Selective inhibition of glycoprotein and membrane protein of vesicular stomatitis virus from interferon-treated cells.

A 200-fold inhibition in the titer of infectious vesicular stomatitis virus (VSV) was produced in cultures of Ly cells treated with 30 reference units of interferon per milliliter. Virus particle production, as measured by VSV particle-associated transcriptase, or nucleocapsid protein was inhibited by a maximum of tenfold. The glycoprotein and membrane protein content was reduced in VSV derived from interferon-treated cells. Thus interferon-treated cells may have produced VSV particles with low infectivity, which may be related to the reduced amount of glycoprotein incorporated into such particles. These findings resemble those reported in interferon-treated cells infected with murine leukemia viruses.

Acyl-GPC and alkenyl/alkyl-GPC:acyl-CoA acyltransferases.

In mammalian tissues, phosphatidylcholine, or 1,2-diacyl-glycerophosphocholine (GPC), is the most abundant form of choline-containing phospholipids. In some electrically active tissues, a significant portion of the choline-containing phospholipids is 1-alkenyl-2-acyl-GPC (plasmenylcholine). The 1-alkyl-2-acyl-GPC is found in significant amounts in circulating cells such as neutrophils and macrophages but in low amounts in other tissues. Structural studies of phosphatidylcholine indicate that there is an asymmetric distribution of acyl groups on the molecule. Saturated fatty acids are usually esterified at the sn-1 position of the glycerol backbone, whereas unsaturated fatty acids are esterified at the sn-2 position. Similarly, unsaturated acyl groups are usually found in the sn-2 position of plasmenylcholine. The remodelling of the sn-2 acyl group in phosphatidylcholine by the deacylation-reacylation process has been demonstrated in a number of tissues. Phospholipase A2 is responsible for the hydrolysis of the acyl group at the sn-2 position, whereas 1-acyl-GPC:acyl-CoA acyltransferase is responsible for the reacylation reaction. The acyltransferase is located in the microsomal fraction and displays specificity towards the polyunsaturated acyl groups. The enzyme can be solubilized by detergent, but the enzyme activity in soluble form is difficult to maintain. The acyltransferase for the reacylation of 1-alkenyl-GPC is also located in the microsomal fraction and is somewhat specific towards polyunsaturated acyl groups. In guinea pig heart mitochondria, however, a new form of 1-alkenyl-GPC acyltransferase was identified which appeared to be different from the microsomal form. The acyltransferase for the acylation of 1-alkyl-GPC into platelet-activating factor has been studied in several tissues including human neutrophils. At present, the contribution of the acyltransferase in attaining the observed molecular composition of the choline-containing phospholipids in the tissue has not been defined. We postulate that the intrinsic acyl-CoA specificity of the acyltransferase, the flux of 1-acyl-GPC, 1-alkenyl-GPC and 1-alkyl-GPC, as well as the pool size of acyl-CoA are major factors in producing the final composition of the molecular species of the choline-containing phospholipids.

Limiting dilution analysis of antigen stimulated IL-4 and IFN-gamma production in human mononuclear cell populations.

Limiting dilution analysis (LDA) of fresh human mononuclear cell populations has previously been used to estimate the frequency of specific B cells, CTL, proliferative T cells, or cells capable of IL-2 production in various clinical situations. Such approaches evaluate the intensity of the response but provide little information concerning the balance between Th1- vs. Th2-like patterns of cytokine gene expression. Here, we describe development of an LDA method to obtain quantitative estimates of the frequency of antigen-specific IFN-gamma or IL-4 producing cells in human peripheral blood. The approach utilizes 3-4 day antigen-mediated restimulation of mononuclear cell populations freshly derived from grass pollen sensitive allergic rhinitis subjects. IFN-gamma and IL-4 production in culture supernatants are determined by ELISA and CT.h4S bioassay. Cytokine production elicited in this assay is CD4 dependent and antigen specific. As such, it provides a useful non-invasive approach for rapid evaluation of low frequency, antigen-induced cytokine production in the circulating repertoire. This method can readily be extended to analysis of other cytokines in other immunologic disorders or in infectious disease states, allowing longitudinal analysis of individuals and facilitating efforts to establish clear correlations between in vivo patterns of cytokine gene expression and disease exacerbation and remission.

Identification of highly conserved and species-specific polypeptides of Haemophilus ducreyi.

Chancroid is a sexually transmitted diseased caused by Haemophilus ducreyi. The pathological manifestations of chancroid are unique among Haemophilus species and the virulence factors of H. ducreyi that account for these features have not been identified. Some of these virulence factors may be unique components of H. ducreyi, but attempts to identify H. ducreyi-specific components have been unsuccessful. Four polypeptides--A, B, C and D of 83, 77, 56 and 28 kDa, respectively--were identified with a panel of nine H. ducreyi-specific monoclonal antibodies (MAbs). Polypeptide C was one of the five major proteins in H. ducreyi and demonstrated micro-heterogeneity in SDS-PAGE. Polypeptides A, B and D were present in only small amounts in whole-cell lysates of H. ducreyi. The relative amounts of A and B varied, suggesting that they may be precursor molecules. The unique polypeptides C and D were not exposed on the surface. Polypeptide C was highly soluble and did not appear to be membrane-bound, whereas polypeptide D appeared to partition with the cytoplasmic membrane and was soluble in Sarkosyl. All four polypeptides appeared to be unique to H. ducreyi since MAbs directed against them did not cross-react with H. influenzae, H. parainfluenzae or Neisseria gonorrhoeae. The mol. wts of all of these polypeptides were conserved throughout 35 clinical isolates collected from 15 cities in eight countries and one reference strain of H. ducreyi that were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane alterations following interferon treatment.

Interferon treatment appears to induce a number of changes in the plasma membrane of uninfected cells. Interferon treatment altered the surface exposure of gangliosides of both Ly and KB cell membranes. The differences were found in the amount and pattern of incorporation of tritium after galactose oxidase treatment. In AKR,C- (AKR-2B) mouse cells, not only was there an apparent increase in the number of intramembranous particles in response to treatment with interferon but also the kinetics of the increase followed that of the establishment of the antiviral activity. The buoyant density of plasma membrane was also found to be significantly increased in interferon-treated cells. Moloney murine leukemia virus produced in interferon-treated mouse thymus and bone marrow cells had a high particle to infectivity ration. This virus contained a prominent glycoprotein with a molecular weight of about 85,000. This large glycoprotein was only a very minor component of Moloney leukemia virus produced in control TB cells and might be an uncleaved precursor to gp 69-71.

Production of monoclonal antibodies against recombinant human interferon-gamma: screening of hybridomas without purified antigen.

A panel of mouse monoclonal antibodies (MAbs) against the recombinant human gamma interferon (HuIFN-gamma) has been produced for the study of the structure-function relationships of this important lymphokine. Enzyme linked immunosorbent assay (ELISA) is the current method of choice to screen hybridomas for specific MAb production. The purity of the antigen used for screening dictates the specificity of the ELISA. As often is the case in many systems, adequately purified biologically active HuIFN-gamma was not readily available for this purpose. A sandwich ELISA which allowed the use of unpurified HuIFN-gamma for hybridoma screening was developed. A rabbit antiserum against the denatured HuIFN-gamma purified by SDS-PAGE was prepared and the nonspecific binding activity was removed by adsorption to control cell proteins immobilized on Sepharose. The adsorbed immunoglobulin fraction was bound to the ELISA plate: (i) to trap HuIFN-gamma specifically from the whole cell lysate, thus providing specificity for MAb detection, and (ii) to avoid direct adsorption of the HuIFN-gamma to the ELISA plate because others have found that this prevented detection of neutralizing MAb. The sandwich ELISA detected both neutralizing and non-neutralizing MAbs with relatively low false positive reactions. This approach to the development of an ELISA method to screen hybridomas without purified antigen should be applicable to the production of MAbs to other proteins.

Production of monoclonal antibodies specific for Haemophilus ducreyi: a screening method to discriminate specific and cross-reacting antibodies.

Haemophilus ducreyi is the etiological agent of chancroid. The organism shares extensive immunological cross-reactivity with other Haemophilus species. This presents substantial difficulties for the production of specific monoclonal antibodies (MAbs). A competition ELISA was devised for hybridoma screening which allowed the detection of H. ducreyi-specific antibody-producing hybridoma cultures during the initial screening process. With this screening method, seven MAbs specific for H. ducreyi were obtained in a single cell fusion exercise. The specificities of the 7 MAbs were demonstrated by direct ELISA and dot immunobinding assays against several strains each of H. influenzae, H. parainfluenzae and Neisseria gonorrhoeae. Five of the MAbs reacted against all ten strains of H. ducreyi. These MAbs may permit the development of rapid and efficient immunodiagnostics for chancroid. The principle of the competition ELISA for hybridoma screening should be widely applicable to the development of specific MAbs to other organisms in which immunological cross-reactivity is an impediment to hybridoma screening by conventional methods.

Distinct domains of recombinant human IFN-gamma responsible for anti-viral effector function.

A panel of 21 independently isolated neutralizing mAb directed against the human rIFN-gamma (rHuIFN-gamma) was used to characterize those epitopes that are involved in the antiviral function of the rHuIFN-gamma. A sandwich competition assay was developed to determine the cross-reactivities between the mAb. The 125I-labeled mAb were allowed to compete with varying amounts of unlabeled mAb for binding to rHuIFN-gamma under Ag-limiting conditions, and the 50% inhibition endpoints were determined for each of the 21 mAb. The competition of each heterologous mAb relative to the competition of the homologous mAb was determined. By grouping the competition patterns of the 21 mAb, it was apparent that at least two epitopes (E1 and E2) were important to the antiviral function of rHuIFN-gamma. The possibility of the separation of the receptor binding site and signal transduction effector site(s) is discussed.

The E1 functional epitope of the human interferon gamma is a nuclear targeting signal-like element. Mapping of the E1 epitope.

Eight neutralizing monoclonal antibodies (mAbs) directed against the human interferon gamma (HuIFN-gamma) that were classified in the E1 epitope group were mapped by the synthetic peptide approach. A set of 136 octapeptide homologs of the 143-residue primary sequence of the HuIFN-gamma, each one with a 7-residue sequence overlap with successive peptide, was synthesized. Based on the similar reactivity patterns of all the mAbs with this set of synthetic peptides, the E1 functional epitope was localized to residues 84-94 on the HuIFN-gamma. The epitope sequence is: Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Gln-Lys. The fact that eight independently isolated mAbs binding to the same domain can neutralize the HuIFN-gamma activity suggests that the E1 domain must be at or adjacent to a functional site. Within this domain is a sequence element, Lys-Lys-Lys-Arg, that resembles the nuclear location signals known to effect the intracellular transportation of a number of nuclear proteins, such as the large tumor antigen (T antigen) of simian virus 40 (SV40) and polyoma virus and steroid hormone receptors. This observation suggests that the HuIFN-gamma molecule and/or its complex with the receptor must function in the nucleus to effect transcription regulation that results in the various biological activities. The signal for that intracellular transportation must be provided by the HuIFN-gamma molecule.

Purification, characterization and modulation of a microsomal carboxylesterase in rat liver for the hydrolysis of acyl-CoA.

A carboxylesterase containing long-chain acyl-CoA hydrolase activity was purified to apparent homogeneity from rat liver microsomes. Palmitoyl-CoA was the most preferred substrate, followed by stearoyl-CoA and oleoyl-CoA. Arachidonoyl-CoA, linoleoyl-CoA and acetyl-CoA were not hydrolysed by the enzyme. The purified enzyme had no activity on the hydrolysis of phospholipids and neutral lipids. The molecular mass of the enzyme was found to be 56 kDa by SDS/PAGE and 64 kDa by gel-filtration chromatography. On isoelectric focusing, the purified enzyme behaved like the ES-4 type, with a pI of 6.15. Determination of the amino acid sequence revealed that its N-terminal sequence is 100% homologous with the only other known N-terminal sequence for a rat carboxylesterase isoenzyme (ES-10). Enzyme activity was inhibited by lysophosphatidic acid and activated by lysophosphatidylcholine. The modulation of enzyme activity by these lysophospholipids might represent a plausible mechanism for the physiological control of acyl-CoA concentrations.

Infectivity-deficient vesicular stomatitis virus produced in the presence of interferon has a functional virion core.

Interferon induces two antiviral actions against vesicular stomatitis virus by (i) inhibiting viral protein synthesis which leads to a reduction in virion production, and (ii) producing progeny which are deficient in infectivity (VSVIF). At low or physiological concentrations of interferon, while the virion production was decreased by less than 10-fold, the virion infectivity yield was suppressed more than 1000-fold. The VSVIF was found to be deficient (quantitatively) in envelop glycoprotein G and protein M. Tryptic peptide mapping indicated that there was no detectable structural abnormality in the G, M, and N proteins of VSVIF. The virion cores, lacking only the envelop G protein, isolated from VSVIF and control VSV have essentially identical specific infectivity. This indicated that the virion proteins L, N, NS, and M, as well as viral RNA that make up the virion core, must be functionally normal, and the observed deficiency in G protein was likely to be the cause of the functional deficiency of the virion. Low concentrations of DEAE-dextran, which is known to partially overcome the virion's dependence on the G protein for adsorption to the cell during infection, were found to enhance the infectivity of VSVIF more than the control virion. These results together indicated that the loss of infectivity in the VSVIF was due to the deficiency of the surface glycoprotein G.

Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells.

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labelings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labelings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labeling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.

The separation of ribonucleic acids from Escherichia coli on lysin-agarose.

Lysine-agarose provides a simple means of separating RNA species of different moleecular weight. When RNA from Escherichia coli is added to a small column of lysine-agarose and elution is carried out at neutral pH with a shallow linear gradient of NaCl the RNA species are eluted according to size; 4S and 5S RNA species are completely separated and after a delay the 16S and 23S species are eluted as separate peaks. The process is very reproducible and the different species are eluted at a fixed salt concentration even when changes are made in the gradient, provided other conditions, under which the column is run remain constant.

Interferon induces the production of membrane protein-deficient and infectivity-defective vesicular stomatitis virions through interference in the virion assembly process.

The reduced rate of synthesis, maturation and degradation as well as the level of accumulation of the intracellular virus proteins in VSV-infected cells may account for the overall reduction (less than 10-fold) of progeny virion yield due to interferon (IFN); however, the deficiency of the virions proteins, G and M, which apparently caused a drastic loss of infectivity of these progeny virions (about 1000-fold) cannot be easily explained, because the concentrations of G and M proteins relative to other virus proteins were not reduced in the cell. In fact, intracellular M protein was significantly increased. Moreover, the virus proteins in IFN-treated and control cells were synthesized and accumulated in large excess of the amount incorporated into the released virions. The reduction in the intracellular activity of GlcNac-P-P-Dol transferase did not appear to play a direct role in the antiviral mechanism in this system. Our results, however, do suggest that the deficiency of G and M proteins in the virion is related to specific inhibition of the incorporation of either or both of these proteins in the virus assembly process.

Eukaryotic translational control: adeno-associated virus protein synthesis is affected by a mutation in the adenovirus DNA-binding protein.

Growth of adeno-associated virus (AAV) requires expression of certain adenovirus (Ad) genes in the same cell. AAV particles contain three proteins, VP1 (Mr 85,700), VP2 (Mr 72,000), and VP3 (Mr 61,500). These proteins have overlapping peptide maps. We recently reported that AAV RNAs make up a 3'-coterminal family of overlapping molecules. We report here that the most abundant AAV mRNA, a 2.3-kilobase spliced RNA, codes for all three proteins--VP1, VP2, and VP3--when translated in an in vitro reticulocyte lysate. This shows that the AAV capsid proteins are coded by the genome sequence between map positions 48.0 and 96.0 (1 map unit is 1% of the genome or 47 base pairs). When AAV was grown in human KB cells with the Ad temperature-sensitive mutant Ad5ts125 at the nonpermissive temperature (40 degrees C), the accumulation in vivo of AAV capsid proteins VP1, VP2, and VP3 was decreased to less than 1/50th. However, normal amounts of the 2.3-kilobase mRNA were accumulated, and this RNA could be efficiently translated in an in vitro reticulocyte lysate system to yield VP1, VP2, and VP3. These experiments suggest that in infected cells control is exerted upon the AAV 2.3-kilobase mRNA at the translational level and that this control can be influenced by mutations in Ad. These Ad mutations map in the region 2 early gene for the Ad DNA-binding protein. The temperature-sensitive system that we have studied may be useful for analysis of translational control of a eukaryotic mRNA.

The use of a dipolar ion-exchanger for the fractionation of transfer ribonucleic acid.

Transfer ribonucleic acid is well fractionated on columns of arginine-agarose, whose properties appear in general to be similar to those of DEAE-Sephadex. However, the amino acid acceptor species are separated into sharper peaks and in several instances, notably for methionine, glycine, serine, leucine and aspartate accepting tRNAs from Escherichia coli, isoaccepting species are well resolved. In the case of methionine accepting tRNA from E. coli the tRNA Met-m species is eluted before the tRNA Met-f species and since it is also eluted prior to the bulk of the tRNA it is obtained in a high degree of purity. By comparing the properties of columns of arginine-agarose and its methyl ester in which the carboxylate anion is blocked, it is seen that the carboxylate ion plays a role in the fractionation of the tRNA Met species.

Physical, morphological, and biochemical alterations in the membrane of AKR mouse cells after interferon treatment.

Interferon treatment of AKR,C- cells was followed by the establishment of an antiviral state and apparently concomitant morphological, physical, and biochemical alterations of the cell plasma membrane. The density of the plasma membrane was significant altered, and the concentration of some plasma membrane glycoproteins and the number of intramembranous particles observed in freeze-fracture electron micrographs were increased. A parallel increase in the concentration of intramembranous particles and the resistance to viral infection during interferon treatment as well as their parallel decrease upon removal of interferon suggests a relationship between the particle density and the establishment of the antiviral state.

Structure-function analysis of the human interferon gamma. The COOH terminus is not essential for functional activity.

The structure-function relationships for the human interferon gamma (HuIFN-gamma) were studied using recombinant variants that had various deletions at the carboxyl terminus. Four COOH-terminal deletion variants were constructed that contained the amino-terminal 122, 117, 111, and 106 amino acid residues. These variants were constructed by specific DNA modifications and were expressed in Escherichia coli. The deletion of 21 amino acid residues resulted in only 2- and 3-fold reduction in the antiviral and antiproliferative specific activities, respectively. Thus, the carboxyl-terminal 21 residues are not directly involved in the function of the HuIFN-gamma. The level of intracellular accumulation was also decreased by 3-5-fold. Further deletions of 26, 32, and 37 residues from the COOH terminus resulted in the lack of detectable activity as well as in 50-100-fold reduction in the level of accumulation in the bacterial cell. However, each of the modified plasmids was found to have comparable efficiency in directing the production of the respective variant molecules relative to the full-length HuIFN-gamma molecule in an in vitro transcription-translation assay. Thus, the failure of some of the deletion variant molecules to accumulate in the E. coli cell is likely due to their instability in vitro. The loss of the COOH-terminal 21 amino acid residues of HuIFN-gamma also resulted in a substantial reduction in the ability of the molecule to be renatured in vitro from the treatment with chaotrophic agents, a method frequently used to extract and purify recombinant polypeptides from E. coli host. The latter result may account for some earlier reports which inferred the involvement of the COOH terminus in the functions of the HuIFN-gamma molecule due to their failure to detect activity upon deletions of only 11-18 residues.