Showing papers by "David Baltimore published in 1979"

A normal cell protein cross-reactive to the major Abelson murine leukaemia virus gene product

Abstract: Abelson murine leukaemia virus (A-MuLV) is a replication-defective retrovirus capable of rapid in vivo and in vitro transformation of bone marrow lymphoid target cells. A-MuLV was isolated from a steroid treated BALB/c mouse inoculated with the replication competent Moloney-MuLV (M-MuLV)1–4. The A-MuLV genome (5.5 kilobases) retains regions of precise homology to M-MuLV at its 5′ end (1,320 bases) and 3′ end (730 bases). The centre of the A-MuLV genome (3,500 bases) is non-homologous to M-MuLV and presumably represents unique sequences derived from the mouse genome by a recombinational event5. The only known protein encoded by the A-MuLV genome is one of MW 120,000, called P120 (refs 6, 7). About one-quarter of this protein is encoded by the 5′-terminal M-MuL V-related sequences and the rest is encoded by the A-MuLV unique sequences. P120 has been found in all cell lines transformed by the strain of A-MuLV that originally derived from ANN-1 cells2, including transformed non-producer cells. P120 can be translated in vitro using A-MuLV genomic RNA as messenger RNA. P120 is a phosphoprotein but there is no evidence for glycosylation6,8,9. We have used an anti-A-MuLV syngeneic tumour regressor serum with defined specificities to search for cross-reacting normal cellular proteins. We have identified a protein of MW 150,000 (NCP150) in metabolically labelled thymus and other lymphoid organs. NCP150 by absorption and competition analysis is closely related to the unique region of the A-MuLV P120 protein.

Preparation of syngeneic tumor regressor serum reactive with the unique determinants of the Abelson murine leukemia virus-encoded P120 protein at the cell surface.

Abstract: Antisera reactive with the Abelson murine leukemia virus (A-MuLV)-specified P120 (anti-AbT sera) were produced in C57L/J mice. Of many strains tested, only C57L/J reproducibly rejected syngenic A-MuLV-induced tumor cells; after multiple immunizations their sera would immunoprecipitate both P120 and Moloney-MuLV (M-MuLV) proteins. Using labeled A-MuLV-induced nonproducer cells, only P120 could be detected by anti-AbT sera, suggesting that it may be the only A-MuLV-specified protein. Reactivity of anti-AbT sera with P120 was not blocked by M-MuLV virion proteins, implying that the sera recognize a portion of P120 that is not homologous to any M-MuLV product. Anti-AbT sera stained the surface of live, A-MuLV-transformed nonproducer cells in a two-stage immunofluorescence assay, and such staining was not blocked by M-MuLV protein. Also, intact A-MuLV-transformed cells absorbed much of the reactivity of certain anti-AbT sera for P120. Thus a portion of P120 appears to be exposed on the surface of transformed cells. P120 lacks detectable carbohydrate, is not affected by endoglycosidase H, and cannot be labeled by lactoperoxidase-catalyzed iodination. Thus P120 is an unusual surface protein.

Poliovirus Polyuridylic Acid Polymerase and RNA Replicase Have the Same Viral Polypeptide

Abstract: A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.

Poliovirus replicase: a soluble enzyme able to initiate copying of poliovirus RNA.

Abstract: The soluble phase of the cytoplasm of poliovirus-infected cells contains an enzymatic activity able to copy RNA without an added primer. This replicase activity has been purified 60-fold; it is absent from uninfected cells. Poly(U) polymerase activity copurifies with replicase activity. Although less pure replicase fractions copy a variety of RNAs, purer fractions respond better to poliovirus RNA than to other viral RNAs. Even the less pure fractions make a specific copy of the added template, as shown by hybridization of the product to its template RNA but not to other RNAs. Among homopolymers only poly(A)-oligo(U) was copied by the replicase; other primed homopolymer templates were inactive.

Virus-like 30S RNA in mouse cells.

Abstract: Uninfected JLS-V9 mouse cells are known to express high levels of viral sequences that hybridize to complementary DNA made by the BrdU-induced virus of JLS-V9 cells. The genome in the BrdU-induced virus has been found to consist mainly of an RNA species that migrates as 30S RNA material during electrophoresis through agarose gels. This virus-like 30S RNA, designated VL30 RNA, apparently represents a new class of endogenous defective retroviruses that are not generally evident because of their defectiveness and lack of biological function. Fingerprint analysis and hybridization studies show that VL30 RNA does not have homology with the standard nondefective murine leukemia viruses. Upon superinfection with a nondefective murine leukemia virus, or upon induction of endogenous virus with BrdU, VL30 RNA is rescued into virions by phenotypic mixing. When VL30 RNA is rescued by BrdU induction, the VL30 RNA is mainly organized as a 50S complex, but when VL30 is rescued by superinfection, VL30 is also found in 70S RNA. Rescued VL30 RNA sequences can be reverse transcribed by the virion-associated DNA polymerase in an endogenous reaction. Many mouse cells express the sequences, whereas heterologous cells such as rat or rabbit cells do not contain them. By using hybridization of a complementary DNA probe to cellular RNA immobilized on paper, no subgenomic RNA related to the VL30 RNA could be found in cells expressing the VL30 sequences. From 20 to 50 copies of these sequences were found to be contained in the mouse genome. VL30 RNA is probably present in most stocks of leukemia and sarcoma viruses made in mouse cells.

Transformation of Immature Lymphoid Cells by Abelson Murine Leukemia Virus

Abstract: Leukemia viruses have a special interest for Immunologists because many of them transform cells of the lymphocyte lineage into contitiuously growing permanent lines of cells. One of the most useful of such viruses is the Abelson murine leukemia virus (A-MuLV) (Rosenberg & Baltimore 1979). This virus is able to generate lines of cells with properties that suggest the cells are counterparts of various stages of B-lymphocyte differentiation. The ability of a virus to transform a specific lineage of cells raises a wide range ofquestions. These questions include: What genetic elements of the virus play a role in Its transformation ability? What proteins are made by the virus that play a role in transformation? How do these proteins work? What kind of cell is a target for the virus? What characteristics can be used to define the differentiation state of the resulting tumor cells? Can such tumor cells be used to understand the normal process of differentiation? In this review we will attempt to summarize the present state of knowledge about these various questions in relation to A-MuLV and suggest the types of information that may be derived from future studies. A-MuLV came to light during an experiment in which Abelson & Rabstein (1970 a,b) infected steroid-treated BALB/cCr mice with Moloney murine leukemia virus (M-MuLV) (Fig. 1). One of the mice in the study developed a lymphosarcoma 37 days after virus infection. This tumor involved the cervical and inguinal lymph nodes, the lower vertebral column, the marrow of the calvaria and the meninges. No evidence of a thymic tumor was present. Histologically, the tumor cells were indistinguishable from other virus-induced lymphoma cells. The lymphoblasticcells were oflarge size with a high nucleus to cytoplasm ratio. The nucleus was characterized by diffuse chromatin and a prominent nucleolus and the cytoplasm had abundant free ribosomes (Rabstein etal. 1971, Siegler etal. 1972).

Synthetic phospholipid vesicles containing a purified viral antigen and cell membrane proteins stimulate the development of cytotoxic T lymphocytes.

Abstract: Synthetic phospholipid vesicles (liposomes) containing the purified glycoprotein (G) of vesicular stomatitis virus (VSV) and solubilized membrane proteins from cells of the appropriate H-2 haplotype elicited H-2-restricted cytotoxic T lymphocytes (CTL) that lysed VSV-infected target cells. The CTL were elicited by intact liposomes, not by released components. Thus, when spleen cells from VSV-primed H-2d X H-2b hybrid mice were stimulated with liposomes having G protein + membrane proteins from cells with one of the parental H-2 haplotypes, the resulting CTL lysed only VSV-infected target cells with that parent's H-2 type. This result argues against the view that T cells in general recognize only processed antigenic fragments on macrophages. Moreover, liposomes were only effective when G protein and cell membrane proteins were included in the same vesicles. This result suggests that for effective interaction with CTL precursors the antigen (G protein) and products of the H-2 complex must be closer to each other than 600--1,000 angstrom, the diameter of the lipid vesicles used in this study.

Synthesis of a 600-nucleotide-long plus-strand DNA by virions of Moloney murine leukemia virus

Abstract: A discrete, 600-nucleotide-long plus-strand DNA has been identified among the products of reverse transcription by virions of Moloney murine leukemia virus. Its polarity was shown by hybridization to minus-strand DNA. It appears to be copied from the right end of minus-strand DNA because (i) its restriction endonuclease cleavage pattern corresponds to the redundant 600-base segment found at either end of the ultimate double-stranded reverse transcription products, (ii) its synthesis is actinomycin D sensitive, and (iii) its synthesis begins during the first hour of a reverse transcription reaction when only the right-hand end of minus-strand DNA is available as template. We therefore call this DNA plus-strong-stop DNA by analogy with the minus-strong-stop DNA copied from the left end of the viral RNA. Both strong-stop DNAs are made early during in vitro reactions and decline in concentration later, consistent with postulated roles as initiators of long minus- and plus-strand DNA. Unlike minus-strong-stop DNA, plus-strong-stop DNA remains as a double-stranded nucleic acid after its synthesis, as shown by S1 nuclease resistance. A primer to initiate plus-strong-stop DNA synthesis has not been identified; the product found thus far has no detectable RNA attached to it.

Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.

Abstract: A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.

Production of a discrete, infectious, double-stranded DNA by reverse transcription in virions of Moloney murine leukemia virus.

Abstract: One of the most unique viral replication schemes is that of the retroviruses (a class that includes the RNA tumor viruses) These viruses synthesize a double-stranded (DS) DNA copy of their single-stranded (SS) RNA genome as the initial event following infection of susceptible cells (see Weinberg 1977) The details of this process—called reverse transcription—are still obscure, but the general outlines have become clear during the last few years

Virus production by Abelson murine leukemia virus-transformed lymphoid cells.

Abstract: Cell lines obtained by in vitro transformation of bone marrow with Abelson murine leukemia virus (A-MuLV) can be divided into three classes: producers, releasing reverse transcriptase-containing particles and infectious virus; nonproducers, releasing no viral particles; and defective producers, the most common phenotype, releasing particulate reverse transcriptase in the absence of infectious virus. When such cell lines were analyzed 1 to 2 weeks after their isolation, however, all produced infectious virus. Because these cell lines were carried in culture, many ceased to release infectious virus but produced defective virions. One defective producer, SWR4, has been extensively studied. The particles it produces have the same density as that of virions of Moloney murine leukemia virus (M-MuLV). The particles contain no 35 to 70S RNA, as determined by analysis of [3H]uridine-labeled particles, and exhibit no endogenous reverse transcriptase activity. Although the reverse transcriptase enzyme is of normal size, the major structural protein of the defective virions has a molecular weight of 28,000 (p28), in contrast to the p30 of M-MuLV, and no viral glycoprotein was evident. The defective particles do not appear to arise either from the helper virus or from Abelson virus. An alteration of the protein of the helper virus is an unlikely source of p28 because particles produced by lymphoid cells transformed with another strain of M-MuLV as helper (M-MuLV-TB) contained p28 with an unaltered cleavage pattern, although M-MuLV-TB p30 differs from M-MuLV p30. The A-MuLV genome lacks the capacity to code for the reverse transcriptase virions. Clones of fibroblasts infected with A-MuLV only occasionally produce defective particles. The defective particles therefore probably arose from an endogenous virus that is preferentially expressed in the class of lymphoid cells transformed by A-MuLV. This interpretation implies that the majority of A-MuLV-transformed lymphoid cells completely lose expression of the helper virus genome.

Synthesis by Lymphoid Cells Transformed in Vitro by Abelson Murine Leukemia Virus

Abstract: Edward J. Siden and David Baltimore Department of Biology and Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Daniel Clark and Naomi E. Rosenberg Department of Pathology and Cancer Research Center Tufts University School of Medicine Boston, Massachusetts 02111 Summary The majority of cell lines derived by infection of murine bone marrow cells with Abelson murine leukemia virus (A-MuLV) synthesize a p chain but no detectable light chain. Aside from this p-only phenotype, lines that make only light chain, both chains or no immunoglobulin-related polypep- tides have also been found. Two lines have been studied in detail: one that makes only p chain and one that makes only