Ronald C. Montelaro

An analysis of type-C retrovirus polypeptides and their associations in the virion.

Abstract The polypeptide compositions of [3H]leucine-labeled Prague-Rous sarcoma virus-subgroup C (Pr-RSV-C) and Friend murine leukemia virus (FLV) were investigated using guanidine hydrochloride gel filtration and a high-resolution SDS-polyacrylamide-gel system. These techniques resolved seven major structural components in Pr-RSV-C (gp85, gp35, p27, p19, p12, p15, and p10), as reported previously for other avian leukosis-sarcoma viruses. However, in the case of FLV two previously unresolved proteins (p15E and p12E) were clearly demonstrated in addition to gp71, p30, p15C, p12, and p10. FLV p15E, p12E, and gp71 were removed when intact virions were digested with bromelain, whereas the remaining components were not affected. These and other studies support the notion that gp71, p15E, and p12E are situated on the surface of the virion. The linkages (covalent and noncovalent) between viral polypeptides were also examined. The results of these studies indicate that more than 90% of avian gp85 and gp35 are disulfide linked as a viral glycoprotein complex (VGP). The data also suggests that p19 exists as a network of disulfide-linked molecules, some of which may be further linked to VGP. In contrast to the avian system, only about 10–15% of FLV gp71 is disulfide linked to p15E in the VGP complex; the remaining gp71 is apparently loosely attached to the virus, perhaps by a noncovalent interaction with p12E. The implications of these associations to virus structure and assembly are discussed.

Structure and morphogenesis of type-C retroviruses.

Publisher Summary This chapter describes the models for the structure and morphogenesis of avian and murine type-C retroviruses. These are based on the molecular arrangement of virion structural components in the particle as well as on available data relating to the biosynthesis of these products. Since the avian and murine retroviruses are the most thoroughly characterized, the chapter focuses mainly on these systems. The visualization of the morphology of type-C viruses by electron microscopy has been generally impaired because of their labile structure and artifacts generated by various staining methods. However, gentle purification procedures combined with improved sample preparation and staining techniques have permitted a visualization of the fine structure of type-C viruses from a variety of species. Recent electron microscopic investigations with murine and avian type-C viruses revealed the presence of a thin layer, the inner coat (IC), located just beneath the viral envelope. The IC is apparently attached to the envelope since it remains in its normal configuration even in instances where the inner components of the virus are well separated from the envelope. The viral envelope and IC surround the virus core that consists of an outer core shell (CS) of hexagonally arranged subunits, enclosing the internal electron-dense ribonucleoprotein complex (RNP). The RNP appears as a filamental strand in the form of a spiral. The localization of the analogous avian and murine structural components in the virion is also summarized.

Further characterization of the oncornavirus inactivating factor in normal mouse serum.

Abstract The nature of the oncornavirus inactivating factor (OIF) contained in the normal sera of certain mouse strains was investigated by various biochemical and biological techniques. The OIF copurified quantitatively with the lipoprotein fraction of STU mouse serum subjected to flotation through sucrose density gradients. The buoyant material consisted of a heterogenous population of spherical particles (200–1200 A) which had a density 70%) lipid moiety. All of these properties are characteristic of serum very low-density lipoproteins (VLDL) and, therefore, suggest that OIF is contained in the VLDL fraction of normal mouse serum. In contrast to whole serum which specifically inactivated xenotropic and polytropic murine oncornaviruses, the isolated VLDL fraction was also able to inactivate murine ecotropic viruses and feline leukemia virus. Fractionation of STU lipoproteins or whole serum by extraction with ether:ethanol (3:1) removed the OIF into the organic solvent. This factor extract inactivated the same spectrum of viruses as serum VLDL. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of unlabeled and radioiodinated factor extracts revealed no detectable protein components. Thus, the inactivating capacity of normal serum apparently can be mediated by lipid micelles devoid of protein. NIH Swiss mouse sera, previously reported not to have OIF, did harbor high titers of factor in an inactive form which displayed activity upon flotation or after extraction with ether: ethanol. However, this phenomenon was specific for NIH mouse sera, since VLDL and ether:ethanol extracted fractions of sera from a number of mammalian species were not found to be active against xenotropic or recombinant murine leukemia viruses (MuLVs). In view of the potential role of recombinant MuLVs in murine leukemia, sera of individual AKR mice were serially examined for a diminution of OIF in the preleukemia or leukemic stages of the disease. Also, serial bleedings were tested from individual C57B1 mice prior to and after irradiation and then monthly until the development of radiation-induced leukemia. In both cases, no apparent significant reduction of OIF titer was observed.

Normal chicken cells (chf−) express a surface antigen which cross-reacts with determinants of the major envelope glycoprotein (gp85) of avian myeloblastosis virus☆

Abstract Using rabbit antisera raised against the purified major envelope glycoprotein (gp85) of avian myeloblastosis virus (AMV), we were unable to differentiate between normal chicken cells carrying the chicken-helper factor (chf+) from those lacking this endogenous virus information (chf−) in several serological assays, including competition radioimmunoassay and membrane immunofluorescence. Radioimmunoprecipitation analysis of the material recognized in uninfected chf(+) and chf(-) chicken cells by the rabbit anti-AMV gp85 antisera revealed the presence in immune precipitates of both cell types of a high molecular weight glycoprotein (∼125,000), while low amounts of gp85 were precipitated only from the chf(+) cells. In contrast, antisera raised against the gp85s of three other avian oncornaviruses did not recognize the 125,000-dalton molecule in either cell type, although gp85 was again precipitated from the chf(+) chicken cells. The high molecular weight glycoprotein (denoted NCG) appears to represent a normal chicken cell surface glycoprotein which is antigenically cross-reactive with unique determinants of AMV gp85, presumably dependent on the carbohydrate portion of the molecules. The significance of this cross-reactivity is discussed, as well as the interpretation of previous results obtained through the use of these rabbit anti-AMV gp85 antisera.

A procedure for the preferential radioiodination of small amounts of protein in the presence of excess lipids

Abstract The chloramine-T, iodine monochloride, and lactoperoxidase radioiodination procedures were evaluated for their ability to label proteins in the presence of large amounts of reactive lipid. Mouse serum very low density lipoproteins (VLDL) (10% protein, 90% lipid) and detergent-disrupted Friend murine leukemia virus (FLV) (60% protein, 33% lipid) were used as model systems. Based on the distribution of label between protein and lipid, as well as the total amount of label incorporated by each procedure, only a modification of the iodine monochloride procedure preferentially labeled proteins to a high specific activity in both VLDL and FLV. The technique, which is described in detail, is technically simple, rapid, and causes no detectable degradation of proteins.

A model for assembly of type-C oncomaviruses

Direct evidence for many aspects of what we propose is not available, or as best fragmentary, especially with regard to the temporal sequence of the events described above. In most instances relevant studies have not been carried out in a single virus system and suggestions are made on the basis that morphogenesis of avain and mammalian type-C viruses is a uniform process. Nonetheless, we have already found this to be a good working model for virus assembly and propose it to others in an effort to stimulate new approaches to this old problem.

Transfer of radioiodine from iodinated oncornavirus proteins to unlabeled protein carrier during routine procedures for peptide mapping

Abstract Denaturation of radioiodinated oncornavirus proteins by performic acid oxidation, a routine procedure in peptide mapping studies, results in the unexpected release of 60–80% of the iodine label. In addition, if unlabeled carrier protein is present during oxidation, the iodine label is efficiently transferred to the carrier protein. Radioiodine release and transfer was not detected when proteins were denatured by reduction and carboxymethylation. These results indicate that reduction and carboxymethylation are the preferred procedures for denaturing proteins prior to chemical or enzymatic cleavage and peptide analysis.

A Single Genetic Locus Determines the Efficacy of Serum Therapy Against Murine Adenocarcinoma 755a

We have recently described a unique model system for immunotherapy where immunological management of a highly lethal tumor, the ascites form of the Adenocarcinoma 755 (AD755a), could be carried out reproducibly and with remarkable efficiency [7]. Some of the basic features of this model are summarized in Table 1. The studies to be described in this report will concern themselves primarily with a more in depth analysis of the phenomenon of strain variation in relation to the protective capacity of the serum, and were done in an effort to gain some insight into the mechanism by which the serum mediates its powerful effect.