Hana Schmeisser

Mini ReviewHuman Interferons Alpha, Beta and Omega

Type I interferons (IFNs), IFN-α, IFN-β, IFN-ω, IFN-δ and IFN-τ are a family of structurally related, species-specific proteins found only in vertebrates. They exhibit a variety of biological functions, including antiviral, antiproliferative, immunomodulatory and developmental activities. Human Type I IFNs interact with the human IFN alpha receptor (IFNAR), which is composed of two identified subunits (IFNAR-1 and IFNAR-2). The interaction of IFN-α/β with its receptor components results in the activation of a number of signaling pathways. The regulation of specific genes and proteins contributes to the numerous biological functions of Type I IFNs.

Identification of Alpha Interferon-Induced Genes Associated with Antiviral Activity in Daudi Cells and Characterization of IFIT3 as a Novel Antiviral Gene

ABSTRACT 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.

New Function of Type I IFN: Induction of Autophagy

Autophagy is a highly conserved cellular process responsible for recycling of intracellular material. It is induced by different stress signals, including starvation, cytokines, and pathogens. Type I interferons (IFN) are proteins with pleiotropic functions, such as antiviral, antiproliferative, and immunomodulatory activities. Several recent studies showed type I IFN-induced autophagy in multiple cancer cell lines as evidenced by autophagic markers, for example, the conversion of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B, also known as LC3-I) to LC3-II and the formation of autophagosomes by electron microscopy. In addition, studies suggest the involvement of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT) and mechanistic target of rapamycin, serine/threonine kinase (mTOR) pathways in the induction of autophagy. This review highlights a new function of type I IFN as an inducer of autophagy. This new function of type I IFN may play an important role in viral clearance, antigen presentation, inhibition of proliferation, as well as a positive feedback loop for the production of type I IFN.

Type I interferons induce autophagy in certain human cancer cell lines.

Autophagy is an evolutionarily conserved cellular recycling mechanism that occurs at a basal level in all cells. It can be further induced by various stimuli including starvation, hypoxia, and treatment with cytokines such as IFNG/IFNγ and TGFB/TGFβ. Type I IFNs are proteins that induce an antiviral state in cells. They also have antiproliferative, proapoptotic and immunomodulatory activities. We investigated whether type I IFN can also induce autophagy in multiple human cell lines. We found that treatment with IFNA2c/IFNα2c and IFNB/IFNβ induces autophagy by 24 h in Daudi B cells, as indicated by an increase of autophagy markers MAP1LC3-II, ATG12–ATG5 complexes, and a decrease of SQSTM1 expression. An increase of MAP1LC3-II was also detected 48 h post-IFNA2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines exposed to type I IFN was confirmed by electron microscopy analysis. Increased expression of autophagy markers correlated with inhibition of MTORC1 in Daudi cells, as well as inhibition of cancer cell proliferation and changes in cell cycle progression. Concomitant blockade of either MTOR or PI3K-AKT signaling in Daudi and T98G cells treated with IFNA2c increased the level of MAP1LC3-II, indicating that the PI3K-AKT-MTORC1 signaling pathway may modulate IFN-induced autophagy in these cells. Taken together, our findings demonstrated a novel function of type I IFN as an inducer of autophagy in multiple cell lines.

A Novel Role for IFN-Stimulated Gene Factor 3II in IFN-γ Signaling and Induction of Antiviral Activity in Human Cells

Type I (e.g., IFN-α, IFN-β) and type II IFNs (IFN-γ) have antiviral, antiproliferative, and immunomodulatory properties. Both types of IFN signal through the Jak/STAT pathway to elicit antiviral activity, yet IFN-γ is thought to do so only through STAT1 homodimers, whereas type I IFNs activate both STAT1- and STAT2-containing complexes such as IFN-stimulated gene factor 3. In this study, we show that IFN-stimulated gene factor 3 containing unphosphorylated STAT2 (ISGF3II) also plays a role in IFN-γ–mediated antiviral activity in humans. Using phosphorylated STAT1 as a marker for IFN signaling, Western blot analysis of IFN-α2a–treated human A549 cells revealed that phospho-STAT1 (Y701) levels peaked at 1 h, decreased by 6 h, and remained at low levels for up to 48 h. Cells treated with IFN-γ showed a biphasic phospho-STAT1 response with an early peak at 1–2 h and a second peak at 15–24 h. Gene expression microarray following IFN-γ treatment for 24 h indicated an induction of antiviral genes that are induced by IFN-stimulated gene factor 3 and associated with a type I IFN response. Induction of these genes by autocrine type I and type III IFN signaling was ruled out using neutralizing Abs to these IFNs in biological assays and by quantitative RT-PCR. Despite the absence of autocrine IFNs, IFN-γ treatment induced formation of ISGF3II. This novel transcription factor complex binds to IFN-stimulated response element promoter sequences, as shown by chromatin immunoprecipitation analysis of the protein kinase R promoter. STAT2 and IFN regulatory factor 9 knockdown in A549 cells reversed IFN-γ–mediated IFN-stimulated response element induction and antiviral activity, implicating ISGF3II formation as a significant component of the cellular response and biological activity of IFN-γ.

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

ABSTRACT Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics.

IRF9 is a key factor for eliciting the antiproliferative activity of IFN-α

A number of tumors are still resistant to the antiproliferative activity of human interferon (IFN)-α. The Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT) pathway plays an important role in initial IFN signaling. To enhance the antiproliferative activity of IFN-α, it is important to elucidate which factors in the JAK-STAT pathway play a key role in eliciting this activity. In human ovarian adenocarcinoma OVCAR3 cells sensitive to both IFN-α and IFN-γ, only IFN regulatory factor 9 (IRF9)-RNA interference (RNAi) completely inhibited the antiproliferative activity of IFN-α among the intracellular JAK-STAT pathway factors. Conversely, Stat1-RNAi did not inhibit the antiproliferative activity of IFN-α, whereas it partially inhibited that of IFN-γ. As a cell death pathway, it is reported that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through TRAIL-receptor (R) 1 and TRAIL-R2. In IFN-α-treated OVCAR3 cells, IRF9-RNAi inhibited transcription of TRAIL whereas Stat1-RNAi did not, suggesting that the transcription of TRAIL induced by IFN-α predominantly required IRF9. Furthermore, IFN-stimulated response element-like motifs of TRAIL bound to IFN-stimulated gene factor 3 (ISGF3) complex after IFN-α treatment. Subsequently, TRAIL-R2-RNAi inhibited both antiproliferative activities of IFN-α and TRAIL, suggesting that TRAIL-R2 mediated both IFN-α and TRAIL signals to elicit their antiproliferative activities. Finally, IRF9 overexpression facilitated IFN-α-induced apoptosis in T98G (human glioblastoma multiforme) cells, which were resistant to IFN-α. Thus, this study suggests that IRF9 is the key factor for eliciting the antiproliferative activity of IFN-α and TRAIL may be one of the potential mediators.

Incidence of Autoantibodies Against Type I and Type II Interferons in a Cohort of Systemic Lupus Erythematosus Patients in Slovakia

Autoantibodies against interferon (IFN) can be found in patients with systemic lupus erythematosus (SLE). However, detailed information about the occurrence of type-specific antihuman IFN antibodies is not available. In this study, we investigated the incidence of autoantibodies specifically recognizing various type I IFNs (alpha1, alpha2, beta, omega) and type II IFN (gamma). Sera from 100 SLE patients were screened for the presence of IFN-binding antibodies by ELISA, using various types of recombinant IFNs as antigen. On the whole, autoantibodies against type I or type II or both IFNs were detected in 45% (45 of 100) of the serum samples investigated. More than half (56%) of the positive samples (25 of 45) contained antibodies specific only for type I IFNs, and 36% of positive sera (16 of 45) had autoantibodies only against type II IFN. Antibodies against both type I and type II IFNs were detected in 8% (4 of 45) of the positive samples. Among autoantibodies to type I IFNs, the most abundant were those against the type IFN-omega (15%) and the subtype IFN-alpha2 (11%). Autoantibodies binding subtype IFN-alpha1 and type IFN-beta were detected at a relatively lower incidence of about 3%-4%. The highest occurrence (20%) showed autoantibodies to the proinflammatory cytokine, IFN-gamma. We did not find any correlation between the production of autoantibodies against particular IFN species and an antibody response to other IFN species. We further observed that 84% (38 of 45) of the positive sera bound only one IFN species, and 13% (6 of 45) of positive samples contained antibodies against two IFN species of five different combinations (alpha1/beta, alpha1/omega, alpha2/omega, alpha2/gamma, omega/gamma). One sample uniquely showed reactivity with three IFN species (alpha2/omega/gamma). Our findings suggest that formation of autoantibodies could reflect humoral immune responses to increased spontaneous production of the respective IFN species in SLE patients.

Structural variants of IFNα preferentially promote antiviral functions.

IFNα, a cytokine with multiple functions in innate and adaptive immunity and a potent inhibitor of HIV, exerts antiviral activity, in part, by enhancing apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (APOBEC3) family members. Although IFNα therapy is associated with reduced viral burden, this cytokine also mediates immune dysfunction and toxicities. Through detailed mapping of IFNα receptor binding sites, we generated IFNα hybrids and mutants and determined that structural changes in the C-helix alter the ability of IFN to limit retroviral activity. Selective IFNα constructs differentially block HIV replication and their directional magnitude of inhibition correlates with APOBEC3 levels. Importantly, certain mutants exhibited reduced toxicity as reflected by induced indoleamine 2,3-dioxygenase (IDO), suggesting discreet and shared intracellular signaling pathways. Defining IFN structure and function relative to APOBEC and other antiviral genes may enable design of novel IFN-related molecules preserving beneficial antiviral roles while minimizing negative effects.

A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis

Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.