Severe acute respiratory syndrome (SARS) coronavirus is highly pathogenic in humans and evades innate immunity at multiple levels. It has evolved various strategies to counteract the production and action of type I interferons, which mobilize the front-line defense against viral infection. In this study we demonstrate that SARS coronavirus M protein inhibits gene transcription of type I interferons. M protein potently antagonizes the activation of interferon-stimulated response elementdependent transcription by double-stranded RNA, RIG-I, MDA5, TBK1, IKK⑀, and virus-induced signaling adaptor (VISA) but has no influence on the transcriptional activity of this element when IRF3 or IRF7 is overexpressed. M protein physically associates with RIG-I, TBK1, IKK⑀, and TRAF3 and likely sequesters some of them in membrane-associated cytoplasmic compartments. Consequently, the expression of M protein prevents the formation of TRAF3⅐TANK⅐TBK1/IKK⑀ complex and thereby inhibits TBK1/IKK⑀-dependent activation of IRF3/IRF7 transcription factors. Taken together, our findings reveal a new mechanism by which SARS coronavirus circumvents the production of type I interferons. Severe acute respiratory syndrome (SARS)2 coronavirus causes a highly lethal infectious disease in humans characterized by an aberrant immune response (1). The production and action of type I interferons, which are major components of antiviral innate immunity (2, 3), are inhibited at multiple levels by SARS coronavirus (4, 5). This inhibition is thought to be mediated through viral structural and nonstructural proteins N, ORF3b, ORF6, nsp1, and papain-like protease (6 -12).The signaling pathways through which viruses induce the production of type I interferons have been well characterized (13)(14)(15). In response to double-stranded RNA (dsRNA) produced during viral replication, endosomal Toll-like receptor 3 (TLR3) and cytoplasmic retinoic acid-inducible gene I (RIG-I) trigger the activation of two different pathways adapted to downstream kinases through TRIF (TLR adaptor inducing interferon ␤) and VISA, respectively. These pathways converge on the formation of TRAF3⅐TANK⅐TBK1/IKK⑀ complex, which catalyzes the phosphorylation of IRF3 and IRF7 transcription factors, leading to the activation of type I interferon promoters (15-17).SARS coronaviral proteins counteract the production of type I interferons at multiple steps. Although IRF3 phosphorylation was inhibited in cells expressing ORF3b, ORF6, or N protein (7), papain-like protease could physically interact with IRF3 and prevent its phosphorylation and nuclear translocation in a protease-independent manner (11). In addition, nsp1 suppressed the synthesis of host proteins including interferons by inducing mRNA degradation (6, 12). Meanwhile, viral proteins such as nsp1 and ORF6 were multifunctional (10,18,19) and could also inhibit interferon signaling. For example, both nsp1 and ORF6 inhibited the activity of STAT1, a key regulator of interferon-responsive genes (8, 9). Whereas nsp1 attenuated phosphorylati...

A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine. In 2003, Farzan and colleagues successfully identified the receptor of SARS-CoV, angiotensin-converting enzyme 2 (ACE2) (7), and a 193-amino-acid fragment in the spike (S) protein (residues 318 to 510) as the receptor-binding domain (RBD) (8). We found that SARS-CoV S-RBD contains a critical neutralizing site (9) which induces potent neutralizing antibodies and protection against SARS-CoV infection in an animal model (10).Since MERS-CoV is genetically related to SARS-CoV (1), we compared their S protein sequences and predicted that the RBD of MERS-CoV might be located in the region spanning residues 377 to 662 of the S1 subunit (Fig. 1). Using the Swiss-Model Workplace homology modeling server (11) and basing our work on the X-ray crystal structure of the SARS-CoV S-RBD (Protein Data Bank [PDB] identification no. 2DD8) (12), we predicted the conformational structure of the region consisting of residues 377 to 662 in the S1 subunit of the MERS-CoV S protein (13). We noticed that the SARS-CoV S-RBD and the predicted MERS-CoV S-RBD possessed similar core structures but had an extended secondary structure consisting predominantly of the receptor-binding motifs (RBM) (12,14). The extended region in MERS-CoV S-RBD is much longer than that in SARS-CoV S-RBD, suggesting that MERS-CoV and SARS-CoV use different receptors. Indeed, it has been proven that dipeptidyl peptidase-4 (DPP4; also known as CD26) is the functional receptor of MERS-CoV (15).We then constructed MERS-CoV S-RBD based on the synthesized codon-optimized MERS-CoV S sequences (GenBank accession no. AFS88936.1) and fused it to Fc of human IgG using pFUSE-hIgG1-Fc2 expression vector (here named Fc) (InvivoGen, San Diego, CA). The SARS-CoV S-RBD-Fc was constructed by fusing RBD of codon-optimized SARS-CoV S sequence into the Fc vector referred to above as a control (Fig. 1) (16). The S-RBD-Fc proteins were expressed in 293T cell culture supernatant and purified by protein A affinity chromatography (GE Healthcare, Piscataway, NJ) (17). We found that both MERS-CoV and SARS-CoV S-RBD-Fc proteins were highly purified from transfected culture supernatants ( Fig. 2A, panel a). MERS-CoV S-RBD-Fc could be recognized by an MERS-CoV S-specific polyclonal antibody (1:1,000), while SARS-CoV S-RBD-Fc could not react with this antibody, as detected by Western blotting (Fig. 2A, panel b).Using analysis performed by Western blotting, we found that DPP4 was highly express...

BackgroundEvidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors.MethodsIn this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors.ResultsOur results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377–662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection.ConclusionTaken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.

Numerous biological processes involve the recognition of a specific pattern of binding sites on a target protein or surface. Although ligands displayed by disordered scaffolds form stochastic rather than specific patterns, theoretical models predict that recognition will occur between patterns that are characterized by similar or "matched" statistics. Endowing synthetic biomimetic structures with statistical pattern matching capabilities may improve the specificity of sensors and resolution of separation processes. We demonstrate that statistical pattern matching enhances the potency of polyvalent therapeutics. We functionalized liposomes with an inhibitory peptide at different densities and observed a transition in potency at an interpeptide separation that matches the distance between ligand-binding sites on the heptameric component of anthrax toxin. Pattern-matched polyvalent liposomes inhibited anthrax toxin in vitro at concentrations four orders of magnitude lower than the corresponding monovalent peptide, and neutralized this toxin in vivo. Statistical pattern matching also enhanced the potency of polyvalent inhibitors of cholera toxin. This facile strategy should be broadly applicable to the detection and neutralization of toxins and pathogens.

Interleukin-17 (IL-17), a member of the IL-17 cytokine family, plays a crucial role in mediating the immune response against extracellular bacteria and fungi in the lung. Although there is increasing evidence that IL-17 is involved in protective immunity against H1 and H3 influenza virus infections, little is known about the role of IL-17 in the highly pathogenic H5N1 influenza virus infection. In this study, we show that H5N1-infected IL-17 knockout (KO) mice exhibit markedly increased weight loss, more pronounced lung immunopathology and significantly reduced survival rates as compared with infected wild-type controls. Moreover, the frequency of B cells in the lung were substantially decreased in IL-17 KO mice after virus infection, which correlated with reduced CXCR5 expression in B cells and decreased CXCL13 production in the lung tissue of IL-17 KO mice. Consistent with this observation, B cells from IL-17 KO mice exhibited a significant reduction in chemokine-mediated migration in culture. Taken together, these findings demonstrate a critical role for IL-17 in mediating the recruitment of B cells to the site of pulmonary influenza virus infection in mice.

The design of polyvalent molecules, consisting of multiple copies of a biospecific ligand attached to a suitable scaffold, represents a promising approach to inhibit pathogens and oligomeric microbial toxins. Despite the increasing interest in structure-based drug design, few polyvalent inhibitors based on this approach have shown efficacy in vivo. Here we demonstrate the structure-based design of potent biospecific heptavalent inhibitors of anthrax lethal toxin. Specifically, we illustrate the ability to design potent polyvalent ligands by matching the pattern of binding sites on the biological target. We used a combination of experimental studies based on mutagenesis and computational docking studies to identify the binding site for an inhibitory peptide on the heptameric subunit of anthrax toxin. We developed an approach based on copper-catalyzed azide-alkyne cycloaddition (click-chemistry) to facilitate the attachment of seven copies of the inhibitory peptide to a β-cyclodextrin core via a polyethylene glycol linker of an appropriate length. The resulting heptavalent inhibitors neutralized anthrax lethal toxin both in vitro and in vivo and showed appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent inhibitors show considerable promise as anthrax anti-toxins.

Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: (a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. (b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) to intact cells significantly increased lawnassociated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P 3 causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.Insulin promotes the uptake of glucose into muscle and fat tissues through a rapid gain in surface-bound glucose transporters (1-3). The muscle-and fat-specific glucose transporter GLUT4 1 cycles continuously between the plasma membrane and intracellular stores, with the steady-state distribution largely favoring the latter. Insulin changes this steady-state resulting in a net gain in surface GLUT4 (4 -6) largely as a result of enhancing the exocytic rate of GLUT4 cycling (7,8). Of significance, insulin resistance and diabetes are accompanied by defective GLUT4 gain at the plasma membrane of muscle and fat cells (9 -11).It is well established that signaling from class IA phosphatidylinositol (PI) 3-kinase is required for the insulin-dependent net gain in surface GLUT4 (12-15), but the specific step(s) in GLUT4 cycling that are regulated are not elucidated. We have recently shown that class IA PI 3-kinase is required for the insulin-dependent acceleration of GLUT4 transit through the recycling endosome (16). A second input of class IA PI 3-kinase in muscle cells is the spatial-temporal actin remodeling and its possible contribution to segregating specific signaling molecules (17, 18). However, it is not known whether fusion of insulin-sensitive GLUT4 vesicles with the plasma membrane is a regulated step, nor w...

Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthraxtoxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.antimicrobial resistance ͉ phage display ͉ therapeutics P athogens can develop resistance to drugs directed against microbial targets by modifying the drug, by lowering the concentration of drug that reaches the target, or by mutating the target (1, 2). There is also an increasing concern that therapeutics developed for bioterrorism agents may be rendered ineffective if the microbial target is altered intentionally. This problem could be overcome, however, by designing inhibitors that block host proteins used by the pathogen or its toxins to cause disease.Microbial pathogens and their products interact with host structures to facilitate colonization or to promote cellular uptake. Many of these interactions are polyvalent, meaning that they involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another (3). The design of synthetic polyvalent (4-8) or oligovalent (9, 10) molecules also represents a promising approach to enhance the potency of inhibitors of microbial pathogens and toxins. Current examples of this approach have involved the design of molecules that bind directly to the pathogen or toxin. Inhibitors that bind host proteins would represent an effective way to attenuate virulence that may be less susceptible to resistance mechanisms, and the use of polyvalency could provide a significant enhancement in the potency of these inhibitors.ANTXR1 and ANTXR2 are host receptors that bind and internalize anthrax toxin (11,12). These proteins are likely important for anthrax pathogenesis because the toxin impairs the immune response and is responsible for the major symptoms and death associated with anthrax. Thus, blocking these receptors could represent a promising approach to anthrax therapy.ANTXR1 and ANTXR2 are widely expressed type I membrane proteins that bind components of the extracellular matrix (13). They both contain an extracellular I domain, which binds the protective antigen (PA) component of anthrax toxin. The two proteins are 40% identical overall and share 60% identity within ...