Lawrence W. Leung

Ebola Virus VP24 Binds Karyopherin α1 and Blocks STAT1 Nuclear Accumulation

ABSTRACT Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-α/β) and the ability of cells to respond to IFN-α/β or IFN-γ. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-α/β production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-α/β and IFN-γ signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-α/β and IFN-γ signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin α1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin α2, α3, or α4. Overexpression of VP24 results in a loss of karyopherin α1-PY-STAT1 interaction, indicating that the VP24-karyopherin α1 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.

Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency

A genetic cause for severe influenza Although chicken soup and plenty of rest get most kids through an influenza virus infection, some require hospitalization. Ciancanelli et al. report on one child who suffered severely from influenza because of null mutations in the gene for transcription factor IRF7. Cells isolated from this patient could not make enough secreted antiviral proteins, called interferons, to halt viral replication. The requirement for IRF7 seems quite specific, because this patient recovers normally from other common childhood viral infections. Science, this issue p. 448 A mutation that reduces antiviral interferons underlies certain cases of severe influenza in children. 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.

Ebolavirus VP24 binding to karyopherins is required for inhibition of interferon signaling.

ABSTRACT The Ebolavirus VP24 protein counteracts alpha/beta interferon (IFN-α/β) and IFN-γ 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 (KPNα) 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-β-induced gene expression and PY-STAT1 nuclear accumulation. We demonstrate that loss of function correlates with loss of binding to KPNα proteins. Thus, the VP24 IFN antagonist function requires the ability of VP24 to interact with KPNα.

Novel Inhibitors of InhA Efficiently Kill Mycobacterium tuberculosis under Aerobic and Anaerobic Conditions

ABSTRACT Drug resistance in Mycobacterium tuberculosis has become a serious global health threat, which is now complicated by the emergence of extensively drug-resistant strains. New drugs that are active against drug-resistant tuberculosis (TB) are needed. We chose to search for new inhibitors of the enoyl-acyl carrier protein (ACP) reductase InhA, the target of the first-line TB drug isoniazid (also known as isonicotinoic acid hydrazide [INH]). A subset of a chemical library, composed of 300 compounds inhibiting Plasmodium falciparum enoyl reductase, was tested against M. tuberculosis. Four compounds were found to inhibit M. tuberculosis growth with MICs ranging from 1 μM to 10 μM. Testing of these compounds against M. tuberculosis in vitro revealed that only two compounds (CD39 and CD117) were bactericidal against drug-susceptible and drug-resistant M. tuberculosis. These two compounds were also bactericidal against M. tuberculosis incubated under anaerobic conditions. Furthermore, CD39 and CD117 exhibited increased bactericidal activity when used in combination with INH or rifampin, but CD39 was shown to be toxic to eukaryotic cells. The compounds inhibit InhA as well the fatty acid synthase type I, and CD117 was found to also inhibit tuberculostearic acid synthesis. This study provides the TB drug development community with two chemical scaffolds that are suitable for structure-activity relationship study to improve on their cytotoxicities and bactericidal activities in vitro and in vivo.

Ebolavirus VP35 suppresses IFN production from conventional but not plasmacytoid dendritic cells

Ebolaviruses naturally infect a wide variety of cells including macrophages and dendritic cells (DCs), and the resulting cytokine and interferon‐α/β (IFN) responses of infected cells are thought to influence viral pathogenesis. The VP35 protein impairs RIG‐I‐like receptor‐dependent signaling to inhibit IFN production, and this function has been suggested to promote the ineffective host immune response characteristic of ebolavirus infection. To assess the impact of VP35 on innate immunity in biologically relevant primary cells, we used a recombinant Newcastle disease virus encoding VP35 (NDV/VP35) to infect macrophages and conventional DCs, which primarily respond to RNA virus infection via RIG‐I‐like pathways. VP35 suppressed not only IFN but also tumor necrosis factor (TNF)‐α secretion, which are normally produced from these cells upon NDV infection. Additionally, in cells susceptible to the activity of VP35, IRF7 activation is impaired. In contrast, NDV/VP35 infection of plasmacytoid DCs, which activate IRF7 and produce IFN through TLR‐dependent signaling, leads to robust IFN production. When plasmacytoid DCs deficient for TLR signaling were infected, NDV/VP35 was able to inhibit IFN production. Consistent with this, VP35 was less able to inhibit TLR‐dependent versus RIG‐I‐dependent signaling in vitro. These data demonstrate that ebolavirus VP35 suppresses both IFN and cytokine production in multiple primary human cell types. However, cells that utilize the TLR pathway can circumvent this inhibition, suggesting that the presence of multiple viral sensors enables the host to overcome viral immune evasion mechanisms.

The role of antigen-presenting cells in filoviral hemorrhagic fever: gaps in current knowledge

Abstract The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), are highly lethal zoonotic agents of concern as emerging pathogens and potential bioweapons. Antigen-presenting cells (APCs), particularly macrophages and dendritic cells, are targets of filovirus infection in vivo. Infection of these cell types has been proposed to contribute to the inflammation, activation of coagulation cascades and ineffective immune responses characteristic of filovirus hemorrhagic fever. However, many aspects of filovirus–APC interactions remain to be clarified. Among the unanswered questions: What determines the ability of filoviruses to replicate in different APC subsets? What are the cellular signaling pathways that sense infection and lead to production of copious quantities of cytokines, chemokines and tissue factor? What are the mechanisms by which innate antiviral responses are disabled by these viruses, and how may these mechanisms contribute to inadequate adaptive immunity? A better understanding of these issues will clarify the pathogenesis of filoviral hemorrhagic fever and provide new avenues for development of therapeutics.

Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis

Significance Tuberculosis (TB) patients would greatly benefit from shorter treatment options. The treatment of drug-susceptible TB, a disease caused by the bacillus Mycobacterium tuberculosis, is a lengthy and strenuous process. This long therapy is because of the ability of a small population of cells to become drug-tolerant. Here, we demonstrate that the addition of small thiols to drug-treated M. tuberculosis prevents the emergence of drug-tolerant but also drug-resistant cells leading to sterilization of the cultures in vitro. The thiols potentiate drug activity by preventing the cells from entering a persister state and shutting down their metabolism while generating an oxidative burst. This dual mechanism of killing could lead to novel approaches to shorten TB chemotherapy. 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 Mycobacterium tuberculosis (Mtb) 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 Mtb 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 N-acetylcysteine to isoniazid treatment potentiated the killing of Mtb. Furthermore, we demonstrate that the addition of small thiols to Mtb drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates Mtb 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.

A Five-Amino-Acid Deletion of the Eastern Equine Encephalitis Virus Capsid Protein Attenuates Replication in Mammalian Systems but Not in Mosquito Cells

ABSTRACT Eastern equine encephalitis virus (EEEV) is a human and veterinary pathogen that causes sporadic cases of fatal neurological disease. We previously demonstrated that the capsid protein of EEEV is a potent inhibitor of host cell gene expression and that this function maps to the amino terminus of the protein. We now identify amino acids 55 to 75, within the N terminus of the capsid, as critical for the inhibition of host cell gene expression. An analysis of stable EEEV replicons expressing mutant capsid proteins corroborated these mapping data. When deletions of 5 to 20 amino acids within this region of the capsid were introduced into infectious EEEV, the mutants exhibited delayed replication in Vero cells. However, the replication of the 5-amino-acid deletion mutant in C710 mosquito cells was not affected, suggesting that virus replication and assembly were affected in a cell-specific manner. Both 5- and 20-amino-acid deletion mutant viruses exhibited increased sensitivity to interferon (IFN) in cell culture and impaired replication and complete attenuation in mice. In summary, we have identified a region within the capsid protein of EEEV that contributes to the inhibition of host gene expression and to the protection of EEEV from the antiviral effects of IFNs. This region is also critical for EEEV pathogenesis.

A Mutation in the Ebola Virus Envelope Glycoprotein Restricts Viral Entry in a Host Species- and Cell-Type-Specific Manner

ABSTRACT Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans. A single viral glycoprotein (GP) mediates viral attachment and entry. Here, virus-like particle (VLP)-based entry assays demonstrate that a GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral entry into mouse CD11b+ APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88, including L111, I113, L122, and F225. GP mutants in which these residues are mutated to alanine displayed preferential and often impaired entry into several cell types, although not in a species-specific manner. Niemann-Pick C1 (NPC1) protein is an essential filovirus receptor that binds directly to GP. Overexpression of NPC1 was recently demonstrated to rescue GP-F88A-mediated entry. A quantitative enzyme-linked immunosorbent assay (ELISA) demonstrated that while the F88A mutation impairs GP binding to human NPC1 by 10-fold, it has little impact on GP binding to mouse NPC1. Interestingly, not all mouse macrophage cell lines permit GP-F88A entry. The IC-21 cell line was permissive, whereas RAW 264.7 cells were not. Quantitative reverse transcription (RT)-PCR assays demonstrate higher NPC1 levels in GP-F88A permissive IC-21 cells and mouse peritoneal macrophages than in RAW 264.7 cells. Cumulatively, these studies suggest an important role for NPC1 in the differential entry of GP-F88A into mouse versus human APCs.

Ebola Virus Failure to Stimulate Plasmacytoid Dendritic Cell Interferon Responses Correlates With Impaired Cellular Entry

We examined the ability of the Ebola virus to elicit an antiviral response from plasmacytoid dendritic cells (pDCs). Exposure of pDCs to Ebola virus did not result in significantly higher levels of interferon-α production than the levels in mock-infected cells. After inoculation with Ebola virus under the same conditions, conventional dendritic cells expressed viral proteins whereas pDCs did not, suggesting that the latter cells were not infected. Assessment of the entry of Ebola virus-like particles into pDCs revealed that pDCs are highly impaired for viral entry in comparison with conventional dendritic cells. These observations identify a novel means by which Ebola virus can avoid triggering an antiviral response.