Long Huw Lee

National Chung Hsing University, Taichung, Taiwan, Taiwan

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Publications (17)34.27 Total impact

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    ABSTRACT: A cDNA encoding a 7 transmembrane (7TM) receptor gene from the adherent cells of chicken peripheral blood mononuclear cells (PBMC) was cloned and characterized. The open reading frame of the chicken-7TM (Ch-7TM) receptor gene was 1008 nucleotides long, encoding a protein of 335 amino acid residues with a molecular mass of approximately 37.1 kDa. Hydrophobic stretches indicated the presence of 7 TM domains. Moreover, the complete nucleotide sequences encoding 7TM of duck (Du-7TM) and goose (Go-7TM), corresponding to the open reading frame of Ch-7TM, were determined. Each of the Du- and Go-7TM encoding regions comprised 990 nucleotides, representing an 18-nucleotide deletion in alignment with the Ch-7TM encoding region, resulting in a 6-amino-acid deletion at the 3'-end. No signal peptides were predicted. Six phosphorylation sites were predicted and conserved for all three 7TMs. The proteins of the three 7TMs were similar, with 11 conserved cysteine residues. No glycosylation sites could be predicted. The results of the pairwise comparisons indicated that the Ch-7TM encoding region and Ch-7TM protein were the least similar to those of Du- and Go-7TMs. These results were in accordance with those of the phylogenetic analysis, which indicated that the Du- and Go-7TM encoding regions clustered, but were separated from the Ch-7TM encoding region. Monoclonal antibody B28D5 was prepared from spleens of mice immunized with the bacterially expressed N-terminal (55 amino acid residues) region of the Ch-7TM protein for further use. Double staining with B28D5 and KUL01 suggested that Ch-7TM was expressed in subsets of the adherent cells, among which a subset that was recognized with both antibodies was likely of monocyte and macrophage lineage. However, the fluorescence intensities of B28D5 and, particularly, KUL01 decreased after the adherent cells were incubated for additional 48 h.
    PLoS ONE 01/2014; 9(1):e86880. · 3.53 Impact Factor
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    ABSTRACT: 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.
    Veterinary Immunology and Immunopathology 06/2011; 141(3-4):283-92. · 1.88 Impact Factor
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    ABSTRACT: Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge.
    Veterinary Microbiology 02/2011; 151(3-4):220-8. · 3.13 Impact Factor
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    ABSTRACT: 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.
    Veterinary Immunology and Immunopathology 10/2010; 139(2-4):167-75. · 1.88 Impact Factor
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    ABSTRACT: The single chain chicken interleukin-12 (chIL-12)- or mature chIL-18-encoding region was cloned into the nonessential gene F11L of fowlpox virus (FPV) to generate the recombinant (r) FPV/chIL-12 (rFPV/chIL-12) or rFPV/chIL-18 for rchIL-12 or rchIL-18 production. Splenocytes of chickens were cultured with various dilutions of binary-ethylenimine (BEI)-inactivated rFPV/chIL-12 or rFPV/chIL-18-infected cell lysate for 48 h for interferon-γ (IFN-γ) determination. It was found that 1:10,000 chIL-12 or 1:10 chIL-18 cell lysate stimulated the highest levels of IFN-γ production. When chickens given BEI-treated 1:10 rchIL-12 or rchIL-18 cell lysate, intraperitoneally, or rFPV (0.2 × 105 TCID50) by wing-web puncture, the highest level of IFN-γ was detected in sera on day 3 postinoculation (dpi). In the splenocyte culture supernatants, the highest level of IFN-γ was detected at 14 dpi or 21 dpi in responses to rchIL-12 or rchIL-18, respectively. The results indicated that rchIL-12 or rchIL-18 could induce IFN-γ production both in vitro and in vivo assays, suggesting that both are biologically active and may allow them to be used in the future as the biological adjuvant in the poultry vaccine development, particularly co-administering with vaccine antigens.
    Process Biochemistry. 01/2010; 45(7):1057-1064.
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    ABSTRACT: Inhibitors of viral disassembly or RNA and protein synthesis, viral disassembly intermediates (infectious subviral particles, ISVP), binary ethylenimine-inactivated virions, and viral particles lacking genomic double-stranded (ds) RNA (empty particles) were used to assess the expression of interleukin-1beta (IL-1beta) mRNA in chicken (chIL-1beta) macrophages in response to avian reovirus. The results demonstrate that two distinct expression patterns of chIL-1beta mRNA mediated by different steps in viral replication were found. Viral disassembly was required for the induction of a rapid, transient expression pattern of chIL-1beta mRNA that was rapidly induced at 30 min, with maximal levels reached by 2 h, and fell to a low level within 6 h post-inoculation, while viral RNA synthesis rather than protein translation, which was subsequent to membrane penetration, was required to induce a stable, sustained expression pattern of chIL-1beta mRNA that occurred at and after 6 h post-inoculation. In addition, the induction of chIL-1beta mRNA expression by the empty particles and ISVP was extremely weak, compared with the active dsRNA(+) virions or binary ethylenimine-inactivated virions, suggesting that the presence of dsRNA, even if transcriptionally inactive, may be an important factor in this response.
    Journal of General Virology 05/2008; 89(Pt 4):1059-68. · 3.13 Impact Factor
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    ABSTRACT: Interleukin (IL)-8-encoding regions of five avian species were cloned, sequenced and characterized. Each IL-8-encoding region is 312 nucleotides long and encodes IL-8 which is 103 amino acids. Pairwise sequence analysis showed that sequence identities of IL-8-encoding regions ranged from 87% to 100%. The IL-8 protein identities varied from 84% to 100%. Phylogenetic analysis indicated that IL-8-encoding regions and encoded proteins of chicken, duck, goose and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from binding reactivities of antiserum against each recombinant IL-8 (rIL-8) protein to homologous or heterologous rIL-8 proteins, chemotactic activities of each rIL-8 protein or reduction levels of the chemotactic activity of rIL-8 protein which was pretreated with homologous or heterlogous antiserum have suggested that all five IL-8 proteins were functionally active, and shared structural and functional identity with each other.
    Veterinary Immunology and Immunopathology 04/2008; 125(3-4):205-15. · 1.88 Impact Factor
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    ABSTRACT: Sequence and phylogenetic analysis of lambdaA and lambdaC protein encoding genes of 12 avian reoviruses is described. The sequence of lambdaA possesses a variable region (residues 19-51) located within a conserved hydrophilic region (residues 1-110) and a C(2)H(2) zinc-binding motif (residues 182-202). lambdaC shows the two conserved K residues at positions 169 and 188 indicative of guanylyltransferase activity, an ATP/GTP-binding site motif A (residues 379-386), and a conserved S-adenosyl-l-methionine-binding motif (residues 822-830). Pairwise sequence comparisons show that the mean sequence identities of lambdaA encoding genes and lambdaA proteins are 92% and 98%, respectively, and those of lambdaC encoding genes and lambdaC proteins are 91% and 95%, respectively. Phylogenetic analysis of lambdaA and lambdaC encoding genes reveals that both encoding genes have diverged into three distinct lineages, respectively, and that there is no correlation between lineages and viral serotypes or pathotypes. Also, reassortment of gene segments L1 and L3 has been observed between viruses.
    Research in Veterinary Science 01/2008; 83(3):394-402. · 1.77 Impact Factor
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    ABSTRACT: Analysis of the amino acid sequence of core protein muA of avian reovirus has indicated that it may share similar functions to protein mu2 of mammalian reovirus. Since mu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant muA ( muA) was designed and used to test these activities. muA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that muA possessed NTPase activity that enabled the protein to hydrolyse the beta-gamma phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k(cat) values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of muA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein muA. The activity of muA is dependent on the divalent cations Mg(2+) or Mn(2+), but not Ca(2+) or Zn(2+). Optimal NTPase activity of muA was achieved between pH 5.5 and 6.0. In addition, muA enzymic activity increased with temperature up to 40 degrees C and was almost totally inhibited at temperatures higher than 55 degrees C. Tests of phosphate release from RNA substrates with muA or K408A/K412A muA indicated that muA, but not K408A/K412A muA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of muA might be carried out at the same active site, and that protein muA could play important roles during viral RNA synthesis.
    Journal of General Virology 07/2007; 88(Pt 6):1797-805. · 3.13 Impact Factor
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    ABSTRACT: Interleukin (IL)-1beta-encoding regions of chicken, duck, goose, turkey and pigeon were cloned and sequenced. Each IL-1beta-encoding region of chicken, duck, goose and turkey is 804 nucleotides long and encodes IL-1beta protein that is 268 amino acids. Pigeon IL-1beta-encoding region is 810 nucleotides long and encodes IL-1beta protein that is 270 amino acids. Two one-nucleotide and one four-nucleotide insertions of pigeon IL-1beta-encoding region sequence were found, resulting in two amino acid insertions in pigeon IL-1beta. Pairwise sequence analysis showed that the sequence identities of IL-1beta-encoding genes ranged from 77% to 99%, which were also found for IL-1beta protein sequence identities, with an average level of both sequence identities of 89%. Phylogenetic analysis indicated that IL-1beta-encoding regions and the encoded proteins of chicken, duck, goose and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from the binding reaction of antiserum against each recombinant IL-1beta (r IL-1beta) protein to homologous or heterologous rIL-1beta, the enhancement levels of K60 mRNA expression in rIL-1beta-treated DF-1 cells or the reduction levels of K60 mRNA expression in DF-1 cells treated with rIL-1beta that was preincubated with homologous or heterologous antiserum showed that all five rIL-1beta were functional active and shared significantly structural and functional homology.
    Veterinary Immunology and Immunopathology 04/2007; 116(1-2):37-46. · 1.88 Impact Factor
  • Ya Ling Lin, Jui Huang Shen, Long Huw Lee
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    ABSTRACT: An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based c-ELISA) for detection of antibody against avian reovirus protein sigmaA in chicken is described. After the conditions for MAb-based c-ELISA had been optimized, sera collected from birds that received live and inactivated avian reovirus vaccines in different combinations were tested for antibody response against virus protein sigmaA. The results show a high level of antibody against sigmaA was in both vaccinated specific pathogenic free (SPF) and vaccinated commercially reared birds as long as one of the vaccines administered was in an inactivated form. The high level of antibody production is indicated by a high percentage inhibition (PI) values in the sera of the birds; but no antibody production was found in birds which received live vaccine only, as indicated by the low PI values. In serum samples from SPF birds receiving vaccines that include an inactivated form of the vaccine, there is a good correlation between the PI values and serum neutralizing antibody (SN) titers. Again, this correlation was not observed in birds that received only live vaccine. The PI values of commercially reared birds receiving inactivated vaccine were significantly different from those of the mock-treated birds, but this was not the case when the birds received only live vaccine. Taken together, the results suggest that MAb-based c-ELISA may provide an alternative choice for determining the immune status of a vaccinated chicken flock as long as one of the vaccines used was inactivated, and thus would allow a more precise way to predict the appropriate time for vaccination.
    Journal of Virological Methods 10/2006; 136(1-2):71-7. · 1.90 Impact Factor
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    ABSTRACT: The sequences and phylogenetic analyses of the M-class genome segments of 12 avian reovirus strains are described. The S1133 M1 genome segment is 2283 base pairs long, encoding a protein muA consisted of 732 amino acids. Each M2 or M3 genome segment of 12 avian reovirus strains is 2158 or 1996 base pairs long, respectively, encoding a protein muB or muNS consisted of 676 and 635 amino acids, respectively. The S1133 genome segment has the 5' GCUUUU terminal motif, but each M2 and M3 genome segment displays the 5' GCUUUUU terminal motif which is common to other known avian reovirus genome segments. The UCAUC 3'-terminal sequences of the M-class genome segments are shared by both avian and mammalian reoviruses. Noncoding regions of both 5'- and 3'-termini of the S1133 M1 genome segment consist of 12 and 72 nucleotides, respectively, those of each M2 genome segment consist of 29 and 98 nucleotides, respectively, and those of each M3 genome segment are 24 and 64 nucleotides, respectively. Analysis of the average degree of the M-class gene and the deduced mu-class protein sequence identities indicated that the M2 genes and the muB proteins have the greatest level of sequence divergence. Computer searches revealed that the muA possesses a sequence motif (NH(2)-Leu-Ala-Leu-Asp-Pro-Pro-Phe-COOH) (residues 458-464) indicative of N-6 adenine-specific DNA methylase. Examination of the muB amino acid sequences indicated that the cleavage site of muB into muBN and muBC is between positions 42 and 43 near the N-terminus of the protein, and this site is conserved for each protein. During in vitro treatment of virions with trypsin to yield infectious subviral particles, both the N-terminal fragment delta and the C-terminal fragment phi were shown to be generated. The site of trypsin cleavage was identified in the deduced amino acid sequence of muB by determining the amino-terminal sequences of phi proteins: between arginine 582 and glycine 583. The predicted length of delta generated from muBC is very similar to that of delta generated from mammalian reovirus mu1C. Taken together, protein muB is structurally, and probably functionally, similar to its mammalian homolog, mu1. In addition, two regions near the C-terminal and with a propensity to form alpha-helical coiled-coil structures as previously indicated are observed for each protein muB. Phylogenetic analysis of the M-class genes revealed that the predicted phylograms delineated 3 M1, 5 M2, and 2 M3 lineages, no correlation with serotype or pathotype of the viruses. The results also showed that M2 lineages I-V consist of a mixture of viruses from the M1 and M3 genes of lineages I-III, reflecting frequent reassortment of these genes among virus strains.
    Journal of Virological Methods 06/2006; 133(2):146-57. · 1.90 Impact Factor
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    ABSTRACT: Crude antigen preparations from avian reovirus (ARV)-infected chicken embryo fibroblasts (sigmaNS) or from bacterially expressed protein sigmaNS (esigmaNS) were captured by monoclonal antibody 1E1(MAb 1E1) against ARV nonstructural protein sigmaNS immobilized on the ELISA plates and were used as the MAb capture ELISA for antibody detection. Sixty one-week-old specific pathogenic free (SPF) chickens were divided into six groups and were vaccinated with live or inactivated ARV vaccine preparations in different combinations or inoculated with a virulent ARV strain. Sera collected from the birds were tested for their antibody responses to ARV nonstructural protein sigmaNS. Using the MAb capture ELISAs, the level of nonspecific binding reactions was tested on the serum samples obtained weekly from mock-infected SPF chickens from 1 to 25 weeks and compared to the results tested by the conventional ELISA. The results indicated that both MAb capture ELISAs had lower nonspecific bindings than those in the conventional ELISA, even in older birds. Antibody responses against ARV sigmaNS of the birds which received the inactivated vaccine twice (group I), inactivated vaccine followed by a live vaccine (group II), or a live vaccine followed by boosting with an inactivated vaccine (group III) were detected by MAb captured ELISA with sigmaNS crude antigens. The absorbance values increased rapidly at 1-2 weeks after boosting, approximated a peak at 5-6 weeks of age, and maintained this throughout the length of the experiment. The absorbance values of the MAb capture ELISA showed a good correlation to the SN titers ( r value > 0.85). On the other hand, serum samples from the birds which received the live vaccine twice (group IV) or were inoculated with a virulent ARV (group V) did not show antibody responses to sigmaNS, similar to those from the mock-infected birds (group VI), as the absorbance values maintained at a low level (below 0.5) throughout the length of the experiment. Similar results were obtained in the sera detected by MAb capture ELISA with crude esigmaNS antigens, except that the absorbance values in the sera from the birds in group III were gradually increased and later approximated a peak at 11 weeks of age and maintained this throughout the length of the experiments. The results suggest that MAb capture ELISAs can be readily used to detect antibody responses of the birds against ARV nonstructural protein sigmaNS which may reflect an immune status of a chicken flock, receiving ARV vaccine as long as including an inactivated vaccine.
    Research in Veterinary Science 06/2004; 76(3):219-25. · 1.77 Impact Factor
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    ABSTRACT: Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.
    Veterinary Microbiology 03/2003; 91(4):309-23. · 3.13 Impact Factor
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    Hsien Sheng Yin, Yu Pin Su, Long Huw Lee
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    ABSTRACT: Both avian reovirus core protein sigma A purified from virus-infected cell extracts and the purified bacterially expressed protein sigma A (e sigma A) were characterized for their nucleoside triphosphate (NTP) hydrolysis activity by thin-layer chromotography. Protein sigma A from both preparations has a nonspecific nucleotidyl phosphatase activity that hydrolyzes four types of NTP to their corresponding nucleoside di- and monophosphates and free phosphate. The divalent cation requirement for this activity of e sigma A was further examined by the addition of Mn(2+), Mg(2+), Ca(2+), and Zn(2+) ions. NTP hydrolysis by e sigma A was maximal when Mn(2+), Mg(2+), or Ca(2+) concentrations were 5, 4, or 1 mM, respectively. Addition of Mn(2+) or Mg(2+) stimulated the reactions up to 4- or 3-fold, respectively, higher than Ca(2+) (2.2-fold). However, Zn(2+) ion inhibited this activity of e sigma A. The results suggest that nucleotidyl phosphatase activity of e sigma A is absolutely dependent on the divalent cations Mn(2+), Mg(2+), or Ca(2+), but not Zn(2+). Similar results were obtained from the analysis of divalent cation requirements for the protein sigma A nucleotidyl phosphatase activity. Optimal pH for nucleotidyl phosphatase activity of protein sigma A from both preparations was determined using reaction mixtures buffered at different pH. The results show that the optimal activities of both proteins were similar and were achieved between pH 7.5 and 8.5.
    Virology 03/2002; 293(2):379-85. · 3.37 Impact Factor
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    ABSTRACT: The double-stranded RNA genome segment S3 of avian reovirus (ARV) S1133 was cloned following polyadenylation of both strands and cDNA synthesis of S3 RNA. The complete segment S3 nucleotide sequence was determined. S3 is 1196 base pairs long with one long open reading frame (ORF). The ORF possesses the AUG initiation codon in an optimum context for translation and starts at the first initiation codon (residue 24) and extends for 367 codons, sufficient to encode a protein of the same size as the known S3 gene product, protein σB, one of the major outer capsid proteins of avian reovirus (Mr 41 471). Protein σB was subsequently expressed in Escherichia coli. The expressed protein σB was indistinguishable from virion protein σB as judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblot assay, and N-terminal amino acid sequencing of several peptides generated by Staphylococcus aureus V8 protease digestion. ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3′-terminus of its plus strand. The pentanucleotide sequence is common to the other genome segment S1 of ARV and to ten genome segments of mammalian reovirus at the 3′-terminus of their plus strands. Amino acid sequence analysis revealed that ARV σB does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus. The results of amino acid sequencing suggest that the most susceptible cleavage sites of σB to V8 protease are located in a hydrophilic area between amino acids 95 and 140.
    Journal of Virological Methods - J VIROL METH. 01/1997; 67(1):93-101.
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    ABSTRACT: The double-stranded RNA genome segment S3 of avian reovirus (ARV) S1133 was cloned following polyadenylation of both strands and cDNA synthesis of S3 RNA. The complete segment S3 nucleotide sequence was determined. S3 is 1196 base pairs long with one long open reading frame (ORF). The ORF possesses the AUG initiation codon in an optimum context for translation and starts at the first initiation codon (residue 24) and extends for 367 codons, sufficient to encode a protein of the same size as the known S3 gene product, protein σB, one of the major outer capsid proteins of avian reovirus (Mr 41 471). Protein σB was subsequently expressed in Escherichia coli. The expressed protein σB was indistinguishable from virion protein σB as judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblot assay, and N-terminal amino acid sequencing of several peptides generated by Staphylococcus aureus V8 protease digestion. ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3′-terminus of its plus strand. The pentanucleotide sequence is common to the other genome segment S1 of ARV and to ten genome segments of mammalian reovirus at the 3′-terminus of their plus strands. Amino acid sequence analysis revealed that ARV σB does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus. The results of amino acid sequencing suggest that the most susceptible cleavage sites of σB to V8 protease are located in a hydrophilic area between amino acids 95 and 140.
    Journal of Virological Methods - J VIROL METH. 01/1997; 67(1):93-101.