Detection of proton movement directly across viral membranes to identify novel influenza virus M2 inhibitors.

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.

In vitro and in vivo replication of influenza A H1N1 WSN33 viruses with different M1 proteins.

The M1 protein is a major structural protein that has multiple functions in various steps within the life cycle of the influenza A virus (IAV). However, little is currently known about the role of M1 in IAV replication in vivo and the associated pathogenesis. In this study, six isogenic H1N1 WSN33 viruses, constructed to express unique M1 proteins derived from various strains, subtypes or WSN33 itself, were tested to determine in vitro and in vivo functional exchangeability of M1 proteins in the replication and pathogenesis of the WSN33 virus. Despite five chimeric M1 viruses replicating to levels similar to those of the parental WSN33 virus in cell cultures, all M1 chimeras exhibited improved replication and enhanced virulence in mice when compared with the WSN33 virus. Interestingly, M1 proteins derived from swine viruses caused more severe clinical diseases than those from human or quail. These data indicate that the M1 protein is an important determinant of viral replication and pathogenic properties in mice, although the functions of M1 observed in vivo are not adequately reflected in simple infections of cultured cells. Chimeric M1 viruses that are variable in their clinical manifestations described here will aid future understanding of the role of M1 in IAV pathogenesis.

Preparation of carboxymethyl cellulose/graphene composite aerogel beads and their adsorption for methylene blue.

Carboxymethyl cellulose/ graphene composite aerogel beads (CMC/GAs) were prepared by the easily scaling-up method, i.e., wet spinning- environmental pressure drying method. The influences of the type and concentration of coagulating bath on the formation of aerogel beads were discussed, and the forming mechanism was analyzed. The CMC/GAs was characterized through SEM, XRD, FI-IR, Raman, XPS, electronic universal testing machine and other methods. The CMC/GAs-30 has an average particle size and a mean pore diameter of 3.83 mm and 82 µm, respectively. The analysis results indicated that the adsorption mechanisms of CMC/GAs on methylene blue (MB) are mainly through the electrostatic interaction. The adsorption process conforms to the Langmuir model (R2 = 0.9964) and pseudo-second-order kinetic model (R2 is higher than 0.99). When the particle size of CMC/GAs-30 decreases, the equilibrium adsorption capacity for MB increases. Under the experimental conditions explored, the Langmuir maximum adsorption capacity of CMC/GAs-30 for MB is 222.72 mg.g-1. The CMC/GAs-30 show good recycle performance in MB adsorption. The removal rate of MB from water by CMC/GAs-30 remained at about 90% after 30-times adsorption- regeneration cycles.

The cellular endosomal sorting complex required for transport pathway is not involved in avian metapneumovirus budding in a virus-like-particle expression system.

Avian metapneumovirus (AMPV) is a paramyxovirus that principally causes respiratory disease and egg production drops in turkeys and chickens. Together with its closely related human metapneumovirus (HMPV), they comprise the genus Metapneumovirus in the family Paramyxoviridae. Little is currently known about the mechanisms involved in the budding of metapneumovirus. By using AMPV as a model system, we showed that the matrix (M) protein by itself was insufficient to form virus-like-particles (VLPs). The incorporation of M into VLPs was shown to occur only when both the viral nucleoprotein (N) and the fusion (F) proteins were co-expressed. Furthermore, we provided evidence indicating that two YSKL and YAGL segments encoded within the M protein were not a functional late domain, and the endosomal sorting complex required for transport (ESCRT) machinery was not involved in metapneumovirus budding, consistent with a recent observation that human respiratory syncytial virus, closely related to HMPV, uses an ESCRT-independent budding mechanism. Taken together, these results suggest that metapneumovirus budding is independent of the ESCRT pathway and the minimal budding machinery described here will aid our future understanding of metapneumovirus assembly and egress.

The potent and broadly neutralizing human dengue virus-specific monoclonal antibody 1C19 reveals a unique cross-reactive epitope on the bc loop of domain II of the envelope protein.

UNLABELLED: Following natural dengue virus (DENV) infection, humans produce some antibodies that recognize only the serotype of infection (type specific) and others that cross-react with all four serotypes (cross-reactive). Recent studies with human antibodies indicate that type-specific antibodies at high concentrations are often strongly neutralizing in vitro and protective in animal models. In general, cross-reactive antibodies are poorly neutralizing and can enhance the ability of DENV to infect Fc receptor-bearing cells under some conditions. Type-specific antibodies at low concentrations also may enhance infection. There is an urgent need to determine whether there are conserved antigenic sites that can be recognized by cross-reactive potently neutralizing antibodies. Here, we describe the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) directed to the DENV domain II fusion loop (FL) envelope protein region from subjects following vaccination or natural infection. Most of the FL-specific antibodies exhibited a conventional phenotype, characterized by low-potency neutralizing function and antibody-dependent enhancing activity. One clone, however, recognized the bc loop of domain II adjacent to the FL and exhibited a unique phenotype of ultrahigh potency, neutralizing all four serotypes better than any other previously described MAb recognizing this region. This antibody not only neutralized DENV effectively but also competed for binding against the more prevalent poor-quality antibodies whose binding was focused on the FL. The 1C19 human antibody could be a promising component of a preventative or therapeutic intervention. Furthermore, the unique epitope revealed by 1C19 suggests a focus for rational vaccine design based on novel immunogens presenting cross-reactive neutralizing determinants. IMPORTANCE: With no effective vaccine available, the incidence of dengue virus (DENV) infections worldwide continues to rise, with more than 390 million infections estimated to occur each year. Due to the unique roles that antibodies are postulated to play in the pathogenesis of DENV infection and disease, there is consensus that a successful DENV vaccine must protect against all four serotypes. If conserved epitopes recognized by naturally occurring potently cross-neutralizing human antibodies could be identified, monovalent subunit vaccine preparations might be developed. We characterized 30 DENV cross-neutralizing human monoclonal antibodies (MAbs) and identified one (1C19) that recognized a novel conserved site, known as the bc loop. This antibody has several desirable features, as it neutralizes DENV effectively and competes for binding against the more common low-potency fusion loop (FL) antibodies, which are believed to contribute to antibody-mediated disease. To our knowledge, this is the first description of a potent serotype cross-neutralizing human antibody to DENV.

Application of a split luciferase complementation assay for the detection of viral protein-protein interactions.

Intraviral protein-protein interactions are critical for virus survival in the host. Discovery of such interactions is important to understand molecular mechanisms of viral replication and pathogenesis. The development of a cell-based assay that can be employed to examine systematically viral protein interactions is described. The method, known as the split luciferase complementation assay (SLCA), is based on the principle that N- and C-terminal domains of luciferase alone do not emit luminescence; however, if fused to interacting proteins the two non-functional halves can be brought into close enough proximity through a specific protein-protein interaction to restore the functions of the enzyme and emit detectable light. The well-studied influenza B polymerase acidic protein (PA) and basic protein 1 (PB1) interaction was used as a model system to develop the assay. Consistent with previous studies, a strong PA-PB1 interaction was demonstrated in the assay. The PA-PB1 interaction was also disrupted by single amino acid mutations in the N-terminal domain of PB1 that is responsible for binding PA. The described SLCA is highly specific and easy to perform, and thus may be useful for studying protein-protein interactions in viral diseases.

Nuclear localization of influenza B polymerase proteins and their binary complexes.

The viral RNA-dependent RNA polymerases of influenza A and B are trimeric complexes of PA, PB1, and PB2 subunits that are crucial for both transcription and replication of the viral genome. Unlike the significant progress made recently in understanding nuclear transport and molecular assembly of influenza A polymerase, little is known about the influenza B polymerase, although influenza B viruses cause severe upper respiratory disease in humans. The aim of this study was to characterize nuclear localization of the influenza B RNA polymerase proteins and binary complexes. We demonstrated that each polymerase protein has a nuclear localization function, and among them, the PB2 protein exclusively locates to the nucleus while PA and PB1 proteins are associated with the cytoplasm and the nucleus. Furthermore, we show that pairwise binary complexes are formed among the influenza B subunits (PA-PB1, PA-PB2, and PB1-PB2) and both PB1-PB2 and PA-PB2 complexes are predominantly associated with the nucleus while the PA-PB1 complex exhibits both nuclear and cytoplasmic fluorescence signals. Results of our studies represent the first step toward the understanding of nuclear transport and molecular assembly within the influenza B polymerase complex.

Torque teno virus (SANBAN isolate) ORF2 protein suppresses NF-kappaB pathways via interaction with IkappaB kinases.

Since the first discovery of Torque teno virus (TTV) in 1997, many researchers focused on its epidemiology and transcriptional regulation, but the function of TTV-encoded proteins remained unknown. The function of the TTV open reading frame (ORF) in the nuclear factor kappaB (NF-kappaB) pathway has not yet been established. In this study, we found for the first time that the TTV ORF2 protein could suppress NF-kappaB activity in a dose-dependent manner in the canonical NF-kappaB pathway. By Western blot analysis, we proved that the TTV ORF2 protein did not alter the level of NF-kappaB expression but prevented the p50 and p65 subunits from entering the nucleus due to the inhibition of IkappaBalpha protein degradation. Further immunoprecipitation assays showed that the TTV ORF2 protein could physically interact with IKKbeta as well as IKKalpha, but not IKKgamma. Luciferase assays and Western blot experiments showed that the TTV ORF2 protein could also suppress NF-kappaB activity in the noncanonical NF-kappaB pathway and block the activation and translocation of p52. Finally, we found that the TTV ORF2 protein inhibited the transcription of NF-kappaB-mediated downstream genes (interleukin 6 [IL-6], IL-8, and COX-2) through down-regulation of NF-kappaB. Together, these data indicate that the TTV ORF2 protein suppresses the canonical and noncanonical NF-kappaB pathways, suggesting that the TTV ORF2 protein may be involved in regulating the innate and adaptive immunity of organisms, contributing to TTV pathogenesis, and even be related to some diseases.