Further studies on the glycosylated gag gene products of Rauscher murine leukemia virus: identification of an N-terminal 45,000-dalton cleavage product

A glycosylated 45,000-Mr protein containing Rauscher murine leukemia virus p15 and p12 antigenic sites and tryptic peptides was identified in Rauscher murine leukemia virus-infected cells. This glycoprotein, termed gP45gag, was also shown to contain a single tryptic peptide also present in gPr80gag and its unglycosylated apoprotein precursor Pr75gag, but lacking in Pr65gag or Pr40gag. The presence of this peptide only in viral precursor proteins containing the so-called leader (L) sequence strongly suggests that gPr45gag is an N-terminal fragment of larger glycosylated gag polyproteins, composed of L sequences in addition to p15 and p12. The kinetics of appearance of radiolabeled gPr45gag and its disappearance during chase-incubation is suggestive of a precursor-like role for this intermediate gene product. An observed 27,000-Mr glycosylated polypeptide, termed gP27gag and containing p15 but not p12, p30, or p10 antigenic determinants, is a candidate cleavage product derived from gPr45gag. These observations suggest that gPr45gag and its putative cleavage product gP27gag represent an authentic pathway for intracellular processing of glycosylated core proteins.     

Fucose plays a role in the clearance and uptake of glucocerebrosidase by rat liver cells

Upon treatment of avian oncovirions with 1% Triton X-100 in a buffer pH 5.0 of low ionic strength containing 1% 2-mercaptoethanol, p27, p19 and p15 internal virion proteins have been quantitatively and selectively solubilized. These proteins have been separated by ion-exchange chromatography on a SP-Sephadex column, yielding well-resolved peaks of p27, non-phosphorylated p19, phosphorylated p19 and p15 proteins. The identity and purity of the isolated proteins has been documented by gel electrophoresis and amino acid composition. High proteolytic activity of the p15 protein indicates that the isolated proteins had not been denatured. This method may be useful for the preparation of pure oncoviral proteins suitable for studies of their chemical, immunological and especially functional properties.     

Polyproteins related to the major core protein of mouse mammary tumor virus.

The mouse mammary tumor virus (MuMTV) contains several low-molecular-weight proteins which, together with the genomic RNA, constitute the core structure of the virion. The most abundant protein in the core is the 27,000-dalton protein (p27), and, by analogy to the type C viruses, this protein probably forms the core shell. In mouse mammary tumor cell lines (GR and Mm5MT) producing MuMTV the major p57 antigenic specificity resides in a large protein, which migrates in polyacrylamide gels as a doublet of 77,000 and 75,000 daltons (p 77/75). A series of lower-molecular-weight proteins, p61, p48, p38, and p34, is also present in small amounts and is probably derived by proteolytic cleavage of the p 77/75. These proteins have been identified by immunoprecipitation with monospecific antiserum, and their sequence relatedness to p27 has been determined by an analysis of the peptides after trypsin digestion. After a 15-min pulse with [35S]-methionine, all of the p27-related proteins in these cell lines were labelled and, during a subsequent chase, progressively disappeared. The p27 was labeled poorly during the pulse, but the amount of label in this protein increased during the chase. A quantitation of these experiments suggested that the majority of the p27-related proteins were quite rapidly turned over in these cell lines. Hence, if p27 is derived by a progressive proteolytic cleavage mechanism, then the process is inefficient in the GR cells and only moderately efficient in the Mm5MT cells. When MuMTV was isolated from the culture medium of these cells harvested at 5-min intervals, the major p27-related protein was p34. The p27 accounted for only 29% of the anti-p27 serum immunoprecipitable proteins compared to 95% in virus isolated from an 18-h harvest. Incubation of the rapid-harvest virus at 37 degrees C for 2 h resulted in some conversion of p34 to p27. These results suggest that some of the p27 in MuMTV is formed in the virions by proteolytic cleavage of p34.     

Analysis of translational products of Friend strain of spleen focus-forming virus.

We have analyzed normal rat kidney cells nonproductively infected with the Friend strain of spleen focus-forming virus (SFFV) for the presence of murine leukemia virus-specific type C viral proteins. SFFV was found to code for the p15 and p12 proteins of Friend-murine leukemia virus as determined by immunological typing of their antigenic determinants. Molecular-size analysis of p15 and p12 proteins in SFFV nonproductively infected normal rat kidney cells indicated that these proteins are translated as parts of polyprotein molecules. The apparent molecular weights of the polypeptides bearing p12 antigenic determinants revealed the presence of translational products of the SFFV genome that could not be accounted for by know type C virus helper structural proteins.     

Immune response of the mouse to the major core protein (p30) of ecotropic leukemia viruses.

Sera from normal C57BL/6 mice contained low titers of antibodies against proteins of MuLV. Sera from C57BL/6 mice that were immunized with allogeneic leukemia cells sometimes contained high-titered antibodies against the p15 protein of MuLV; these antibodies detected group-specific antigenic determinants of the p15 protein, since reactions were observed with the p15 proteins of both AKR and Moloney viruses. In contrast, antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted strongly with the p30 protein of MuLV, as well as with p15. Antibodies in the C57BL/6 anti-AKR K36 sera detected group-specific antigenic determinants of the p30 protein; reactions were observed with the C57BL/6 anti-AKR K36 serum and the p30 proteins of both AKR and Moloney viruses. It was concluded that mice do have the capacity to respond immunologically to antigenic determinants of the MuLV p30 protein, although in most circumstances this is not observed.     

Evolutionary relationships between gag gene-coded proteins of murine and primate endogenous type C RNA viruses.

Several low molecular weight proteins of endogenous type C viruses of the RD114/baboon group are compared with the gag gene translational products of endogenous type C viruses of murine origin. The p10 proteins of each virus group are shown to be immunologically and biochemically related, while the p12 proteins of RD114/baboon viruses are demonstrated to share antigenic determinants with murine viral p15. Moreover, highly type-specific phosphoproteins, p15 of RD114/baboon viruses and p12 of murine viruses, are shown to possess very similar biochemical properties. These findings, along with previous studies indicating immunologic cross-reactivity between their major internal antigens, p30, demonstrate that each of the gag gene-coded proteins of murine type C viruses has a analogue in viruses of the RD114/baboon group. The immunologic and biochemical relatedness of their gag gene translational products supports the concept of a common progenitor in the evolution of these endogenous viruses.     

Structural protein markers in the avian oncoviruses.

The proteins of purified avian oncoviruses were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing. Certain members of the avian leukosis-sarcoma viruses (ALSV) had group-specific antigens with altered electrophoretic properties. (i) The p27 protein of Rous-associated virus 0 (RAV-0) had a lower electrophoretic mobility in SDS gels and a lower isoelectric point than the p27 of other ALSV. (ii) The p19 proteins of RAV-1, RAV-2, and the Bryan high-titer strain of Rous sarcoma virus had higher mobilities in SDS gels than did the corresponding protein of other viruses. This altered electrophoretic mobility was correlated with specific differences in the tryptic peptides of radioiodinated p19s. (iii) The p15 protein of RAV-7 had a lower mobility in SDS gels than did the p15 of other ALSV. These markers were used in a study of the structural proteins of subgroup E RAV-60 produced after infection of chicken embryo cells by exogenous ALSV. Although exogenous group-specific protein markers could often be identified in the subgroup E isolates, one RAV-60 had a p27 that comigrated with the p27 of RAV-0. The p19s of two other RAV-60 isolates had electrophoretic properties that were different than those of p19s from either RAV-0 or the exogenous viruses. These results support the hypothesis that RAV-60 is generated by recombination between endogenous and exogenous oncoviruses and indicate that at least the p27 encoded by RAV-0 is closely related to a protein specified by endogenous viral information in chicken cells.     

Monoclonal antibodies of African swine fever virus: antigenic differences among field virus isolates and viruses passaged in cell culture.

An analysis of the binding properties of a collection of monoclonal antibodies to African swine fever virus particles showed that virus field isolates passaged in porcine macrophages changed antigenically more than a strain of a cell-adapted virus passaged in Vero cells. From seven clones isolated from the spleen of a field-infected pig, we found four clones that had the same antigenic properties, one clone that had large changes in proteins p150 and p27 and small changes in proteins p37 and p14, and two clones that had minor changes in proteins p150 and p27, respectively. An analysis of the binding properties of the monoclonal antibodies to 23 field isolates from Africa, Europe, and America showed that the African isolates differed among themselves more than the European and the American isolates; in this study we found changes in 8 of the 10 virus proteins tested. The most variable proteins in the African isolates were p150, p27, p14, and p12. In contrast to the African isolates, protein p12 from the non-African viruses did not change. The clustering of the field virus isolates in six antigenic homology groups indicated the existence of a complex variety of African swine fever virus serotypes.     

Purification of viral proteins from avian sarcoma virus QV2.

A procedure was established whereby most of the major viral proteins were isolated to apparent homogeneity in biologically and immunologically active forms from a single batch of avian sarcoma virus QV2. For the initial step of purification, gently disrupted virions were fractionated by CsCl centrifugation into envelope proteins, RNA-dependent DNA polymerase, and viral core proteins. Further purification of envelope glycoproteins and DNA polymerase was performed by affinity chromatography on agarose columns cross-linked with plant lectins and poly(C), respectively. On the other hand, core proteins were fractionated by a combination of gel filtration and ion-exchange column chromatography into components p27, p19, and p15. The core protein p15 thus isolated retained proteolytic activity even after storage for 6 months. The present study also demonstrated that QV2 p19 is structurally altered from the corresponding protein of avian myeloblastosis virus (AMV), a reference avian leukosis-sarcoma virus having a well-characterized polypeptide composition.     

Peroxisome proliferator-activated receptor-gamma agonists suppress the production of IL-12 family cytokines by activated glia

The IL-12 family of cytokines, which include IL-12, IL-23, and IL-27, play critical roles in the differentiation of Th1 cells and are believed to contribute to the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Relatively little is known concerning the expression of IL-12 family cytokines by cells of the CNS, the affected tissue in MS. Previously, we and others demonstrated that peroxisome proliferator-activated receptor (PPAR)-gamma agonists suppress the development of EAE, alter T cell proliferation and phenotype, and suppress the activation of APCs. The present studies demonstrated that PPAR-gamma agonists, including the naturally occurring 15-deoxy-Delta(12,14)-PGJ(2) and the synthetic thiazoladinedione rosiglitazone, inhibited the induction of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 proteins by LPS-stimulated primary microglia. In primary astrocytes, LPS induced the production of IL-12p40, IL-23, and IL-27p28 proteins. However, IL-12p70 production was not detected in these cells. The 15-deoxy-Delta(12,14)-PGJ(2) potently suppressed IL-12p40, IL-23, and IL-27p28 production by primary astrocytes, whereas rosiglitazone suppressed IL-23 and IL-27p28, but not IL-12p40 in these cells. These novel observations suggest that PPAR-gamma agonists modulate the development of EAE, at least in part, by inhibiting the production of IL-12 family cytokines by CNS glia. In addition, we demonstrate that PPAR-gamma agonists inhibit TLR2, MyD88, and CD14 expression in glia, suggesting a possible mechanism by which these agonists modulate IL-12 family cytokine expression. Collectively, these studies suggest that PPAR-gamma agonists may be beneficial in the treatment of MS.     

Interstrain Variation of the Major Internal Structural Component (p30gag) of Two Murine Oncornaviruses: Comparative Immunochemical, Biochemical, and Biophysical Analysis

The major internal structural protein (p30(gag)) of the Moloney leukemia virus and the endogenous Y-1 murine oncornavirus was examined for biochemical and biophysical manifestations of interstrain antigenic variation. Although the two viral proteins share murine group-specific antigenic determinants, the Y-1 virus p30 appeared to have both a lower relative number of such determinants and a decreased affinity at the cross-reactive sites for Moloney virus p30 monospecific antibodies. Further, immunological analysis indicated the presence of unique antigenic sites on the Moloney virus p30 not shared by the analogous Y-1 virus molecule. The two polypeptides copurified and had similar isoelectric points (pH 6.2 to 6.3) and sedimentation coefficients (2.47S). However, equilibrium sedimentation yielded a significant mass difference between the two proteins, 28,300 +/- 600 and 31,000 +/- 300 daltons for the Moloney and Y-1 virus molecules, respectively. Amino acid analysis indicated a concomitant increase in total residues for the Y-1 virus p30, although a number of residues appeared to have been conserved between the two viral proteins. Conformational studies and hydrodynamic calculations demonstrated marked secondary and tertiary structural differences; with the Y-1 virus p30 being an asymmetric prolate ellipsoid containing 27 to 28% alpha-helix and Moloney virus p30 being somewhat more spherical and possessing an alpha-helical content of 50 to 55%. Two-dimensional mapping of (125)I-labeled tryptic peptides of each p30 suggested that considerable sequence heterogeneity is responsible for many of the biophysical, biochemical, and immunochemical differences in these two analogous structural proteins.     

Endoplasmic reticulum targeted Bcl2 confers long term cell survival through phosphorylation of heat shock protein 27

Overexpression of anti-apoptotic Bcl2 family proteins is often seen in cancers rendering them insensitive to apoptosis inducing anticancer strategies. Anti-apoptotic Bcl2 family proteins are associated with different organelles like mitochondria and endoplasmic reticulum (ER) and exert their anti-apoptotic activity by inhibiting the release of Cyt.C from mitochondria irrespective of its localization. Here, we have identified a long term survival function for Bcl2 targeted at ER in mammalian system compared to wild type Bcl2 that is mediated by enhanced phosphorylation of heat shock protein 27 at ser 15, 78 and 82 sites with inhibition of caspase9 activity. Phosphorylation of hsp27 was prevented and the survival of ER-Bcl2 cells was reversed by inhibiting p38 and MEK suggesting that these kinases can act as the upstream targets for hsp27 phosphorylation. The results suggest that Bcl2 possess additional survival function in the regulation of apoptosis which is primarily regulated by its association with the ER in an hsp27 dependent manner. The interplay of both hsp27 and ER-Bcl2 in providing long term survival to cancer cells is interesting since both of these proteins are overexpressed in tumors with aggressive phenotype. The results suggest that spatial localization of Bcl2 family proteins also play a key role in long term survival of cancers indicating another level of functional regulation of Bcl2 in cancer cell survival.     

Detection of polymorphism in BALB/c leukemia viruses with mouse antisera.

Antisera produced in mice recognize primarily type-specific antigenic determinants on both the major core protein, p30, and the major envelope proteins, gp70 and p15(E), of the endogenous leukemia viruses (MuLV) of BALB/c mice. Three different mouse sera were investigated in detail. (i) Antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted with the gp70, p15(E), and p30 proteins of MuLV. Certain pools of the C57BL/6 anti-AKR K36 serum contained antibodies which serologically distinguished the p30 proteins of N-ecotropic, B-ecotropic, and xenotropic BALB/c MuLV. (ii) Antisera prepared in BALB/c mice against the BALB/c sarcoma 1315 contained antibodies that reacted with a type-specific antigen of the 1315 MuLV gp70 that is not found on other BALB/c MuLV. (iii) The normal sera of multiparous BALB/c mice contained antibodies that reacted with gp70 and p15(E) proteins of ecotropic MuLV. Sera from some of these mice contained antibodies that serologically distinguished the gp70 of N-ecotropic and B-ecotropic BALB/c viruses. These results emphasize the utility of mouse antisera in the serological typing of MuLV. Furthermore, the antigenic differences observed in the p30 and gp70 proteins should be of particular use in the future analysis of recombinant BALB/c MuLV.     

Taenia solium: a two-dimensional Western blotting method combined with the use of an EST-library for the identification of immunogenic proteins recognized by sera from neurocysticercosis patients

Commercial antigens used to diagnose human neurocysticercosis (NCC) are obtained from either a soluble parasite extract or a parasite-derived glycoprotein fraction. The aim of the present study was to identify antigenic proteins as potential diagnostic candidates in this context. Soluble immunogenic proteins from Taenia solium cysticerci were identified by two-dimensional electrophoresis Western blotting using human sera from Nicaragua confirmed to be positive for NCC by computer tomography. Six antigenic proteins were identified and sequenced by liquid chromatography–mass spectrometry. Among these immunogenic proteins, a novel sequence was found and named Tsol-p27. To determine the antigenicity of Tsol-p27, the previously reported antigen TsolHSP36 and the new Tsol-p27 were expressed as recombinant proteins and evaluated serologically. Immunoblotting demonstrated that Tsol-p27 was recognized by sera from 13 NCC-positive humans, whereas TsolHSP36 was identified by only two of those 13 positive sera. None of the antigens were recognized by negative control sera. Despite the limited number of serum samples evaluated in this study, the results indicate that Tsol-p27 might be a suitable candidate for diagnosis of human NCC.     

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.     

Simultaneous Purification of Murine Mammary Tumor Virus Structural Proteins: Analysis of Antigenic Reactivities of Native gp34 by Radioimmunocompetition Assays

All the structural proteins (gp47, gp34, p27, p23, p16, and p12) of the murine mammary tumor virus (MuMTV) were simultaneously purified utilizing alkylagarose chromatography as the initial fractionation step. Least-hydrophobic MuMTV polypeptides (p23, p16) and the slightly hydrophobic p27 were separated from moderately hydrophobic proteins gp47 and p12 by passage through octylimino (C(8))-agarose; the gp47 and p12 could be removed from the matrix by elution with ethylene glycol, whereas the most hydrophobic MuMTV protein, gp34, was eluted using nonionic detergent together with ethylene glycol. Subsequent purification steps involved ion-exchange or gel filtration chromatography. The resulting protein preparations appeared near-homogeneous on analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Recoveries of MuMTV proteins, based on their approximate individual contribution to total virus protein, ranged from about 20% for gp47 to greater than 100% for the minor structural component p23, the major phosphoprotein of MuMTV. Antiserum against purified C3H MuMTV gp34, together with purified, radioiodinated gp34, was used to develop a radioimmunoassay which showed that from 13 to 14% of total MuMTV protein by weight is gp34. Using this assay system, the group-specific antigenic reactivity of gp34 was also demonstrated. When solubilized preparations of C3H, RIII, and GR MuMTV's were used as competing antigens in gp34 radioimmunoassays with anti-C3H MuMTV serum, both group- and type-specific differences in antigenic reactivity were found.     

In vivo proteins of defective interfering particles of poliovirus

DX particles of poliovirus are deletion mutants that do not induce synthesis of capsid proteins or the precursor of capsid proteins (NCVPla) during infection. However, cells infected with DX particles synthesize two proteins, p68 and p25, that are not detected during growth of standard virus, and a protein of 27 000 (p27) which is comparable in molecular weight to VP3. Peptide maps of these proteins were obtained by partial digestion with Staphylococcus aureus V8 protease and elastase. The peptide map of p68 corresponded approximately 70% with the peptide map of NCVPla, and antiserum against virions reacted with p68. These data suggest that p68 is a large fragment of NCVPla. Digestion of purified structural proteins VP1, VP2, and VP3 yielded distinct peptide maps, but p25 was resistant to both V8 protease and elastase and did not react noticeably with anticapsid antibody. Peptide maps obtained for in vivo viral proteins migrating with a molecular weight of 27 000 were complex, indicating the presence of at least two and possibly three proteins. Cells infected with standard gs and gr viruses produced authentic VP3, but cells infected with defective interfering particles did not. However, one gr variant of standard virus contained a mutation in structural protein VP2.