Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-␣Further, VP24 is found to specifically interact with karyopherin ␣1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin ␣2, ␣3, or ␣4. Overexpression of VP24 results in a loss of karyopherin ␣1-PY-STAT1 interaction, indicating that the VP24-karyopherin ␣1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.The filoviruses, Ebola virus (EBOV) and Marburg virus, cause periodic outbreaks of severe hemorrhagic fever in humans. In EBOV outbreaks consisting of more than 10 reported cases, mortality rates have ranged from 40 to 90% (41), and Marburg virus outbreaks have had reported case fatality rates ranging from 25 to 80% (13). This extreme virulence has made Ebola and Marburg viruses of concern both as naturally emerging pathogens and as potential bioweapons (41).The molecular mechanisms contributing to the severe pathogenesis of filovirus infection are poorly understood. Several potential mechanisms contributing to EBOV virulence have been reviewed (41). These include cytotoxicity of the viral glycoprotein, the production of proinflammatory cytokines, and the dysregulation of the coagulation cascade due to the production of tissue factor (14,20,21,62,64). Infection also appears to induce a general immune suppression (11, 53). Possible mechanisms contributing to this suppression include inhibition of dendritic cell activation and an induction of lymphocyte apoptosis (2,8,18,22,43). Each of these pathogenic processes likely occurs as a result of the active replication of the virus. Thus, the ability of the virus to counteract early antiviral responses, including those of the host's interferon system, likely plays an important role in EBOV virulence (41).EBOV encodes mechanisms to counteract the host interferon (IFN) response by blocking both production of IFN-␣/␤ and cellular responses to IFN-␣/␤ or -␥ treatment (6,24,26,27). We previously demonstrated that the EBOV VP35 protein suppresses IFN-␣/␤ production by inhibiting the activation of interferon regulatory factor 3 (IRF-3) (5, 7, 51), and subsequent studies confirm that VP35 exerts this function (8, 28). However, the manner in which EBOV blocks signaling from the IFN-␣/␤ or -␥ receptor has remained incompletely defined.IFN-␣/␤, a family of structurally related proteins, and IFN-␥ bind to two distinct receptors but activate similar signaling pathways (reviewed in reference 38). For both pathways, ligand binding activates receptor-associated Jak family tyrosine kinases. These undergo auto-and transphosphorylation and phosphorylate the cytoplasmic domains of the receptor subunits. The receptor-associated phosphotyrosine residues then serve as docking sites for the SH2 domains of STAT proteins. The receptor-associated STATs then undergo tyrosine-phosphorylation and form homo-or heterodimers via reciprocal SH2 domai...

Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient’s leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient’s dermal fibroblasts and induced pluripotent stem cell (iPSC)–derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.

Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing () cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either isoniazid or rifampicin prevents the formation of drug-tolerant and drug-resistant cells in cultures. This effect was concentration- and time-dependent, relying on increased oxygen consumption that triggered enhanced production of reactive oxygen species. In infected murine macrophages, the addition of-acetylcysteine to isoniazid treatment potentiated the killing of Furthermore, we demonstrate that the addition of small thiols to drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates respiration and converts persister cells to metabolically active cells. This prevention of both persister cell formation and drug resistance leads ultimately to mycobacterial cell death. Strategies to enhance respiration and initiate oxidative damage should improve tuberculosis chemotherapies.

Daily phagocytosis of spent photoreceptor outer segments is a critical maintenance function performed by the retinal pigment epithelium (RPE) to preserve vision. Aging RPE accumulates lipofuscin, which includes N-retinylidene-N-retinylethanolamine (A2E) as the major autofluorescent component. We studied the effect of physiological levels of A2E in RPE cultures on their ability to phagocytose outer segments. A2E localized to lysosomes in cultured RPE as well as in human RPE in situ. A2E-loaded RPE cells in culture bound and internalized identical numbers of outer segments as control RPE indicating that A2E does not alter early steps of phagocytosis. A2E-loaded RPE degraded outer segment proteins efficiently but, strikingly, failed to completely digest phospholipids within 24 h. Because of the circadian rhythm of RPE phagocytosis in the eye, a delay in lipid degradation would likely result in a build up of undigested material in RPE that could contribute to the development of age-related macular degeneration.A ge-related macular degeneration (ARMD) is a degenerative disease that causes severe visual impairment in one fourth of the population over 65. Some predisposing genetic and environmental factors, such as oxidative stress, low eye pigmentation, excessive light exposure, and smoking, have been identified (1-3). However, the mechanisms underlying the pathogenesis of the disease remain largely obscure.Clinical and histopathological analysis of diseased eyes suggests that alterations in the retinal pigment epithelium (RPE) are key determinants of the disease (4). The most profound change of human RPE with age is the accumulation in the cytoplasm of lipofuscin granules, storage bodies with an autofluorescent mixture of lipids arising from incomplete digestion of phagocytosed photoreceptor (PR) outer segment (OS) membranes (5-7). One of the major components of RPE lipofuscin has been characterized as N-retinylidene-N-retinylethanolamine (A2E), a pyridinium bis-retinoid and photoinducible free-radical generator (8, 9). While human RPE in general accumulates lipofuscin with age, excessive lipofuscin accumulation is associated with a variety of hereditary retinal degenerations (10-12) and may correlate with ARMD (4).The identification and efficient synthetic production of A2E have enabled studies on the consequences of lipofuscin accumulation for RPE function (8, 9, 13). The chemical structure of A2E indicates that it may act as a lysosomotropic agent, abolishing the acidic pH gradient required for normal lysosome function. Indeed, Holz et al. reported that A2E accumulation alone is sufficient to inhibit the lysosomal turnover of endogenous proteins in cultured RPE (14). On the other hand, A2E did not directly reduce activities of a wide range of lysosomal hydrolases in in vitro assays (15). A2E can initiate apoptotic cell death in RPE and other cell types (16, 17) when present at high levels. None of the studies mentioned above measured cytoplasmic levels of A2E. In fact, at concentrations similar to those found in a...

The Ebolavirus VP24 protein counteracts alpha/beta interferon (IFN-␣/␤) and IFN-␥ signaling by blocking the nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1). According to the proposed model, VP24 binding to members of the NPI-1 subfamily of karyopherin alpha (KPN␣) nuclear localization signal receptors prevents their binding to PY-STAT1, thereby preventing PY-STAT1 nuclear accumulation. This study now identifies two domains of VP24 required for inhibition of IFN-␤-induced gene expression and PY-STAT1 nuclear accumulation. We demonstrate that loss of function correlates with loss of binding to KPN␣ proteins. Thus, the VP24 IFN antagonist function requires the ability of VP24 to interact with KPN␣.In the order Mononegavirales, Ebolavirus and Marburgvirus belong to the family Filoviridae (12). Ebolaviruses are responsible for outbreaks in central Africa of severe hemorrhagic fever in humans and nonhuman primates, with human fatality rates of up to 90% (16). Currently, there are no licensed vaccines or approved treatments available for filovirus infections. Ebolavirus counteracts synthesis of alpha/beta interferon (IFN-␣/␤) and cellular responses to IFN-␣/␤/␥ (7,8,10,11,23). VP35 blocks production of IFN-␣/␤ by inhibiting the activation of interferon regulatory factors by blocking interferon regulatory factor 3 (IRF-3) kinases IKKepsilon and TBK-1 (1, 2, 21) and by increasing SUMOylation of IRF-7 (3). VP24 impairs cellular responses to IFN-␣/␤/␥ by blocking nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1). Nuclear translocation of PY-STAT1 is essential for transcriptional activation of numerous IFN-responsive genes and occurs through interaction with karyopherin ␣-1 (KPN␣1) and perhaps other members of the NPI-1 subfamily of KPN␣ proteins (14,15,18,19,22,25). Correspondingly, VP24 interacts with the KPN␣ proteins that mediate PY-STAT1 nuclear accumulation (23, 24). Other pathogenic viruses also impair innate immune signaling via interaction with KPN␣ proteins, suggesting that this may be a common immune evasion strategy. For example, the severe acute respiratory syndrome coronavirus (SARS-CoV) ORF6 protein tethers a karyopherin ␣-2/karyopherin ␤ complex to the endoplasmic reticulum (ER)/Golgi membrane to prevent PY-STAT1 nuclear import complex formation (6), and Hantaan virus nucleocapsid protein inhibits NF-B activation through interaction with KPN␣ (26). Our previous work suggests that the interaction of VP24 with select KPN␣ proteins is responsible for the ability of VP24 to inhibit IFN signaling (23,24).In this study, mutations that impair VP24 inhibition of IFNinduced gene expression were identified. Plasmids expressing wild-type or mutated VP24 proteins were cotransfected into 293T cells together with plasmids expressing Renilla luciferase (expressed from a constitutive cytomegalovirus [CMV] promoter) and firefly luciferase (expressed from an IFN-inducible ISG54 promoter). At 1 day posttransfection, the cells were treated with 1,000 U/ml of human IFN-␤ (PBL Interferon Source) fo...