic cell hybrids (SCH), a single SCH line which supported EIAV transactivation was identified, indicating that the presence of specific horse chromosomes provided cellular factors required for transactivation. Transformed cell lines from several different species were also tested and found to differ in their ability to support EIAV transactivation. A canine cell line, Cf2Th, which was permissive for EIAV transactivation, and a human cell line, HeLa, which was not permissive for EIAV transactivation, were used to map regions of the LTR and Tat that were important in cell-specific transactivation. As expected, the R region of EIAV LTR was required for transactivation by EIAV Tat in all cell lines studied. Similarly, the R region of HIV LTR was necessary for transactivation by HIV Tat. However, the composition of the U3 region also influenced transactivation in a cell-specific manner. In Cf2Th cells, replacement of EIAV U3 sequences with HIV U3 sequences resulted in high basal (nontransactivated) expression, and as a result, only a twofold increase in expression was observed in the presence of EIAV Tat. Similar studies using HIV Tat demonstrated that transactivation occurred in Cf2Th cells when either EIAV or HIV U3 sequences were present in the LTR. In contrast, transactivation by either HIV or EIAV Tat in HeLa cells required the presence of HIV enhancer sequences. These findings suggested that the ability of transactivation to occur in some cell lines may involve interactions between cell-specific transcription factors and the activation domain of Tat. For transactivation in other cell lines, Tat appeared to require more ubiquitious factors that interact with both EIAV and HIV U3 sequences.

Interferon gamma (IFN-gamma), a lymphokine that exerts multiple immunoregulatory effects, has been found to be elevated in the plasma, cerebrospinal fluid, and lymph nodes of human immunodeficiency virus (HIV)-infected individuals and has shown variable effects on HIV replication in acutely infected cells. In the present study, we have demonstrated that IFN-gamma is a potent modulator of HIV expression in persistently infected U1 promonocytic cells in which virus production is characterized by a constitutive state of relative latency. Direct stimulation of U1 cells with IFN-gamma (10-1,000 U/ml) activated HIV expression, as measured by reverse transcriptase (RT) activity in the culture supernatant and increased levels of cell-associated viral protein and mRNAs. These effects on virus expression were not accounted for by the induction of endogenous TNF-alpha secretion, as previously described in U1 cells stimulated with phorbol myristate acetate (PMA). At the ultrastructural level, the stimulatory activity of IFN-gamma was correlated with HIV particle production in intracytoplasmic vacuoles along with the differentiation of U1 into macrophage-like cells. Furthermore, costimulation of U1 cells with IFN-gamma and PMA significantly increased the accumulation of vacuole-associated HIV concomitant with decreasing membrane-associated particles and RT activity production, as compared with cells stimulated with PMA alone. No evidence of spontaneous secretion of intracellular vacuole-associated virus was obtained by kinetic analysis of the RT activity released in the supernatants throughout the culture period unless cells were deliberately disrupted. These findings suggest that vacuole-associated virions likely represent a relatively stable intracellular reservoir of HIV, as previously described in primary macrophages infected in vitro or in infected macrophages in the brains of patients with acquired immune deficiency syndrome. The reduced levels of RT activity observed in the culture supernatants of U1 cells stimulated with PMA in the presence of IFN-gamma were not indicative of a suppressive effect of IFN-gamma on PMA-induced expression of HIV proteins and mRNAs, either directly or mediated by the release of IFN-alpha/beta. This study suggests that IFN-gamma may play an important role as an inducer of HIV expression in infected mononuclear phagocytes.

Expression of human immunodeficiency virus type 1 (HIV-1) can be activated in a chronically infected T-cell line (ACH2 cells) by a cytokine, human tumor necrosis factor alpha (TNF-alpha). TNF-alpha treatment of ACH2 cells resulted in an increase in steady-state levels of HIV RNA and HIV transcription. Gel mobility shift assays demonstrated that the transcriptional activation of the HIV long terminal repeat (LTR) by TNF-alpha was associated with the induction of a nuclear factor(s) binding to the NF-kappa B sites in the LTR. Deletion of the NF-kappa B sites from the LTR eliminated activation by TNF-alpha in T cells transfected with plasmids in which the HIV LTR directed the expression of the bacterial chloramphenicol acetyltransferase gene. Thus, TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.

Addition of exogenous Mg(2+) (2 millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K(+). Addition of K(+) (10-50 millimolar) reversed both stromal acidification and K(+) efflux from the chloroplast caused by Mg(2+). These data suggested that Mg(2+) induced reversible H(+)/K(+) fluxes across the chloroplast envelope. Ca(2+) and Mn(2+) (2 millimolar) were as effective as 4 millimolar Mg(2+) in causing K(+) efflux from chloroplasts and inhibition of O(2) evolution. In contrast, 10 millimolar Ba(2+) induced only a small amount of inhibition. The lack of strong inhibition by Ba(2+) indicated that the effects of divalent cations such as Mg(2+) cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg(2+) was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O(2) evolution (43%) induced by Mg(2+).A variety of ATPase inhibitors were tested for effects on Mg(2+)-induced H(+)/K(+) fluxes. Oligomycin was the only ATPase inhibitor which specifically inhibited photosynthesis in the presence of Mg(2+) + K(+), but had little or no effect in the absence of these cations. In the presence of oligomycin, much higher concentrations (50 millimolar) of exogenous K(+) were required to reverse Mg(2+)-induced acidification and inhibition of O(2) evolution than in its absence. Oligomycin (in the absence of divalent cations) increased the inhibition of photosynthesis caused by sodium acetate, which acts by causing stromal acidification. In addition, the chloroplast envelope ATPase was inhibited partially (45%) by oligomycin. These results suggested that H(+) fluxes across the chloroplast envelope are regulated by two mechanisms: (a) an active, oligomycin-sensitive H(+) efflux and (b) a reversible, Mg(2+)-dependent, oligomycin-insensitive H(+)/K(+) exchange.