Surface-exposed Loops Research Articles

Expression of chimeric proteins based on a backbone of the GII.4 norovirus VP1 and their application in the study of a GII.6 norovirus-specific blockade epitope.

The surface-exposed loop regions of the protruding domain of the norovirus (NoV) major capsid protein VP1 can tolerate the insertion of foreign antigens without affecting its assembly into subviral particles. In this study, we investigated the tolerance of the surface-exposed loop region of the GII.4 NoV VP1 by replacing it with homologous or heterologous sequences. We designed a panel of constructs in which the amino acid sequence from position 298-305 of the GII.4 NoV VP1 was replaced by sequences derived from the same region of GI.3, GII.3, GII.6, and GII.17 NoVs as well as neutralizing epitopes of enterovirus type 71 and varicella-zoster virus. The constructs were synthesized and expressed using a recombinant baculovirus expression system. The expression of target proteins was measured by indirect enzyme-linked immunosorbent assay (ELISA), and the assembly of virus-like particles (VLPs) was confirmed by electron microscopy. Our results showed that all of the constructs expressed high levels of target chimeric proteins, and all of the chimeric proteins successfully assembled into VLPs or subviral particles. An in vitro VLP-histo-blood group antigen (HBGA) binding assay revealed that chimeric-protein-containing VLPs did not bind or showed reduced binding to salivary HBGAs, a ligand for NoV particles. The results of an in vitro VLP-HBGA binding blockade assay indicated that the predicted surface-exposed loop region of the GII.6 NoV VP1 may comprise a blockade epitope. In summary, the surface-exposed loop region of the GII.4 NoV VP1 can be replaced by foreign sequences of a certain length. Using this strategy, we found that the predicted surface-exposed loop region of GII.6 NoV VP1 might contain a blockade epitope.

Biophysical characterization of the homodimers of HomA and HomB, outer membrane proteins of Helicobacter pylori

Helicobacter pylori is a Gram-negative bacterium that causes chronic inflammations in the stomach area and is involved in ulcers, which can develop into gastric malignancies. H. pylori attaches and colonizes to the human epithelium using some of their outer membrane proteins (OMPs). HomB and HomA are the most studied OMPs from H. pylori as they play a crucial role in adherence, hyper biofilm formation, antibiotic resistance and are also associated with severe gastric malignancies. The role of HomA and HomB in pathogenesis concerning their structure and function has not been evaluated yet. In the present study, we explored the structural aspect of HomA and HomB proteins using various computational, biophysical and small-angle X-ray scattering (SAXS) techniques. Interestingly, the in-silico analysis revealed that HomA/B consists of 8 discontinuous N and C terminal β-strands forming a small β-barrel, along with a large surface-exposed globular domain. Further, biophysical experiments suggested that HomA and HomB are dimeric and most likely the cysteine residues present on surface-exposed loops participate in protein–protein interactions. Our study provides essential structural information of unexplored proteins of the Hom family that can help in a better understanding of H. pylori pathogenesis.

The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

Full-length TprK of Treponema pallidum subsp. pallidum in lipid nanodiscs is a monomeric porin

TprK is a key virulence factor of Treponema pallidum subsp. pallidum (T. pallidum) due to its ability to undergo intra-strain antigenic variation through gene conversion. This mechanism can generate millions of tprK gene and protein variants to allow immune evasion and pathogen persistence during infection. In silico structural modeling supports that TprK is an outer membrane β-barrel with porin function and with several surface-exposed loops, seven of which corresponding to the variable regions. No definitive structural of functional data, however, exist for this protein aside from its role in immune evasion. Studies to elucidate TprK biological function as a porin, are hindered by the evidence that TprK is not abundant on T. pallidum outer membrane, and by the fragility of T. pallidum envelope. To gain insight onto TprK structure and possible function as a porin, we used an Escherichia coli - based expression system that yielded highly pure full-length TprK without any intermediate denaturation step, and proceeded to reconstitute it in detergents and lipid nanodiscs. Visualization of TprK in nanodiscs using negative staining electron microscopy supported that TprK is a monomeric porin in an artificial lipid environment mimicking T. pallidum membrane. Our work provided evidence that TprK is a possible porin transporter of T. pallidum, a biological function compatible with its structural models. These results bring us closer to a comprehensive understanding of the function of this important virulence factor in syphilis pathogenesis and T. pallidum biology.

Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane-penetrating loop.

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is one of two major virulence factors of Bacillus anthracis, the causative agent of anthrax disease. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the cytotoxic enzymes of the toxin are extruded. The PA pore is formed by a cooperative conformational change in which the membrane-penetrating loops of PA associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target for monoclonal antibody-based therapy. cAb29 is a highly effective neutralizing antibody against PA. Here, the cryo-EM structure of PA in complex with the Fab portion of cAb29 was determined. It was found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to the static surface-exposed loop of the D3 domain of the same subunit, thereby preventing pore formation. These results provide the structural basis for the antibody-based neutralization of PA and bring into focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

Structure, Antigenic Properties, and Highly Efficient Assembly of PCV4 Capsid Protein.

Porcine circovirus type 4 (PCV4), a recently reported circovirus, was first identified in pigs with clinical signs similar to porcine dermatitis nephropathy syndrome (PDNS), in Hunan province, China, in 2019. More knowledge regarding the assembly of capsid protein (Cap) into virus-like particles (VLPs), their structure and antigenic properties, are needed to provide new knowledge for diagnosis and further characterization of PCV4. In this study, high-level expression of PCV4 Cap was achieved in Escherichia coli with purified Cap self-assembling into VLPs (~20 nm) in vitro. Furthermore, these VLPs were internalized in vitro by PK15 and 3D4/21 cell lines. Significant structural differences between PCV4 and PCV2 capsids were demonstrated among loops (loop BC, CD, DE, EF, and GH), based on comparisons of 3D structures. In addition, five potential B cell epitopes identified in silico were mostly located in surface-exposed loops of PCV4 capsid. Cross-reaction between PCV4 and PCV2 or PCV3 conferred by humoral immune responses was deemed unlikely on the basis of ELISA and Western blotting for assessment of VLPs and using PCV4 or PCV2 VLPs. In conclusion, these studies provided new knowledge regarding PCV4 capsid surface patterns. It is noteworthy that the PCV4 VLPs prepared in our study have much potential for development of serological diagnostics for PCV4 and to further characterize this virus.

Differential Enhancement of Neutrophil Phagocytosis by Anti-Bactericidal/Permeability-Increasing Protein Antibodies.

Bactericidal/permeability-increasing protein (BPI) plays a major role in innate immunity through the ability of the N-terminal domain (NTD) to bind LPS, mediate cytotoxicity, and block LPS-induced inflammation. The C-terminal domain mediates phagocytosis of bacteria bound to the NTD. These two domains are linked by a surface-exposed loop at amino acids 231-249 for human BPI, known as the "hinge region." Autoantibodies to human BPI are prevalent in many chronic lung diseases; their presence is strongly correlated with Pseudomonas aeruginosa and with worse lung function in patients with cystic fibrosis and bronchiectasis. Although prior literature has reported BPI neutralization effect with autoantibodies targeting either NTD or C-terminal domain, the functionality of BPI Ab to the hinge region has never been investigated. Here, we report that Ab responses to the BPI hinge region mediate a remarkably selective potentiation of BPI-dependent phagocytosis of P. aeruginosa with both human and murine neutrophils in vitro and in vivo. These findings indicate that autoantibodies to the BPI hinge region might enhance bacterial clearance.

An engineered cellobiohydrolase I for sustainable degradation of lignocellulosic biomass.

This study provides computational-assisted engineering of the cellobiohydrolase I (CBH-I) from Penicillium verruculosum with simultaneous enhanced thermostability and tolerance in ionic liquids, deep eutectic solvent, and concentrated seawater without affecting its wild-type activity. Engineered triple variant CBH-I R1 (A65R-G415R-S181F) showed 2.48-fold higher thermostability in terms of relative activity at 65°C after 1 h of incubation when compared with CBH-I wild type. CBH-I R1 exhibited 1.87-fold, 1.36-fold, and 1.57-fold higher specific activities compared with CBH-I wild type in [Bmim]Cl (50 g/L), [Ch]Cl (50 g/L), and two-fold concentrated seawater, respectively. In the multicellulases mixture, CBH-I R1 showed higher hydrolytic efficiency to hydrolyze aspen wood compared with CBH-I wild type in the buffer, [Bmim]Cl (50 g/L), and two-fold concentrated seawater, respectively. Structural analysis revealed a molecular basis for the higher stability of the CBH-I structure in which A65R and G415R substitutions form salt bridges (D64 … R65, E411 … R415) and S181F forms π-π interaction (Y155 … F181), leading to stabilize surface-exposed flexible α-helixes and loop in the multidomain β-jelly roll fold structure, respectively. In conclusion, the variant CBH-I R1 could enable efficient lignocellulosic biomass degradation as a cost-effective alternative for the sustainable production of biofuels and value-added chemicals.

Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins

Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.

Diversification of OmpA and OmpF of Yersinia ruckeri is independent of the underlying species phylogeny and evidence of virulence-related selection

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) which causes economically significant losses in farmed salmonids, especially Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss, Walbaum). However, very little is known about the genetic relationships of disease-causing isolates in these two host species or about factors responsible for disease. Phylogenetic analyses of 16 representative isolates based on the nucleotide sequences of 19 housekeeping genes suggests that pathogenic Atlantic salmon and rainbow trout isolates represent distinct host-specific lineages. However, the apparent phylogenies of certain isolates has been influenced by horizontal gene transfer and recombinational exchange. Splits decomposition analysis demonstrated a net-like phylogeny based on the housekeeping genes, characteristic of recombination. Comparative analysis of the distribution of individual housekeeping gene alleles across the isolates demonstrated evidence of genomic mosaicism and recombinational exchange involving certain Atlantic salmon and rainbow trout isolates. Comparative nucleotide sequence analysis of the key outer membrane protein genes ompA and ompF revealed that the corresponding gene trees were both non-congruent with respect to the housekeeping gene phylogenies providing evidence that horizontal gene transfer has influenced the evolution of both these surface protein-encoding genes. Analysis of inferred amino acid sequence variation in OmpA identified a single variant, OmpA.1, that was present in serotype O1 and O8 isolates representing typical pathogenic strains in rainbow trout and Atlantic salmon, respectively. In particular, the sequence of surface-exposed loop 3 differed by seven amino acids to that of other Y. ruckeri isolates. These findings suggest that positive selection has likely influenced the presence of OmpA.1 in these isolates and that loop 3 may play an important role in virulence. Amino acid sequence variation of OmpF was greater than that of OmpA and was similarly restricted mainly to the surface-exposed loops. Two OmpF variants, OmpF.1 and OmpF.2, were associated with pathogenic rainbow trout and Atlantic salmon isolates, respectively. These OmpF proteins had very similar amino acid sequences suggesting that positive evolutionary pressure has also favoured the selection of these variants in pathogenic strains infecting both species.

Modeling the efficiency of filovirus entry into cells in vitro: Effects of SNP mutations in the receptor molecule.

Interaction between filovirus glycoprotein (GP) and the Niemann-Pick C1 (NPC1) protein is essential for membrane fusion during virus entry. Some single-nucleotide polymorphism (SNPs) in two surface-exposed loops of NPC1 are known to reduce viral infectivity. However, the dependence of differences in entry efficiency on SNPs remains unclear. Using vesicular stomatitis virus pseudotyped with Ebola and Marburg virus GPs, we investigated the cell-to-cell spread of viruses in cultured cells expressing NPC1 or SNP derivatives. Eclipse and virus-producing phases were assessed by in vitro infection experiments, and we developed a mathematical model describing spatial-temporal virus spread. This mathematical model fit the plaque radius data well from day 2 to day 6. Based on the estimated parameters, we found that SNPs causing the P424A and D508N substitutions in NPC1 most effectively reduced the entry efficiency of Ebola and Marburg viruses, respectively. Our novel approach could be broadly applied to other virus plaque assays.

Prevalence of antibodies against a cyclic peptide mimicking the FG loop of the human papillomavirus type 16 capsid among Tunisian women

BackgroundIn the past decade, cervical cancer has gone from being the second to the fourth most common cancer in women worldwide, but remains the second most common in developing countries. This cancer is most commonly caused by high-risk types of human papillomavirus (HPV), mainly type 16 (HPV16), which are sexually transmitted. This study aimed to investigate the usefulness of a cyclic synthetic peptide designed from the major L1 capsid protein of HPV16 for detecting anti-HPV16 antibodies.MethodsWe designed and synthetized a peptide that corresponds to the full sequence of the surface-exposed FG loop. We tested the antigenicity of the linear and the cyclic peptides against HPV16 L1 monoclonal antibodies. We used ELISA to detect anti-peptide antibodies in sera and cervical secretions of 179 Tunisian women, and we applied polymerase chain reaction and direct sequencing methods to detect and genotype HPV DNA.ResultsBoth the linear and the cyclic peptides were recognized by the same neutralizing monoclonal antibodies, but the cyclic peptide was more reactive with human sera. The prevalence of the anti-peptide antibodies in sera was higher in women with low-grade squamous intraepithelial lesions (LGSIL) than in women with high-grade squamous intraepithelial lesions (HGSIL) (44% and 15%, respectively). This contrasts with HPV16 DNA prevalence. Compared to women from the general population, systemic IgG prevalence was significantly higher among sex workers (25%; P = 0.002) and women with LGSIL (44%; P = 0.001). In addition, systemic IgA and cervical IgG prevalence was higher among sex workers only (P = 0.002 and P = 0.001, respectively). We did not observe anti-peptide IgG antibodies in women with a current HPV16 infection.ConclusionAnti-peptide IgG in sera or in cervical secretions could be markers of an effective natural immunization against HPV16. This may open novel perspectives for monitoring vaccinated women and for the design of synthetic peptide-based vaccines.

Production and characterization of monoclonal antibodies against GII.6 norovirus virus-like particles

In this study we generated and characterized a series of monoclonal antibodies (mAbs) against GII.6 norovirus (NoV) virus like particles (VLPs). Mice were immunized with purified GII.6 NoV VLPs and peptide-bovine serum albumin (BSA) conjugates with the peptide sequence (31 aa) derived from the trypsin cleavage region. An indirect enzyme-linked immunosorbent assay was used to identify positive cell clones during cloning and subcloning, and an in vitro VLP-histo-blood group antigens (HBGAs) binding blockade assay was used to identify mAbs with blocking ability. A total of seven mAbs comprising five (1F7, 1F11, 2B6, 2C4, and 2E10) reactive with major capsid proteins (VP1) and two (1E5 and 2B2) reactive with both VP1 proteins and the peptide were identified. mAb 1F7, 1F11, and 2B6 were identified as blocking antibodies. Sandwich ELISA indicated that all these mAbs recognized soluble GII.6 NoV VLPs. Cross-reactivities with GI.7, GII.3, and GII.4 NoV VLPs were observed in indirect and sandwich ELISA. Western blot analysis indicated that all non-blocking mAbs recognized denatured GII.6 VP1 proteins and blocking mAbs only recognized non-denatured proteins. The in vitro VLP-HBGA binding blockade assay indicated that the three blocking antibodies exhibited blocking effects against GII.6 NoV VLPs, but not GI.7, GII.3, and GII.4 NoV VLPs. Epitope mapping and HBGA blocking assay indicated that mAbs targeting the predicted surface-exposed loop region did not have blocking effects, suggesting a possible important role of this region in regulating NoV-HBGA interactions. This is the first report regarding the characterization of mAbs with blocking ability against GII.6 NoV VLPs. These mAbs might be useful in facilitating our understanding of this group of viruses.

Surface-exposed loops L7 and L8 of Haemophilus (Glaesserella) parasuis OmpP2 contribute to the expression of proinflammatory cytokines in porcine alveolar macrophages

Outer membrane protein P2 (OmpP2) of the virulent Haemophilus (Glaesserella) parasuis has been shown to induce the release of proinflammatory cytokines. The OmpP2 protein is composed of eight or nine surface-exposed loops, but it is unclear which of them participates in the OmpP2-induced inflammatory response. In this study, we synthesized linear peptides corresponding to surface-exposed loops L1–L8 of OmpP2 from the virulent H. parasuis SC096 strain to stimulate porcine alveolar macrophages (PAMs) in vitro. We found that both L7 and L8 significantly upregulated the mRNA expression of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-17, and IL-23 and the chemokines CCL-4 and CCL-5 in a time- and dose-dependent manner. Additionally, we constructed ompP2ΔLoop7 and ompP2ΔLoop8 mutant SC096 strains and extracted their native OmpP2 proteins to stimulate PAMs. These mutant proteins induced significantly less mRNA expression of inflammatory cytokines than SC096 OmpP2. Next, the amino acid sequences of L7 and L8 from 15 serovars of H. parasuis OmpP2 were aligned. These sequences were relatively conserved among the most virulent reference strains, suggesting that L7 and L8 are the most active peptides of the OmpP2 protein. Furthermore, L7 and L8 significantly upregulated the NF-κB and AP-1 activity levels based on luciferase reporter assays in a dose-dependent manner. Therefore, our results demonstrated that both surface-exposed loops L7 and L8 of H. parasuis OmpP2 induced the expression of proinflammatory cytokines possibly by activating the NF-κB and MAPK signalling pathways in cells infected by H. parasuis.

Genomic Analyses Identify Novel Molecular Signatures Specific for the Caenorhabditis and other Nematode Taxa Providing Novel Means for Genetic and Biochemical Studies.

The phylum Nematoda encompasses numerous free-living as well as parasitic members, including the widely used animal model Caenorhabditis elegans, with significant impact on human health, agriculture, and environment. In view of the importance of nematodes, it is of much interest to identify novel molecular characteristics that are distinctive features of this phylum, or specific taxonomic groups/clades within it, thereby providing innovative means for diagnostics as well as genetic and biochemical studies. Using genome sequences for 52 available nematodes, a robust phylogenetic tree was constructed based on concatenated sequences of 17 conserved proteins. The branching of species in this tree provides important insights into the evolutionary relationships among the studied nematode species. In parallel, detailed comparative analyses on protein sequences from nematodes (Caenorhabditis) species reported here have identified 52 novel molecular signatures (or synapomorphies) consisting of conserved signature indels (CSIs) in different proteins, which are uniquely shared by the homologs from either all genome-sequenced Caenorhabditis species or a number of higher taxonomic clades of nematodes encompassing this genus. Of these molecular signatures, 39 CSIs in proteins involved in diverse functions are uniquely present in all Caenorhabditis species providing reliable means for distinguishing this group of nematodes in molecular terms. The remainder of the CSIs are specific for a number of higher clades of nematodes and offer important insights into the evolutionary relationships among these species. The structural locations of some of the nematodes-specific CSIs were also mapped in the structural models of the corresponding proteins. All of the studied CSIs are localized within the surface-exposed loops of the proteins suggesting that they may potentially be involved in mediating novel protein–protein or protein–ligand interactions, which are specific for these groups of nematodes. The identified CSIs, due to their exclusivity for the indicated groups, provide reliable means for the identification of species within these nematodes groups in molecular terms. Further, due to the predicted roles of these CSIs in cellular functions, they provide important tools for genetic and biochemical studies in Caenorhabditis and other nematodes.

Exogenous Peptide Replacement of Three Loops in Domain II of Cry1Ab Toxin Reduces Its Toxicity against Helicoverpa armigera (Lepidoptera: Noctuidae)

To investigate the contribution of surface-exposed loops (loops 1, 2 and 3) in the toxicity of Cry1Ab against Helicoverpa armigera larvae, three loop-replaced Cry1Ab mutants were prokaryotically expressed in Escherichia coli BL21 (DE3). Proteolytic processing of Cry1Ab toxins with trypsin indicated that the replacement of each surface-exposed loop caused no damage to toxin stability. Results of in vitro binding and competition assay indicated that, similarly to the native Cry1Ab toxin, the mutants retained almost the same binding affinity and specificity with brush border membrane vesicles (BBMV) of H. armigera. Nevertheless, H. armigera larvae fed with each loop-replaced Cry1Ab mutant showed less growth inhibition in comparison with those fed with native Cry1Ab toxin. The most significant decrease in toxicity was observed from larvae fed with loop 2-replaced Cry1Ab mutant, with a c. 12-fold increase in the weight of tested larvae relative to that of larvae fed native Cry1Ab toxin. The results demonstrate that the replacement of three surface-exposed loops located in domain II of Cry1Ab toxin may result in a certain reduction in the toxicity. Relative to loop 1 and loop 3, loop 2 seem to play a more important role in generating toxicity of Cry1Ab against H. armigera larvae.

The surface-exposed loop region of norovirus GII.3 VP1 plays an essential role in binding histo-blood group antigens.

Trypsin digestion promotes disassembly of GII.3 NoV virus-like particles (VLPs) and binding of VLPs to salivary histo-blood group antigens (HBGAs), but it is not clear which specific regions or residues mediate viral attachment to HBGAs. An earlier study indicated that arginine residues in the predicted surface-exposed loop region are susceptible to trypsin digestion. Here, we introduced single or multiple substitutions of four arginine residues located in the predicted surface-exposed loop region of the GII.3 NoV capsid protein (VP1) and observed their effects on susceptibility to trypsin digestion and binding to HBGAs. All of the mutations in VP1, including single substitutions (R287G, R292G, R296G or R307G) and quadruple substitutions (R287G, R292G, R296G and R307G), permitted successful VLP assembly. After tryptic digestion, all VP1 proteins bearing single point mutations were cleaved, resulting in complete digestion or single fragments with various molecular sizes (27-35kDa), while the VP1 protein bearing four substitutions was cleaved into two fragments (27-55kDa). Binding assays using synthetic and salivary HBGAs showed that none of the VP1 mutants (singly or quadruply substituted) exhibited detectable binding to HBGA before or after trypsin cleavage. These results indicated that arginine residues within the predicted surface loop region of GII.3 NoV VP1 were involved directly or indirectly in binding salivary HBGAs and could potentially mediate the HBGA-GII.3 NoV interactions through which host cells become infected.

Novel Mutations in nsP2 Abolish Chikungunya Virus-Induced Transcriptional Shutoff and Make the Virus Less Cytopathic without Affecting Its Replication Rates.

Alphavirus infections are characterized by global inhibition of cellular transcription and rapid induction of a cytopathic effect (CPE) in cells of vertebrate origin. Transcriptional shutoff impedes the cellular response to alphavirus replication and prevents establishment of an antiviral state. Chikungunya virus (CHIKV) is a highly pathogenic alphavirus representative, and its nonstructural protein 2 (nsP2) plays critical roles in both inhibition of transcription and CPE development. Previously, we have identified a small peptide in Sindbis virus (SINV) nsP2 (VLoop) that determined the protein's transcriptional inhibition function. It is located in the surface-exposed loop of the carboxy-terminal domain of nsP2 and exhibits high variability between members of different alphavirus serocomplexes. In this study, we found that SINV-specific mutations could not be directly applied to CHIKV. However, by using a new selection approach, we identified a variety of new VLoop variants that made CHIKV and its replicons incapable of inhibiting cellular transcription and dramatically less cytopathic. Importantly, the mutations had no negative effect on RNA and viral replication rates. In contrast to parental CHIKV, the developed VLoop mutants were unable to block induction of type I interferon. Consequently, they were cleared from interferon (IFN)-competent cells without CPE development. Alternatively, in murine cells that have defects in type I IFN production or signaling, the VLoop mutants established persistent, noncytopathic replication. The mutations in nsP2 VLoop may be used for development of new vaccine candidates against alphavirus infections and vectors for expression of heterologous proteins.IMPORTANCE Chikungunya virus is an important human pathogen which now circulates in both the Old and New Worlds. As in the case of other Old World alphaviruses, CHIKV nsP2 not only has enzymatic functions in viral RNA replication but also is a critical inhibitor of the antiviral response and one of the determinants of CHIKV pathogenesis. In this study, we have applied a new strategy to select a variety of CHIKV nsP2 mutants that no longer exhibited transcription-inhibitory functions. The designed CHIKV variants became potent type I interferon inducers and acquired a less cytopathic phenotype. Importantly, they demonstrated the same replication rates as the parental CHIKV. Mutations in the same identified peptide of nsP2 proteins derived from other Old World alphaviruses also abolished their nuclear functions. Such mutations can be further exploited for development of new attenuated alphaviruses.

Structural and Functional Role of the Surface-Exposed Loops of Ail During Complement-Mediated Evasion by Y. pestis

Yersinia pestis, the causative agent of plague, promotes its proliferation in the host by expressing various bacterial proteins that inhibit the host innate immune system. One of these is attachment invasion locus (Ail), which is an eight-stranded β-barrel outer membrane protein with four surface exposed extracellular loops. Ail is essential for Y. pestis pathogenesis and proliferation as it recruits host serum proteins to inhibit the complement-dependent bacteriolysis and inflammatory response, and regulates host cell attachment and delivery of Yersinia outer proteins to host tissue. Recently, we demonstrated that Y. pestis can also recruit vitronectin (Vn) from the human serum in Ail-dependent manner. Vn is a 459-residue multi-domain protein that interacts with various molecules to promote cell adhesion and migration, and inhibits formation of the membrane attack complex, indicating that the interaction between Ail and Vn may be responsible for promoting Y. pestis survival and pathogenesis. Notably, previous works have indicated that serum proteins such as Vn are recruited by Ail via its extracellular loops. Thus, we hypothesized that Ail's extracellular loops may also be responsible for binding to Vn. To characterize this interaction, amino acids in each extracellular loop of Ail were substituted for alanine to individually characterize the loops for Vn binding. The structure and function of each Ail loop mutant was characterized in a native like environment inside membrane (detergent micelle and nanodiscs) by solution state NMR and ELISA binding assays. These studies identify specific extracellular loops of Ail that are important for binding to Vn, which will guide future developments of therapeutics aimed at combating Y. pestis infections.

An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion

Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface-exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge-dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000-fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single-chain variable fragment (scFv) homolog of IgG12, G12-scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12-scFv to a conformational S epitope shared with IgG12. Despite 20-fold lower affinity, G12-scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50 = 1.8 nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50 = 1.25 nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications.