Reactivity of avian RNA tumor viruses with lectins.

The infectivity of avian RNA tumor viruses was inactivated to varying degrees by treatment with either concanavalin A (Con A) or phytohemagglutinin but not by treatment with wheat germ agglutinin. In general, leukosis viruses reacted preferentially with Con A, whereas sarcoma viruses showed more affinity for phytohemagglutinin. In a more extensive study with subgroup A of Prague Rous sarcoma virus (PR-A), the effect of inactivation by Con A could be specifically prevented by the addition of alpha-methyl-D-mannoside, alpha-methyl-D-glucoside, and N-acetyl-D-glucosamine. These sugars were also capable of eluting [3H]glucosamine-labeled material from disrupted PR-A virus, which was bound to a Con A-sepharose affinity column. A major viral glycoprotein recovered from the column had the same mobility as gp85 in polyacrylamide gel electrophoresis and could be immunoprecipitated with anti-gp85 antiserum. These results suggest that the material reacting with Con A is present on the gp85 component of the viral glycoprotein. The diversity in the reactivity of the glycoproteins of transforming and nontransforming viruses with plant lectins is discussed.     

RNA complementary to the genome of RNA tumor viruses in virions and virus-producing cells.

Cells producing type C (avain sarcoma virus) or type B (mouse mammary tumor virus) RNA tumor viruses contain small amounts of RNA complementary to the viral genomes. The negative strands are complementary to at least 30 to 45% of the viral genomes and are found as RNA-RNA duplexes in the nucleus and cytoplasm of infected cells and in mature virions.     

Rapid purification of 70S RNA from media of cells producing RNA tumor viruses.

Media from cells producing RNA tumor viruses, when treated with sodium dodecyl sulfate and polyvinyl sulfate, yield 70S RNA as the major species binding oligo(dT)-cellulose. The procedure described for purifying 70S RNA requires no special equipment and is suitable for rapidly processing large quantities of media or for purifying RNA from commercially avialable virus, with a 5- to 10-fold higher yield than was obtained using existing methods.     

Regulation of viral and cellular RNA turnover in cells infected by eukaryotic viruses including HIV-1.

The following reviews the role of mRNA stability in the regulation of both viral and cellular gene expression in virus-infected cells. Indeed, several eukaryotic viruses, including the human immunodeficiency virus, HIV-1, regulate cellular protein synthesis via such control mechanisms. The following systems will be discussed: (i) the degradation of viral and cellular mRNAs in cells infected by herpes simplex virus (HSV) and advances made using the HSV virion host shutoff mutant; (ii) the degradation of viral and cellular mRNA and ribosomal RNA in cells infected by vaccinia virus and the possible role of the oligoadenylate synthetase-RNase L pathways; (iii) the turnover of RNAs in cells infected by encephalomyocarditis virus, reovirus, and La Crosse virus; and finally (iv) recent studies from our laboratory on the degradation of cellular mRNAs in cells infected by HIV-1.     

Alterations of acetylcholine enzymes in neuroblastoma cells persistently infected with lymphocytic choriomeningitis virus.

Persistent infection of murine neuroblastoma cells with a relatively non-cytopathic virus, lymphocytic choriomeningitis virus (LCMV), significantly lowered the cells' concentrations of choline acetyl transferase (CAT) and acetylcholine esterase (ACHE), enzymes which make or degrade acetylcholine. Quantities of acetylcholine enzymes remained depressed during the observation period of more than two years. This cellular luxury function was turned off without observable alterations in the cells' vital functions – growth rates, protein and RNA synthesis. Cloning experiments showed that CAT and ACHE levels were altered in the majority of LCMV infected neuroblastoma cells in culture and not limited to a specific subpopulation. Cells persistently infected with virus also contained receptors for neurotoxin A and α bungarotoxin. Six months after becoming infected, neuroblastoma cells having significant alterations in luxury functions stopped making infectious virus. Instead these cells now produced a defective interfering virus component. Similar events to those seen in vitro with neuroblastoma cells also occurred in vivo. Mice inoculated with LCMV at birth carried high titers of LCMV in brain tissues and viral antigens in neuronal cells as adults. Some of these mice also showed significant alterations in their ability to make and degrade acetylcholine when compared to age and sex matched controls.     

Interactions between 125-I-labelled RNA from RNA tumor viruses and RNA of other sources.

Fragmented 125I-labelled RNA from RNA tumor viruses was hybridized to unlabelled RNA from cells, viruses, and homoribopolymers. The viral RNA interacted with all RNA tested, except for certain homoribopolymers. Complex formation with unlabelled RNA was verified by nuclease resistance, buoyant density measurements, and thermal stability in solutions of different ionic strength. The RNAase-resistant complex involved 20-30% of the sequences in the 125I-labelled viral RNA and formed preferentially with nuclear RNA of cells. 125I-labelled hemoglobin mRNA, 125I-labelled immunoglobulin light chain (lambda2) mRNA, or 125I-labelled viral RNA from encephalomyocarditis virus (EMC) dit not from RNAase-resistant complexes with unlabelled cellular RNA.     

The expression and localization of surface neoantigens in transformed and untransformed cultured cells infected with avian tumor viruses

The presence and localization of neoantigens induced in cultured cells, infected or transformed with avian tumor viruses (ATV), were studied ultrastructurally on carbon platinum replicas of cell surfaces. The use of antibody, labeled with hemocyanin molecules, provided sensitive detection and analysis of cell surface antigen distribution. The subgroup-specific antigens of the viral envelope were found in considerable amount in the plasma membranes of ATV-infected chick embryo fibroblasts. The distribution of these antigens over the cell surface, evaluated on cells which were prefixed with glutaraldehyde, was found to be diffuse with a greater density on the cell processes in some cells. Reaction of antibody to viral envelope antigens with living ATV-infected cells resulted in a number of patterns of redistribution of membrane antigen-antibody complexes (AAC). Redistribution occurred in symmetrical or asymmetrical modes. The former consisted of randomly oriented aggregates (patches) of AAC over the cell surface. The latter included: (a) linear accumulation of AAC at cell margins; and (b) condensation of complexes into one or more centers of coalescence. These observations could be made on chick embryo cells infected (but not transformed) by avian leukosis virus, or on cells oncogenically transformed by avian sarcoma virus. The regions of coalescence were suggestive of the “capping” phenomenon seen in other systems, and their formation was temporally correlated with endocytosis of labeled AAC and the gradual loss of AAC from the surface. The effects of several biologically perturbing substances on the processes of redistribution were investigated in ALV-infected fibroblasts. Sodium azide, puromycin, actinomycin D, and colchicine had no effect on either form of asymmetrical redistribution. Cytochalasin B (CB) and iodoacetic acid (IAA) appeared to have some effect on the marginal redistribution, and to completely prevent the condensation into foci of coalescence (FC). When treated with these compounds, reacted with antibody at low temperature, washed free of unbound antibody, and warmed at 37° C, cells rapidly cleared their surfaces of AAC. This was not accompanied by formation of FC or endocytosis. In some of these cells, a distribution was observed which suggested a possible centrifugal flow of antigenic sites – perhaps an alternate route for disposal of AAC. None of the drugs tested affected symmetrical redistribution. Repeated attempts at detection and topographical analysis of a tumor-specific antigen on the surface of Rous sarcoma virus-transformed chicken and rat cells have provided no evidence for antibody to such an antigen in the serum of immunized animals. Autochthonous, homologous, and heterologous immunizations of chickens and rats did not produce a detectable antibody response to a virus-specific tumor surface antigen. Preliminary results, however, suggest the expression of an individual-specific (unique) tumor antigen on the surface of Rous sarcoma cells.     

Differential in vitro growth properties of cells transformed by DNA and RNA tumor viruses

Mammalian cells transformed by DNA and RNA tumor viruses are shown to display consistently different growth properties. All SV40, adenovirus type 7 and polyoma virus (DNA viruses) transformed cells propagated to high densities. The same cells transformed instead by RNA viruses: MSV strain Kirsten (MSV-Ki) or MSV strain Maloney (MSV-M) grew to densities which were consistently lower than DNA virus-transformed cells but greater than that of untransformed cells. The capacity to synthesize DNA at increasing densities also differentiated the RNA and DNA virus-transformed cells. As growing cultures of untransformed cells neared saturation density, the fraction of cells synthesizing DNA was minimal. The RNA virus-transformed cells were also contact-inhibited but at a significantly higher density. In contrast the DNA virus-transformed cells propagated to still greater densities and continued DNA synthesis at a high rate even at very high densities. Therefore the DNA virus-transformed cells truly are not contact inhibited. It is suggested that the capacity to continue DNA synthesis at high densities explains the attainment of much greater densities by DNA virus-transformed cells. There were no clear-cut differences in the ability to form colonies in agar, although a few of the RNA virus-transformed lines could not be propagated in semi-solid medium. These results may be explained as a persistence of the capacity of DNA tumor viruses to stimulate host cell DNA synthesis.     

Virus envelope markers in mammalian tropism of avian RNA tumor viruses.

Pseudotypes of vesicular stomatitis virus were prepared with avian sarcoma viruses and avian leukemia viruses representing five different subgroups. These pseudotypes display a host range restricted to that of the avian tumor virus when assayed on avian cells and are neutralized by subgroup-specific antisera. The efficiency of penetration of mammalian cells was assayed by using these vesicular stomatitis virus pseudotypes. Pseudotypes of avian tumor viruses belonging to subgroup D and of B77 virus were able to plate on mammalian cells with a high efficiency, whereas pseudotypes of other strains were not. The efficiency of penetration of the vesicular stomatitis virus pseudotypes was 10-2-to 10-3-fold higher than the efficiency of transformation of the corresponding avian tumor virus strain assayed on mammalian cells, suggesting that there are postpenetration blocks to the expression of transformation in these cells.     

A continuous-flow system for the growth of RNA tumor viruses.

By continuously pumping tissue culture medium into roller bottles containing infected cells and continuously harvesting the spent medium, RNA tumor virus can be harvested conveniently within 20 min of its release from the infected cells. An apparatus used to collect Moloney mouse leukemia virus in such a way yielded up to 390 μg protein in purified virus (about 1 mg virus) from each liter of tissue culture fluid.     

Analysis of precursors to the envelope glycoproteins of avian RNA tumor viruses in chicken and quail cells.

Immune precipitation with monospecific antiserum was employed to study the intracellular synthesis of viral glycoproteins gp85 and gp37. Labeled gp85 and gp37 were detected from lysates of cells transformed with Rous sacroma virus, strain B77, after long-term labeling with radioactive glucosamine or phenylalanine. Immune precipitates prepared from lysates of cells pulse-labeled for a short time resulted in a glycoprotein of 92,000 molecular weight (gp92). This precursor was stable in B77-transformed Japanese quail cells for several hours, whereas in chicken cells it could be chased within a few hours into virion glycoproteins gp85 and gp37. Similarly, the precursor for the structural viral proteins, pr76, persisted in quail cells much longer than in chicken cells. During very short pulses or in the presence of a glucosamine block (25 mM glucosamine), the antiserum against the viral envelope glycoproteins detected a precursor of higher electrophoretic mobility of approximately 70,000 molecular weight, "p70." Fucose label entered gp92 and gp85 as well as "p70." Proteolytic treatment of virion-bound gp85 in vitro generated two discrete glycoproteins of 62,000 and 45,000 molecular weight, but did not result in an increase in the amount of gp37.