Expression of endogenous xenotropic retrovirus by methylcholanthrene-induced squamous cell carcinoma of the mouse respiratory tract.

As a model for human lung cancer, squamous cell carcinomas were induced by 3-methylcholanthrene in mouse tracheas which had been explanted to a subcutaneous site. The tumors that developed were examined for both ecotropic and xenotropic infectious murine leukemia virus (MuLV). From all squamous carcinomas--six out of six--a xenotropic MuLV was isolated. From some of the fibrosarcomas that occurred incidentally in our induction system, ecotropic MuLV was isolated. However, in the fibrosarcomas, no xenotropic MuLV at all was found.

Identification of tryptic peptides unique to a 110,000-molecular weight polyprotein encoded by the T-8 isolate of murine leukemia virus.

Mink cells nonproductively-infected with the weakly-transforming T-8 isolate of murine leukemia virus (MuLV) express a 110,000 mol wt polyprotein designated T-8 P110. By immunoprecipitation analysis, T-8 P110 is shown to contain AKR-MuLV amino terminal gag gene-specific components (p15, p12) but to lack p30, p10, gp70, and p15(E) antigenic determinants. These observations are further substantiated by tryptic peptide analysis indicating T-8 P110 to share approximately six lysine-containing tryptic peptides with AKR-MuLV Pr65gag, and none with AKr-MuLV Pr82env. Furthermore, of seven methionine-containing T-8 P110 tryptic peptides, at least four can be conclusively shown not to be present in either AKr-MuLV Pr180gag/pol or Pr82env. A clonal mink cell line nonproductively infected by T-8, and expressing high levels of P110, although not morphologically transformed, is shown to lack elevated levels of tyrosine-specific protein kinase activity and reduction of epidermal growth factor binding sites characteristic of cells transformed by many other RNA-transforming viruses. These findings argue either that the T-8 viral genome contains acquired cellular sequences encoding a portion of P110, or that T-8 P110 represents an inphase deletion of AKR-MuLV Pr180gag/pol with extensive posttranlational modification and that an as yet unidentified protein is responsible for T-8 associated transformation.

Relationships of gp70 of MuLV envelopes to gp70 components of mouse lymphocyte plasma membranes.

The family of glycoproteins called gp70 includes molecules that are the main constituent of murine C-type viral envelopes, and some that are expressed as mendelian constituents of thymocyte plasma membranes in the absence of virions. To investigate further the relation of viral gp70s to plasma- membrane gp70s we compared peptide maps of gp70s derived by immunoprecipitation from cells infected with chosen viruses and from various thymocytes and leukemiacells known to express one or more of three immunogenetically defined gp70 types: Glx-gp70, X-gp70, and O-gp70. Maps of gp70 from cultured cells infected with ecotropic and xenotropic viruses were distinguishable from one another, and in general resembled gp70 maps prepared directly from ecotropic and xenotropic virions respectively. Maps of gp70s immunoprecipitated from thymocytes of five mouse strains and from two A strain T-cell leukemias also fell into two distinguishable and generally corresponding patterns. Thus peptide-mapping substantiates earlier conclusions that viral gp70s and plasma-membrane gp70s inherited independently of virus-production are highly related or identical molecules. The gp70 maps of thymocytes from B6, B6-G(+IX), 129, and A mice formed a group resembling the map from cultured cells infected with xenotropic virus. Thymocytes from AKR mice, and the two A strain leukemias, gave gp70 maps conforming more to the second pattern, that of cultured cells infected with ecotropic virus. This second pattern probably comprises at least two gp70 types, one of which is X-gp70. Our data indicate that the G(IX)-gp70 and O-gp70 sub-species of gp70 expressed in the cell populations we have studied are coded by xenotropic viral genomes, and X-gp70 by ecotropic viral genomes.

Amino acid sequence homology between histone H5 and murine leukemia virus phosphoprotein p12.

The amino terminal acid sequences of several mouse leukemia virus phosphoproteins (p12) show definite homology with the amino terminal conserved region of H5 histones, the phosphorylated nuclear proteins of nucleated erythrocytes. Differences in the amino acid compositions of the two groups of proteins seem to rule out the possibility that they evolved from a single common ancestral gene. The finding of sequence homology between viral p12's and cellular histones, however, is consistent with evolution of retrovirus structural proteins by a process of differentiation from preexisting cellular genes. The conserved primary and secondary structure at the amino terminal region, common to both groups of proteins, may be related to their common function of nucleic acid binding modulated by phosphorylation.

Molecular relatedness of mammalian RNA tumor viruses as determined by DNA hybridization.

Each of six mammalian C-type viruses-including two feline leukemia viruses, three murine leukemia viruses, and the human "candidate" virus RD-114-can be distinguished from each other by hybridizing DNA synthesized by viral reverse transcriptase with viral RNA. Characterization of the DNA . RNA hybrids by hydroxylapatite chromatography revealed nucleotide sequence diversity among the viruses, detectable both by the amount of cross-hybridization and by the decreased thermal stability of heterologous hybrids.

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses.

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.

Complete amino acid sequence of the nucleic acid-binding protein of bovine leukemia virus.

The complete amino acid sequence of the nucleic acid-binding protein p12 of bovine leukemia virus (BLV) has been determined. Peptides were generated by enzymatic digestion and formic acid cleavage, purified by reversed-phase liquid chromatography and subjected to automated Edman degradation. BLV p12 is a proline-rich linear polypeptide composed of 69 amino acids with Mr 7558. A comparison of the p12 structure to that of the avian and murine type C retroviral nucleic acid-binding proteins shows significant homology only in the putative binding domain. This conserved region is duplicated BLV p12 as in the avian homolog.

Measurement of binding specificity between T cell lymphomas and murine leukemia viruses.

We have previously reported the presence of receptors on radiation leukemia virus (RadLV)-induced thymomas and malignant thymocytes from AKR mice which specifically bind retrovirus produced by these T cell clones. These receptors have been shown to have specificity for virus reminiscent of an immune-specific receptor. Previous studies on T cell lymphoma binding to retroviruses have involved measurement of the interaction of labelled virus with cells using fluorescence-activated cell sorter (FACS) analysis (McGrath et al., J. Virol. (1978) 25, 923; McGrath and Weissman, Cell (1979) 17, 65; Weissman and McGrath, Curr. Top. Microbiol. Immunol. (1982) 98, 103). Here we report development of an assay for measuring lymphoma binding to virus, prepared as an immunoabsorbent adhered to a microtiter plate. Using this assay, we have shown that only T and not B cell lymphomas can bind to T cell-tropic viruses, and some cell lines have greatest specificity for homologous virus. The AKR-derived T cell lymphomas, SL3 and KKT-2, show greater specificity for leukemogenic AKR viruses, than for an AKR xenotropic virus or the recombinant AKR virus, MCF247. The RadLV-induced T cell lines, C6VL/1 and BL/VL3, have been found to bind cross-reactively to several different murine leukemia viruses (MuLVs). RadLV-induced T cell lymphomas do have greater specificity for their cognate retroviruses since free, homologous retrovirus can best block the interaction between cells and virus adhered to the wells of a microtiter plate. Cross-reactive interactions are more easily demonstrated by this assay, probably because low avidity interactions are stabilized as a result of the mode of virus presentation. Binding specificity for retroviral envelope determinants has been demonstrated using a rat anti-retroviral antiserum prepared as an F(ab)1 fragment. This antiserum can inhibit the interaction between the C6VL/1 thymoma and its RadLV virus. Specificity of this antibody for a gp70-like protein was confirmed by NaDodSO4-polyacrylamide gel electrophoresis (PAGE) and by loss of this activity after absorption of antibody on virus. Antibodies specific for RadLV/VL3 gp70 determinants can inhibit the interaction of C6VL/1 with RadLV/VL3 suggesting that cross-reactive binding to heterologous virus is also specific for a gp70 viral env determinant.

[Properties of mouse hemocytoblastosis-reticulosis (Mazurenko) virus]. / Izuchenie nekotorykh svoistv virusa gemotsitoblastoza-retikuleza (Mazurenko) myshei

The results of molecular biological study of the properties of hemocytoblastosis-reticulosis virus (Mazurenko) are presented. The virus was found to have buoyant density of 1.16 g/cm3 in sucrose density gradient. Virions contain 70S RNA possess reverse transcriptase activity.

Spatial resolution and detection sensitivity in microanalysis by electron loss selected imaging.

An electron energy filter of the Castaing-Henry type in a high resolution transmission electron microscopy was tested for sensitivity and spatial resolution at a specific electron energy loss in energy selected images of a murine leukaemia virus. Electron spectroscopic images of phosphorus within the virus membrane bilayer demonstrated a best spatial resolution between 0.3 and 0.5 nm and a sensitivity of 2 X 10(-21) g.

Restriction endonuclease mapping of unintegrated viral DNA of B- and N-tropic BALB/c murine leukemia virus.

Unintegrated linear and closed circular DNAs of B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were extracted from newly infected mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII, SalI, XbaI, KpnI, SmaI, and PstI restriction endonucleases. The DNA fragments were separated by electrophoresis and analyzed by the Southern blot hybridization procedure. EcoRI did not cleave the two genomes. A physical map of 15 cleavage sites on B- and N-tropic genomes was constructed with the other restriction endonucleases. Identical cleavage sites of B- and N-tropic MuLV DNAs were found with all these enzymes. However, the N-tropic linear genome was found to lack about 75 base pairs at each end of the molecule. PstI, KpnI, and SmaI recognize a cleavage site at both ends of the linear molecules. And sequences derived from the 5' end of the RNA genome were found in the third left end of the linear DNA and at its extreme right-end terminus, suggesting the presence of redundant sequences. Two species of closed circular viral DNA were observed. The larger species has the same size as the linear molecule and appears to be a circularized form of linear DNA. The smaller species contains sequences common to both the linear and the larger circular viral DNA but seems to be deleted from sequences present at either one or both ends of the linear DNA. Therefore, the general structure of the linear and circular DNA species of these B- and N-tropic endogenous BALB/c MuLV appears analogous to the structure found with other retroviruses.