[Interactions between Mycoplasma and human and animal viruses. Ultrastructural analysis]. / Vzaimodeistvie mikoplazm s virusami cheloveka i zhivotnykh. Ul'trastrukturnyi analiz.

Presented are the data on the ultrastructural analysis of interaction between mycoplasma and certain cancerogenic and infectious viruses in humans and animals. Revealed are spontaneous associations of mycoplasma with viruses of cattle leukemia, T-cell human leukemia and with a representative of Bunyaviruses. Immediate interaction of these agents is found possible. Simulated complexes of mycoplasma with infectious viruses are developed. Electron microscopy on supramolecular levels revealed immediate interaction of different agents in membranes. Some methodological procedures help to reveal that the interaction of M. pneumoniae and A. laidlawii with orthomyxo-, paramyxo and togavirus is of specific character and is realized as receptor ligand form due to the affinity in the receptor requirements of these pathogens. This property as well as a bequeath distribution and frequent association in respiratory infections enable one to suggest the possibility of their immediate interactions in a host body.

AtT20 pituitary tumour cells contain mouse mammary tumour virus and intracisternal A-type particles in addition to murine leukemia virus.

By electron microscopy and immunocytochemistry we have examined the retroviruses endogenous to AtT20 D16V cells, a cloned line of murine pituitary tumour cells. In addition to the C-type retrovirus particles related to Rauscher murine leukemia virus (MuLV) previously reported to bud from these cells we observed cytoplasmic A-type particles and intracisternal A-type particles (IAP). In the cytoplasm the A-type particles occur in large clusters often associated with sheets of material with a fine structure resembling the shells of the particles. At the plasma membrane individual A-type particles bud to give rise to extracellular virions. The IAP are restricted to the rough endoplasmic reticulum (RER) into which they bud: they are not transported out of the RER to the Golgi apparatus and beyond. We describe a new monoclonal antibody (designated 83E7) which is specific for an epitope of the major core protein (MTVp27) of mouse mammary tumour virus (MMTV). Using immunogold labelling procedures we have specifically labelled both the A-type particles and the associated sheets of material with this antibody. We conclude that the A-type particles and the virions they give rise to are MMTV. The sheets of material must also at least in part be made up of the major core protein of MMTV or its precursor polypeptide. AtT20 cells, therefore, contain endogenous MuLV and MMTV as well as IAP.

Early event of C-type virus budding in cells infected with a Rauscher leukemia virus temperature-sensitive mutant.

Surface replicas of ts25-infected cells reveal the organization of virus-specific knobs prior to and during the early stage of budding, and antibody-mediated ferritin labeling suggests a transmembrane association of viral envelope and core components.

Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes.

A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction.

Morphological conversion of 'immature' Rauscher leukaemia virus cores to a 'mature' form after addition of the P65-70 (gag gene product) proteolytic factor.

When the partially purified P65-70 proteolytic factor was added at increasing concentrations to 'immature' core sub-particles of Rauscher leukaemia virus (RLV), we observed an increased cleavage of P65-70 (the gag gene product) and P40 (an intermediate cleavage product containing p30) to p30, the major group specific antigen. When examined by electron microscopy the immature cores exhibited a linear decrease in number, with a concomitant increase in the number of mature cores after treatment. Various intermediate structures retaining elements of both immature and mature forms were also observed, suggesting that the in vitro conversion from immature cores to mature cores can occur on a I:I basis.

Rauscher leukemia virus populations enriched for "immature" virions contain increased amounts of P70, the gag gene product.

Preparations of Rauscher leukemia virus (RLV) that had relatively low, intermediate, or high levels of P70 (the gag gene product) on sodium dodecyl sulfatepolyacrylamide gel electrophoresis were examined by thin-section electron microscopy. A direct correlation was found between the number of immature virions in the RLV preparation and the amount of P70. The immature core subparticles isolated from these RLV preparations could themselves be further subdivided into two categories, based on their P70 content and negative stain morphology. Those immature cores containing a high P70/p30 ratio predominantly (85%) exhibited a highly coiled internal structure; those with a relatively low level of P70 exhibited less of an internal coiled structure.

Effects of filipin on the structure and biological activity of enveloped viruses.

The interaction of the polyene antibiotic filipin with membrane-bound cholesterol in vesicular stomatitis (VS), influenza, and Rauscher leukemia virions was studied. Exposure of virions to filipin resulted in a series of depressions and ridges in the envelope of VS virions, with a periodicity of 15 to 20 nm perpendicular to the long axis of the particle; similar morphological alterations were observed in negatively stained preparations, in thin-sectioned virions, and in protease-treated virions that lack surface glycoproteins. This morphological effect was specific for filipin, since the envelopes of VS virions that had been treated with another polyene antibiotic, amphotericin B, exhibited markedly different morphology. Morphological alterations induced by filipin in influenza and Rauscher leukemia virions differed from those seen in VS virions. The infectivity of filipin-treated VS virions was reduced up to 500-fold, whereas influenza virions were resistant to filipin treatment. Incorporation of filipin into the virions was demonstrated, and no release of either lipids or proteins from virions was detected after filipin treatment. A stoichiometry of approximately 1 mol of bound filipin per mol of cholesterol was found in both intact and protease-treated VS virions. The equilibrium dissociation constant for filipin-cholesterol interaction was approximately 74-fold larger in intact than in protease-treated VS virions. The initial rate of association of filipin with cholesterol in intact virions was slower than that in protease-treated particles. The fluidity of lipids in VS viral membranes, as probed by a stearic acid derivative spin label, was markedly reduced when either intact or protease-treated virions were treated with filipin.

Properties of a P70 proteolytic factor of murine leukemia viruses.

Murine leukemia viruses, such as Rauscher leukemia virus (RLV), contain a proteolytic factor which becomes activated after detergent treatment of the virus. This factor specifically cleaves P70, the gag precursor polyprotein which is enriched for in preparations of immature virus core subparticles. The factor has been partially purified on Sephadex G-75 columns. It has a molecular weight of 10,000-12,000 daltons but does not coincide in elution position with the major peaks of the viral polypeptides p10 or p12. Under optimal conditions, that is 2% NP-40 (v/v), 10 mM DTT, (pH 7.2) and incubation for 16 hr at 22 degrees C, cleavage of labeled P70 occurs and increasing amounts of the four gag polypeptides p30, p15, p12 and p10 are obtained. The P70 cleavage activity is blocked by TLCK, TAME, CBZ-lysine and other lysyl-containing protease inhibitors. Further, the CBZ-lysine inhibition is reversible, while an inhibition by phenyl-methylsulfonyl fluoride (PMSF) is irreversible. These inhibition studies suggest that a similarity exists between the P70 proteolytic factor and some serine proteases, such as trypsin. The cleavage pattern of P70-rich immature cores treated with trypsin or chymotrypsin is different from that obtained with the P70 proteolytic factor. Thus murine leukemia virions apparently contain a unique, highly specific protease which is present in small amounts and cleaves P70.

Murine leukemia virus morphogenesis: cleavage of P70 in vitro can be accompanied by a shift from a concentrically coiled internal strand ("immature") to a collapsed ("mature") form of the virus core.

Disruption of Rauscher leukemia virus (RLV) with low levels of Nonidet P-40 yielded "immature" cores. These cores have a diameter of about 920 A, as opposed to the 1300-A diameter of RLV, possess knob-like protuberances, and contain a concentrically coiled internal strand apposed to the core shell. The two major polypeptide components of immature cores are (i) p30, the 30,000-dalton group-specific antigen, and (ii) a polypeptide that has the size and antigenic characteristics of P70, the 70,000-dalton precursor protein of the group-specific antigens of murine leukemia virus. Disruption of RLV at high ratios of Nonidet P-40 to virus yielded "mature" cores. These cores have an average diameter of 850 A, a smooth proteinaceous perimeter, and a collapsed internal strand, and they contain predominantly p30. Treatment of RLV with low levels of Nonidet P-40 for 16 hr at 22 degrees yielded cores that showed (I) a 70% decrease in the number of immature forms and concomitant increase in the number of mature forms, (II) a 60-90% decrease of P70, and (iii) a 30% increase in a 40,000- to 42,000-dalton protein. These results suggest that maturation of RLV cores is accomplished by cleavage of P70.

Envelope glycoproteins of Rauscher murine leukemia virus: isolation and chemical characterization.

The envelope glycoproteins (designated gp70 and gp45) of the Rauscher strain of murine leukemia virus were solubilized by osmotic shock and freeze-thawing in chaotropic solutions. The viral glycoproteins were then purified by phosphocellulose chromatography and gel permeation chromatography on Bio-Gel A-1.5m. Yields by this procedure were 6.2% for gp70 and 1.3% for gp45 on a protein input basis. The apparent molecular weights were respectively 67 500 and 47 500 with a polypeptide chain molecular weight of approximately 45 000 for both glycoproteins. Amino acid analysis showed a high degree of similarity for both components, with some differences subject to further evaluation. The total carbohydrate content was approximately 32% for gp70 and 6-9% for gp45. In keeping with the amino acid compositional similarity suggesting relationships, alanine was found to ba the amino-terminal amino acid of both glycoproteins, and cross-reactivity was demonstrated by immunologic tests. The data suggest that the chief difference between gp70 and gp45 lies in the carbohydrate content.

Structural proteins of ribonucleic acid tumor viruses. Purification of envelope, core, and internal components.

Murine type C virus structural proteins, the envelope glycopeptides, 30,000 dalton major core protein, and 15,000 dalton internal protein have each been purified to near homogeneity and in high yield from the smae batch of virus by use of phosphocellulose column chromatography and gel filtration procedures. Evidence that these proteins are specified by the viral genome was obtained by competition radioimmunoassay analysis, comparing these polypeptides from Rauscher virus cultivated in a variety of mammalian cell lines; all of the reactive antigenic determinants of these proteins appeared to be virus-specific.