Hsien-Sheng Yin

Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation

Enveloped viruses have evolved complex glycoprotein machinery that drives the fusion of viral and cellular membranes, permitting entry of the viral genome into the cell. For the paramyxoviruses, the fusion (F) protein catalyses this membrane merger and entry step, and it has been postulated that the F protein undergoes complex refolding during this process. Here we report the crystal structure of the parainfluenza virus 5 F protein in its prefusion conformation, stabilized by the addition of a carboxy-terminal trimerization domain. The structure of the F protein shows that there are profound conformational differences between the pre- and postfusion states, involving transformations in secondary and tertiary structure. The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.

Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers

Mitochondria are key organelles in mammary cells responsible for several cellular functions including growth, division, and energy metabolism. In this study, mitochondrial proteins were enriched for proteomics analysis with the state-of-the-art two-dimensional differential gel electrophoresis and matrix-assistant laser desorption ionization–time-of-flight mass spectrometry strategy to compare and identify the mitochondrial protein profiling changes between three breast cell lines with different tumorigenicity and metastasis. The proteomics results demonstrate more than 1,500 protein features were resolved from the equal amount pooled from three purified mitochondrial proteins, and 125 differentially expressed spots were identified by their peptide finger print, in which, 33 identified proteins belonged to mitochondrial proteins. Eighteen out of these 33 identified mitochondrial proteins such as SCaMC-1 have not been reported in breast cancer research to our knowledge. Additionally, mitochondrial protein prohibitin has shown to be differentially distributed in mitochondria and in nucleus for normal breast cells and breast cancer cell lines, respectively. To sum up, our approach to identify the mitochondrial proteins in various stages of breast cancer progression and the identified proteins may be further evaluated as potential breast cancer markers in prognosis and therapy.

Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens.

A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.

Structural and functional comparison of cytokine interleukin-1 beta from chicken and human

Interleukin-1 beta (IL-1β) is an important cytokine in the immune system. The properties of avian IL-1βs are less well understood than the mammalian IL-1βs, and there is no available structure of avian IL-1βs in the Protein Data Bank. Here, we report the crystal structures of wild-type and Y157F mutant IL-1βs from chicken. Both the wild-type and mutant IL-1βs share a beta-trefoil conformation similar to that of human IL-1β and also have an internal hydrophobic cavity. However, the cavity sizes clearly differ from that of human IL-1β due to the packing of hydrophobic residues. Our studies also reveal that the relative thermal stability of IL-1βs does not correlate with cavity size but rather is dependent on the amino acid residues present around the cavity. This cavity serves as a scaffold for maintaining the structure of the IL-1β core region but does not have a biological function per se. Moreover, we found that human IL-1β cannot induce chemokine expression in chicken fibroblasts or elevate plasma cortisol levels in chickens, implying a lack of cross-species bioactivity. Close examination reveals that significant structural and sequence differences occur in the terminal and some loop regions between human and chicken IL-1βs. These variable regions have been shown to be critical for receptor binding, thus resulting in a lack of species cross-reactivity between human and chicken IL-1β.

Potentiation of cell-mediated immune responses against recombinant HN protein of Newcastle disease virus by recombinant chicken IL-18.

In this study, recombinant fowlpox viruses (rFPV/HN) expressing Newcastle disease virus (NDV) HN protein and rFPV/HN/chIL-18 co-expressing chicken IL-18 (chIL-18) and HN protein have been constructed and characterized. The co-expressed rHN/chIL-18 antigen or rchIL-18, expressed by our previous construct rFPV/chIL-18 and co-administered with NDV rHN, was assessed for its immunostimulatory activities and protection against NDV challenge in 2-week-old chickens. Chickens were vaccinated, intramuscularly, with various amounts of rHN or rHN/chIL-18 mixed with mineral oil. Production of hemagglutination-inhibition (HI) antibody depended on the concentration of the injected rHN or rHN/chIL-18. The lower HI antibody titers were obtained in chickens group rHN/chIL-18/6 and rHN/chIL-18/7, receiving 50 ng rHN/16.5 ng chIL-18 with mineral oil and 20 ng rHN/6.6 ng chIL-18 with mineral oil, respectively, compared to those in chickens rHN/6 and rHN/7, respectively receiving 50 ng and 20 ng rHN with mineral oil alone. However, the same protection rates were obtained from chickens in groups rHN/chIL-18/6 and rHN/6. Chicken groups rHN/chIL-18/7 and rHN/chIL-18/8 showed higher protective achievements than those in groups rHN/7 and rHN/8, respectively. When rchIL-18 was co-injected with 20ng rHN plus mineral oil, low level of HI antibody titer was produced; whereas, higher level of IFN-γ production and full protection rates were obtained. On the other hand, lower levels of IFN-γ production and lower protection rate (67%) were obtained in chickens injected with the same amount of rHN with mineral oil alone. Similar results were obtained when 10 ng rHN was used. Thus, when the concentration of rHN decreased to 50 ng or less, rchIL-18 reduced HI antibody production. The increase in IFN-γ production suggested that the enhancement of the cell-mediated immunity might confer the protection from NDV challenge, even accompanied with low HI antibody induction.

Proteomics Analysis of the DF-1 Chicken Fibroblasts Infected with Avian Reovirus Strain S1133

Background Avian reovirus (ARV) is a member of the Orthoreovirus genus in the Reoviridae family. It is the etiological agent of several diseases, among which viral arthritis and malabsorption syndrome are the most commercially important, causing considerable economic losses in the poultry industry. Although a small but increasing number of reports have characterized some aspects of ARV infection, global changes in protein expression in ARV-infected host cells have not been examined. The current study used a proteomics approach to obtain a comprehensive view of changes in protein levels in host cells upon infection by ARV. Methodology and Principal Findings The proteomics profiles of DF-1 chicken fibroblast cells infected with ARV strain S1133 were analyzed by two-dimensional differential-image gel electrophoresis. The majority of protein expression changes (≥1.5 fold, p<0.05) occurred at 72 h post-infection. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 51 proteins with differential expression levels, including 25 that were upregulated during ARV infection and 26 that were downregulated. These proteins were divided into eight groups according to biological function: signal transduction, stress response, RNA processing, the ubiquitin-proteasome pathway, lipid metabolism, carbohydrate metabolism, energy metabolism, and cytoskeleton organization. They were further examined by immunoblotting to validate the observed alterations in protein expression. Conclusion/Significance This is the first report of a time-course proteomic analysis of ARV-infected host cells. Notably, all identified proteins involved in signal transduction, RNA processing, and the ubiquitin-proteasome pathway were downregulated in infected cells, whereas proteins involved in DNA synthesis, apoptosis, and energy production pathways were upregulated. In addition, other differentially expressed proteins were linked with the cytoskeleton, metabolism, redox regulation, and stress response. These proteomics data provide valuable information about host cell responses to ARV infection and will facilitate further studies of the molecular mechanisms underlying ARV pathogenesis.

Comparative analysis of receptor binding by chicken and human interleukin-1β

Interleukin-1β (IL-1β) is an important cytokine in the immune system. Mammalian and avian IL-1βs share only 31-35% sequence identity, and the function of avian IL-1βs is less well understood by comparison. Although chicken and mammalian IL-1βs have similar tertiary structures, these ILs differ significantly with respect to receptor activation. Analysis of the structures and sequences of IL-1βs reveals that the major differences lie in loops. Modeling docking of chicken IL-1β to its receptor reveals that these variable loops are critical for receptor binding. Molecular dynamics simulations of the IL-1βs reveal significant changes in the dynamic range of motion upon receptor binding. Loops 3 and 9 of the unbound chicken IL-1β had greater fluctuations compared with the other loops. Upon binding, the flexibility of these loops, which directly contact the receptor, markedly decreases. Taken together, these results suggest that receptor binding leads to not only favorable enthalpy but also lower conformational entropy.

Development of ELISA kits for antibodies against avian reovirus using the σC and σB proteins expressed in the methyltropic yeast Pichia pastoris

Both the sigmaC and sigmaB proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 sigmaC, 1071-1 sigmaC, S1133 sigmaB, and S1133 sigmaC-sigmaB fusion genes of ARV were cloned into a secreted vector pPICZalphaA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV sigmaC, sigmaB, and sigmaC-sigmaB fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1sigmaC and S1133 sigmaB and S1133 sigmaC-sigmaB fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1sigmaC/S1133 sigmaB and S1133 sigmaC-sigmaB ELISA kits were 0.24 and 0.12, respectively. Forty-four serum neutralization test-positive and twenty-eight serum neutralization-negative samples from vaccinated and commercial farm chickens were tested by the new ELISA kits and by the conventional ELISA. The new ELISA kits have higher positive rates than the conventional ELISA. The results revealed that the correlation rates for the serum neutralization titer and the absorbance values with the new ELISA kits and the conventional ELISA were 100% and 95.8%, respectively.

Circular permutation of chicken interleukin-1 beta enhances its thermostability

Interleukin-1β is a cytokine critically involved in immune and inflammatory responses. To extend its use as a component of avian vaccines, a circularly permuted chicken interleukin-1β was synthesized that maintains its activity after pre-incubation at high temperatures, unlike wild-type chicken interleukin-1β, which is irreversibly inactivated at high temperatures.

Structural basis for the specific recognition of IL-18 by its alpha receptor.

Interleukin 18 (IL‐18), a member of the IL‐1 family of cytokines, is an important regulator of innate and acquired immune responses. It signals through its ligand‐binding primary receptor IL‐18Rα and accessory receptor IL‐18Rβ. Here we report the crystal structure of IL‐18 with the ectodomain of IL‐18Rα, which reveals the structural basis for their specific recognition. It confirms that surface charge complementarity determines the ligand‐binding specificity of primary receptors in the IL‐1 receptor family. We suggest that IL‐18 signaling complex adopts an architecture similar to other agonistic cytokines and propose a general ligand‐receptor assembly and activation model for the IL‐1 family.