E2 Site Research Articles

Approaching rational epitope vaccine design for hepatitis C virus with meta-server and multivalent scaffolding.

Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314–324) and E2 (residues 412–423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.

The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

Preferable sites and orientations of transgene inserted in the adenovirus vector genome: The E3 site may be unfavorable for transgene position

The adenovirus vector (AdV) can carry two transgenes in its genome, the therapeutic gene and a reporter gene, for example. The E3 insertion site has often been used for the expression of the second transgene. A transgene can be inserted at six different sites/orientations: E1, E3 and E4 sites, and right and left orientations. However, the best combination of the insertion sites and orientations as for the titers and the expression levels has not sufficiently been studied. We attempted to construct 18 AdVs producing GFP or LacZ gene driven by the EF1α promoter and Cre gene driven by the α-fetoprotein promoter. The AdV containing GFP gene at E3 in the rightward orientation (GFP-E3R) was not available. The LacZ-E3R AdV showed 20-fold lower titer and 50-fold lower level of fiber mRNA than the control E1L AdV. Notably, we found four aberrantly spliced mRNAs in the LacZ-E3L/R AdVs, probably explaining their very low titers. Although the transgene expression levels in the E4R AdVs were about threefold lower than those in the E1L AdVs, their titers are comparable with that of E1L AdVs. We concluded that E1L and E4R sites/orientations are preferable for expressing the main target gene and a second gene, respectively.

Evaluation of acidity estimation methods for mine drainage, Pennsylvania, USA.

Eighteen sites impacted by abandoned mine drainage (AMD) in Pennsylvania were sampled and measured for pH, acidity, alkalinity, metal ions, and sulfate. This study compared the accuracy of four acidity calculation methods with measured hot peroxide acidity and identified the most accurate calculation method for each site as a function of pH and sulfate concentration. Method E1 was the sum of proton and acidity based on total metal concentrations; method E2 added alkalinity; method E3 also accounted for aluminum speciation and temperature effects; and method E4 accounted for sulfate speciation. To evaluate errors between measured and predicted acidity, the Nash-Sutcliffe efficiency (NSE), the coefficient of determination (R (2)), and the root mean square error to standard deviation ratio (RSR) methods were applied. The error evaluation results show that E1, E2, E3, and E4 sites were most accurate at 0, 9, 4, and 5 of the sites, respectively. Sites where E2 was most accurate had pH greater than 4.0 and less than 400mg/L of sulfate. Sites where E3 was most accurate had pH greater than 4.0 and sulfate greater than 400mg/L with two exceptions. Sites where E4 was most accurate had pH less than 4.0 and more than 400mg/L sulfate with one exception. The results indicate that acidity in AMD-affected streams can be accurately predicted by using pH, alkalinity, sulfate, Fe(II), Mn(II), and Al(III) concentrations in one or more of the identified equations, and that the appropriate equation for prediction can be selected based on pH and sulfate concentration.

Adsorption of 17β-estradiol by multi-walled carbon nanotubes in natural waters with or without aquatic colloids

The adsorption of 17β-estradiol (E2) by multi-walled carbon nanotubes (MWCNTs) was investigated under environmentally relevant conditions. Six different surface water samples were collected in different areas of China. The effects of aquatic colloids on E2 adsorption by MWCNTs were estimated by separating the colloids using cross-flow filtration. The results show that, in the absence of aquatic colloids, the distribution coefficient (Kd) for E2 adsorption by MWCNTs shows strong correlation with the zeta potential of the MWCNTs and the fluorescence intensities of the water samples, indicating that the binding between dissolved organic matter and E2 and the increasing negative charges of MWCNTs inhibits the E2 adsorption by MWCNTs. In the presence of aquatic colloids, the Kd values for E2 adsorption by MWCNTs are negatively affected by various factors simultaneously, including the colloid content, the intensities of two typical fluorescence peaks, total organic carbon content, conductivity, and zeta potential of MWCNTs. The binding of aquatic colloids and MWCNTs, which was shown using transmission electron microscopy images, may inhibit or promote E2 adsorption by blocking or providing the adsorption sites for E2. These two effects may counteract and result in different effects of various aquatic colloids on E2 adsorption by MWCNTs.

Identification, purification and partial characterization of low molecular weight protein inhibitor of Na⁺/K⁺-ATPase from pulmonary artery smooth muscle cells.

We have identified a novel endogenous low mol wt. (15.6 kDa) protein inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of bovine pulmonary artery smooth muscle cells. The inhibitor showed different affinities toward the α₂β₁ and α₁β₁ isozymes of Na(+)/K(+)-ATPase, where α₂ is more sensitive than α₁. The inhibitor interacted reversibly to the E1 site of the enzyme and blocked the phosphorylated intermediate formation. Circular dichroism study suggests that the inhibitor causes an alteration in the confirmation of the enzyme.

Production of multi‐transgenic pigs tailored for xenotransplantation by using zinc‐finger nucleases and sleeping beauty transposons

Multi‐transgenic pigs are required for prolonging survival of porcine xenografts after transplantation into primate recipients. A homozygous knockout of the alpha1,3‐galactosyltransferase gene (GGTA‐1, encoding for Gal‐epitopes) is critical for controlling the hyperacute rejection response. Nevertheless, the additional expression of complement regulatory proteins such as CD55 (decay accelerating factor, DAF) on a GGTA‐1 KO background is thought to further improve survival after xenotransplantation. The next hurdle is the acute vascular rejection (AVR), which is primarily caused by endothelial cell activation. Previous experiments had shown that expression of anti‐apoptotic/anti‐inflammatory genes, such as human heme oxygenase‐1 (hHO‐1) or human A20 in pig organs may overcome AVR (1, 2). Adult fibroblasts from previously generated hHO‐1 transgenic pigs were transfected with Zinc‐finger nuclease plasmids directed towards knockout of the GGTA‐1 gene. Transfected cells were selected via a magnetic bead selection approach as previously described (3). GGTA‐1‐KO/hHO‐1 cells were used as donor cells in somatic cell nuclear transfer (SCNT). The first pregnancy was terminated at day 25 of gestation and 11 healthy fetuses were obtained. All fetuses were GGTA‐1‐KO and expressed hHO‐1 at similar levels as the hHO‐1 transgenic cell donors. Next, fetal GGTA‐1‐KO/hHO‐1 cells from one fetus (C6F5) were transfected with a Sleeping Beauty (SB) transposon (kindly provided by Dr. Zoltan Ivics) coding for hCD55. Two positive cell clones were pooled and used as donor cells for SCNT. One pregnancy was terminated on day 25 and four healthy fetuses were obtained which showed the desired GGTA‐1 KO/HO‐1/CD55 genotype. Real‐time PCR detected a 0.9 fold expression of CD55 over the housekeeping gene GAPDH. Compared to human umbilical vein cells (HUVEC), GGTA‐1 KO/HO‐1/CD55 fetal fibroblasts expressed CD55 10–15 fold higher than HUVEC. Two fetuses showed a slightly higher expression of CD55 and are currently used as cell donors for recloning to produce live triple transgenic offspring.To solve the problem of transgene segregation occurring after mating of multitransgenic pigs, we generated two tri‐cistronic SB vectors coding for A20 and CD55 separated by the self‐cleaving peptide E2 site. To select for transfected cells, one vector carried a Tomato‐fluorescence cassette whereas the second conferred puromycin resistance. Red fluorescent/puromycin resistant cells served as donor cells for SCNT. Six healthy fetuses were collected on day 25 of pregnancy; all showed high expression of A20 and CD55 as detected by Real‐time PCR and FACS. One of the fetuses was used as cell donor for recloning. Currently, three pregnancies with A20/CD55 fetuses are going on and will be allowed to go to term. These results show that molecular scissors, including ZFNs and TALENS, and expression vectors such as the Sleeping beauty system are valuable tools to enhance and accelerate the generation of multi‐transgenic pigs for xenotransplantation.

An inter‐laboratory study on the variability in measured concentrations of 17β‐estradiol, testosterone, and 11‐ketotestosterone in white sucker: Implications and recommendations

Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies.

Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

Spatial differences in East scotia ridge hydrothermal vent food webs: influences of chemistry, microbiology and predation on trophodynamics.

The hydrothermal vents on the East Scotia Ridge are the first to be explored in the Antarctic and are dominated by large peltospiroid gastropods, stalked barnacles (Vulcanolepas sp.) and anomuran crabs (Kiwa sp.) but their food webs are unknown. Vent fluid and macroconsumer samples were collected at three vent sites (E2, E9N and E9S) at distances of tens of metres to hundreds of kilometres apart with contrasting vent fluid chemistries to describe trophic interactions and identify potential carbon fixation pathways using stable isotopes. δ13C of dissolved inorganic carbon from vent fluids ranged from −4.6‰ to 0.8‰ at E2 and from −4.4‰ to 1.5‰ at E9. The lowest macroconsumer δ13C was observed in peltospiroid gastropods (−30.0‰ to −31.1‰) and indicated carbon fixation via the Calvin-Benson-Bassham (CBB) cycle by endosymbiotic gamma-Proteobacteria. Highest δ13C occurred in Kiwa sp. (−19.0‰ to −10.5‰), similar to that of the epibionts sampled from their ventral setae. Kiwa sp. δ13C differed among sites, which were attributed to spatial differences in the epibiont community and the relative contribution of carbon fixed via the reductive tricarboxylic acid (rTCA) and CBB cycles assimilated by Kiwa sp. Site differences in carbon fixation pathways were traced into higher trophic levels e.g. a stichasterid asteroid that predates on Kiwa sp. Sponges and anemones at the periphery of E2 assimilated a proportion of epipelagic photosynthetic primary production but this was not observed at E9N. Differences in the δ13C and δ34S values of vent macroconsumers between E2 and E9 sites suggest the relative contributions of photosynthetic and chemoautotrophic carbon fixation (rTCA v CBB) entering the hydrothermal vent food webs vary between the sites.

Analysis of Transcriptional Regulation of the Human miR-17-92 Cluster; Evidence for Involvement of Pim-1

The human polycistronic miRNA cluster miR-17-92 is frequently overexpressed in hematopoietic malignancies and cancers. Its transcription is in part controlled by an E2F-regulated host gene promoter. An intronic A/T-rich region directly upstream of the miRNA coding region also contributes to cluster expression. Our deletion analysis of the A/T-rich region revealed a strong dependence on c-Myc binding to the functional E3 site. Yet, constructs lacking the 5′-proximal ~1.3 kb or 3′-distal ~0.1 kb of the 1.5 kb A/T-rich region still retained residual specific promoter activity, suggesting multiple transcription start sites (TSS) in this region. Furthermore, the protooncogenic kinase, Pim-1, its phosphorylation target HP1γ and c-Myc colocalize to the E3 region, as inferred from chromatin immunoprecipitation. Analysis of pri-miR-17-92 expression levels in K562 and HeLa cells revealed that silencing of E2F3, c-Myc or Pim-1 negatively affects cluster expression, with a synergistic effect caused by c-Myc/Pim-1 double knockdown in HeLa cells. Thus, we show, for the first time, that the protooncogene Pim-1 is part of the network that regulates transcription of the human miR-17-92 cluster.

Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

BackgroundMosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.Methods and FindingsCHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36). Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates.ConclusionsThe greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.

Abstract A160: Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complex.

Abstract Background and Objectives: The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. A longstanding question in this field is how a single lysine residue in the nucleosome is singled out for ubiquitin modification and why only one ubiquitin is added rather than a chain of ubiquitins. To better understand how Bmi1/Ring1b controls monoubiquitination of H2A, we structurally characterized the E2 and nucleosome interaction sites on Bmi1/Ring1b. Methods: X-ray crystallography was used to determine the structure of a complex between Bmi1/Ring1b and UbcH5c, its cognate E2 enzyme. Fluorescence-polarization assays were used to measure the binding of Bmi1/Ring1b to a synthetic DNA duplex. Site-directed mutagenesis was used to assess the importance of residues at the potential Bmi1/Ring1b-DNA interface, and in vitro ubiquitin ligase assays were used to measure the catalytic activity of mutant complexes. Molecular modeling studies were performed using HADDOCK v2.0. Results: We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Through site-directed mutagenesis we have been able to uncouple the E2 binding and the DNA binding activities of Bmi1/Ring1b, indicating that Bmi1/Ring1b uses distinct binding surfaces to recognize E2 and the nucleosomal substrate. Computational modeling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific mono-ubiquitination of H2A. Conclusions: Our data show that the direct interaction of the RING domains with nucleosomal DNA is crucial for the ubiquitin ligase activity of Bmi1/Ring1b. To our knowledge, this is the first example of a RING-domain E3 ligase binding directly to its substrate via the RING domain. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A160.

Abstract LB-177: HPV16 CpG and de novo-cytosine methylation is differentially associated with low-grade versus high-grade anal intraepithelial neoplasia in HIV-infected men

Abstract Objective: To determine if cytosine methylation in CpG and de novo sites is differentially associated with high- or low-grade anal intraepithelial neoplasias (HG-, LG-AIN), HPV16 DNA from182 clinical specimens. Background: Intra-anal cancer (IAC) is an emerging health crisis for gay, bisexual, transgender and other men who have sex with men (MSM) and rates have risen sharply among HIV-infected MSM despite introduction of HAART. High-risk HPVs are a causal risk factor for IAC and are especially common where MSM show HIV-coinfection. Further, cytosine methylation has been posited as an epigenetic transcription regulator that is poorly described in intra-anal dysplasias and cancers. Methods: HPV16 DNA was extracted from anal swab specimens and tested using bisulfite modification, PCR, cloning and sequencing of ∼10 clones/sample. Cervical cancer cell lines, CaSki and SiHa, and paraffin-embedded anal cancer specimens were similarly characterized as controls. Genomic sequences were evaluated using CLUSTAL and BiQ Sequence Alignment analysis software. Descriptive, tabular and multivariate analyses were performed using SAS (Version 9.2). Graphical representations were compiled using SIGMAPLOT (Version 9.0). Findings: Overall, methylation was relatively low in clinical AIN specimens. For 25,085 cytosines, the mean prevalence of methylation was 2.7% (SEM=0.4%) across 3′ L1 and LCR; the prevalence me-CpG, -CpA, -CpT, and -CpC was 3.2% (0.2%), 2.6% (0.1%), 2.5% (0.1%), and 2.7% (0.1%), respectively. However, some variation in HPV16 genomes was observed across specific functional gene sequences for HG- and LG-AIN specimens. Specifically, LG-AINs showed 1.30–2.62 times greater mean prevalence of methylation when compared to HG-AINs, across 24 of the 31 cytosines between nt7428–7564 (p-values<0.05) in the 5′ LCR and enhancer. In total, across the 148 cytosine positions, 37 sites showed statistically significantly greater prevalence of meC in LG-AINs and none showed higher methylation in HG-AINs, yielding a false discovery rate of 0.2 (7.4/37). The nt7428–7564 contains binding sites for E2–1, TEF-1, NF-1, YY1, OCT-1, GRE and potential deamination targets for ApoBec3G. Analyses are ongoing, nonetheless, multivariate analyses show risk for HG-AIN decreases by 27% for each cumulative increase of 100% cytosine-methylation across nt7220–7558, even after we controlled for age, CD4+ T-lymphocyte count, and repeated measurements. Conclusions: Data suggest the HPV16 5′LCR/enhancer region contains a large concentration of cytosine-containing transcription regulatory elements where binding interference by methylation might alter expression of the p97 promoter. Methylation in the HPV16 5′LCR/enhancer may decrease risk for HG-AIN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-177. doi:10.1158/1538-7445.AM2011-LB-177

Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Binding cooperativity guides the formation of protein−nucleic acid complexes, in particular those that are highly regulated such as replication origins and transcription sites. Using the DNA binding domain of the origin binding and transcriptional regulator protein E2 from human papillomavirus type 16 as model, and through isothermal titration calorimetry analysis, we determined a positive, entropy-driven cooperativity upon binding of the protein to its cognate tandem double E2 site. This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained. Two homologous E2 domains, those of HPV18 and HPV11, showed that the enthalpic−entropic components of the reaction and DNA deformation can diverge. Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold. This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

Identification of antigen-specific residues on E2 glycoprotein of classical swine fever virus

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. Our previous study revealed that N-terminal 90 residues (domains B/C) of E2 play key roles in differentiating vaccine strain LPC/AHRI (subgroup 1.1) from the two field strains TD/96/TWN (subgroup 2.1) and 94.4/IL/94/TWN (subgroup 3.4) ( Chang et al., 2010). This study further analyzed the reaction patterns between monoclonal antibodies (mAbs) and expressed hybrid N-terminal of E2 of the abovementioned viruses, revealing that mAbs T33 and C2, mAbs V8 and T23, and mAbs L7 and L150 required binding sites specifically at residues 690–714 in domain B, residues 715–740 in domain C, and residues 741–765 in domain C, respectively. Site-directed mutagenesis further demonstrated that residues 713E and 729D were critical for antigenic specificity of field strain (94.4/IL/94/TWN), while residues 705D and 761K were specific for vaccine strain (LPC/AHRI). These specific residues likely mediated in determining the topography of mAb binding sites of E2 to allow for differentiation between strains based on the premise that the structural integrity of the conformational epitope is maintained.

The Human Papillomavirus E7−E2 Interaction Mechanism in Vitro Reveals a Finely Tuned System for Modulating Available E7 and E2 Proteins

Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionic strength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experiments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with a >or=2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed in excess of E2C. A discrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. The N-terminal domain of E7 interacts with E2C with a K(D) of 0.1 muM, where the stretch of residues 25-40 of E7, encompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement of the soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that the protein-protein interface involves the DNA binding helix of E2. The formation of complexes of different sizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that could regulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2 into E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome.

Enhanced Conductivity and Magnetic Ordering in Isostructural Heavy Atom Radicals

Synthetic methods have been developed to generate the complete series of resonance-stabilized heterocyclic thia/selenazyl radicals 1a-4a. X-ray crystallographic studies confirm that all four radicals are isostructural, belonging to the tetragonal space group P42(1)m. The crystal structures consist of slipped pi-stack arrays of undimerized radicals packed about 4 centers running along the z direction, an arrangement which gives rise to a complex lattice-wide network of close intermolecular E2---E2' contacts. Variable temperature conductivity (sigma) measurements reveal an increase in conductivity with increasing selenium content, particularly so when selenium occupies the E2 position, with sigma(300 K) reaching a maximum (for E1 = E2 = Se) of 3.0 x 10(-4) S cm(-1). Thermal activation energies E(act) follow a similar profile, decreasing with increasing selenium content along the series 1a (0.43 eV), 3a (0.31 eV), 2a (0.27 eV), 4a (0.19 eV). Variable temperature magnetic susceptibility measurements indicate that all four radicals exhibit S = 1/2 Curie-Weiss behavior over the temperature range 20-300 K. At lower temperatures, the three selenium-based radicals display magnetic ordering. Radical 3a, with selenium positioned at the E1 site, undergoes a phase transition at 14 K to a weakly spin-canted (phi = 0.010 degrees) antiferromagnetic state. By contrast, radicals 2a and 4a, which both possess selenium in the E2 position, order ferromagnetically, with Curie temperatures of T(c) = 12.8 and 17.0 K, respectively. The coercive fields H(c) at 2 K of 2a (250 Oe) and 4a (1370 Oe) are much larger than those seen in conventional light atom organic ferromagnets. The transport properties of the entire series 1a-4a are discussed in the light of Extended Hückel Theory band structure calculations.

Extended Polypeptide Linkers Establish the Spatial Architecture of a Pyruvate Dehydrogenase Multienzyme Complex

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.

Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins

Recently, the characterization of a cell culture system allowing the amplification of an authentic virus, named hepatitis C virus cell culture (HCVcc), has been reported by several groups. To obtain higher HCV particle productions, we investigated the potential effect of some amino acid changes on the infectivity of the JFH-1 isolate. As a first approach, successive infections of naïve Huh-7 cells were performed until high viral titres were obtained, and mutations that appeared during this selection were identified by sequencing. Only one major modification, N534K, located in the E2 glycoprotein sequence was found. Interestingly, this mutation prevented core glycosylation of E2 site 6. In addition, JFH-1 generated with this modification facilitated the infection of Huh-7 cells. In a second approach to identify mutations favouring HCVcc infectivity, we exploited the observation that a chimeric virus containing the genotype 1a core protein in the context of JFH-1 background was more infectious than wild-type JFH-1 isolate. Sequence alignment between JFH-1 and our chimera, led us to identify two major positions, 172 and 173, which were not occupied by similar amino acids in these two viruses. Importantly, higher viral titres were obtained by introducing these residues in the context of wild-type JFH-1. Altogether, our data indicate that a more robust production of HCVcc particles can be obtained by introducing a few specific mutations in JFH-1 structural proteins.