Induction of Interferon-α by Glycoprotein D of Herpes Simplex Virus: A Possible Role of Chemokine Receptors

AbstractThe induction of type I interferons by most RNA viruses is initiated by virus-derived double-stranded (ds)RNA. However, retro- and DNA-viruses, which do not synthesize dsRNA, must rely on different mechanisms of induction. For human immunodeficiency virus type 1 (HIV-1), recombinant glycoproteins 120 or 160 suffice to induce interferon (IFN)-α in blood-derived lymphocytes [H. Ankel, M. R. Capobianchi, C. Castilletti, and F. Dianzani (1994).Virology205, 34–43]. Here we show that for herpes simplex virus type 1 (HSV-1) recombinant glycoprotein, gD is the major inducer, whereas gB, gC, gE, gG, gI, and the complex of gH and gL are poor inducers. The recombinant extramembrane fragment of gD was sufficient to induce IFN-α levels comparable to that of intact virus. Like with HIV-1, induction was inhibited by a monoclonal antibody that recognizes cerebrosides and sulfatides. Furthermore, monoclonal antibodies specific for the chemokine receptors CCR3 and CXCR4 also blocked induction. We conclude that HSV-1 induces IFN-α by interaction of its glycoprotein gD with appropriate receptors on IFN-producing cells. Based on the known receptor roles of galactosyl cerebrosides and chemokine receptors in HIV infection, such structures on IFN-producing cells could also participate in the induction of IFN-α by HSV-1

Prediction of sequential antigenic regions in proteins

AbstractPrediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363-373]. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B-subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions

Purification of fusion proteins expressed by pEX3 and a truncated pEX3 derivative

AbstractA derivative of the pEX3 expression vector was constructed that codes for the first 407 amino acids of the 1051 amino acids of the pEX3 fusion protein. The amount of truncated fusion protein (40 mg/g cells), obtained by expression in E. coli, was similar to that produced by the original pEX3 vector. The truncated fusion protein was purified more easily from E. coli contaminants than the original fusion protein by washing with 2 M urea and 0.5% Triton X-100

Natural infection with herpes simplex virus type 1 (HSV-1) induces humoral and T cell responses to the HSV-1 glycoprotein H:L complex

The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)H(t(His)):gL complex of HSV-1 were st

Purification strategies for sendai virus membrane proteins

Viral membrane proteins extracted from Sendai virions with the non-ionic detergents decylpolyethyleneglycol-300 and Triton X-100 were used as a model mixture of hydrophobic membrane proteins. The detergent extract contained the fusion protein (F) and the tetrameric and dimeric forms of the hemagglutinin-neuraminidase protein (HN). These proteins were purified by size-exclusion high-performance liquid chromatography (HPLC) in the presence of 0.1% sodium dodecyl sulphate, by ion-exchange and metal chelate affinity HPLC in the presence of 0.1% decylpolyethyleneglycol, and by reversed-phase HPLC without prior removal of the detergent. The tetramer of HN and F could be purified by size-exclusion HPLC after dissociation of a micellar aggregate containing tetrameric HN and multimeric F. The F and HN proteins could be purified by ion-exchange HPLC. Pure F protein could be obtained after metal chelate affinity HPLC. The F protein and the dimer and tetramer of HN could be eluted from a large-pore (100 nm) reversed-phase column, but they were eluted as broad, overlapping peaks. Only after reduction of the virion extract, the relatively small (13–15 kilodaltons) F2 protein could be obtained in pure for

Size-exclusion high-performance liquid chromatography of Sendai virus membrane proteins in different detergents:A comparison of different columns

Four column packings for size-exclusion high-performance liquid chromatography (HPLC) of proteins with particle sizes from 3 to 13 μm were compared, using 0.1% sodium dodecyl sulphate in the solvent. Their suitability for the purification of hydrophobic membrane proteins was studied with Sendai virus proteins as a model. The calibration curves of the two 13-μm column packings were linear up to high molecular weights. In contrast to this, large proteins (> 100–150 kD) were eluted later than expected from the 3- and 6-μm packings. Peak capacities for proteins larger than 20 kD ranged from 4.7 to 5.5. Therefore, purification of complex mixtures of membrane proteins will often require rechromatography by a different mode of HPLC. Non-ionic detergents are suitable for further ion-exchange chromatography. The effect of addition of 0.1% of five non-ionic detergents (three gluco-methylalkanamide detergents, octylglucoside, and decyl-polyethyleneglycol-300) to the solvent was investigated and decyl-polyethyleneglycol-300 was found to be most suitable. Size-exclusion HPLC with this detergent resulted in the separation of micelles of three different sizes, of which the larger two contained exclusively the Sendai virus F protei

Combined size-exclusion and reversed-phase high-performance liquid chromatography of a detergent extract of sendai virus

Virus envelope proteins obtained by Triton X-100 extraction of Sendai virions were purified to a high degree by a combination of high-performance liquid chromatography (HPLC) methods. Size-exclusion HPLC on a TSK 4000 PW column with several concentrations of acetonitrile or ethanol-1-butanol in 0.1% hydrochloric acid as eluent was used as the first chromatographic step. Peak fractions were diluted in water and further fractionated on reversed-phase columns (TMS-250 or Vydac 218 TP). Size-exclusion HPLC with 45% acetonitrile in 0.1% hydrochloric acid, combined with reversed-phase HPLC on either column, was most suitable for obtaining highly purified F2 protein. Antibodies obtained after injection of this protein were reactive with the intact virus

High-performance liquid chromatography of Sendai virus membrane proteins

The three membrane proteins of Sendai virus can be extracted from the membrane by non-ionic detergents. The extracted proteins serve as a model mixture for the development of high-performance liquid chromatographic methods for the purification of hydrophobic membrane protein

Fixed herpes simplex type 1-infected cells as antigen for in vitro lymphocyte proliferation

Fixed herpes simplex virus type 1 (HSV-1)-infected Vero cells were used as antigen in the in vitro lymphocyte reactivity (LR) test and compared with extracellular HSV, HSV-infected cell extract and purified virions. The highest LR was measured after an incubation period of lymphocytes with the fixed HSV-infected Vero cells of 5–7 days. The LR appeared to be dependent on the lymphocyte to fixed HSV-infected cell ration and was found to be optimal at a ration of 10–20. The fixed HSV-infected cells could be stored at 6°C without detectable loss of LR. Addition of high-titered anti-HSV pooled serum to the lymphocyte cultures with the fixed HSV-infected cells as antigen inhibited the LR. The highest reactivity was found using HSV-negative pooled serum. Lymphocytes from seropositive donors were stimulated by the fixed HSV-infected cells and the purified virions. LR to extracellular HSV and an extract of HSV-infected cells were negative for 5 and 2 out of 13 seropositive donors, respectively. Lymphocytes from seronegative donors were not stimulated by any of the HSV-antigen preparations. Fixed HSV-infected cells, which have the advantage that they are easy to prepare and can be stored at 6°C for several months, are a good alternative to purified HSV-1 virions in the LR test

Comparison of reversed-phase column materials for high-performance liquid chromatography of proteins

Nine reversed-phase materials with various bonded phases from different suppliers were studied for the separation of hydrophilic proteins with two solvent systems. Protein retention, resolution and recovery were not correlated with the nature of the hydrocarbonaceous ligand. Peak volumes increased with molecular weight, which led to broad, irregular peaks for the larger proteins on some columns. Four columns that performed equally well were selected for the purification of hydrophobic Sendai virus membrane proteins. In this case, more distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent system