“gag” polyprotein precursors of rauscher murine leukemia virus

Section: Resultsmentioning

confidence: 99%

Epididymis is a principal site of retrovirus expression in the mouse.

Kiessling

Crowell

Fox

1989

“…The lack of gp7O reactivity may be due to specific suppressor cells (46,47 (53,54), and uncleaved precursors can appear on the cell surface as glycosylated products (55). Precursor polyproteins of Mr 180,000-210,000 containing gag and pol gene products have been detected in murine leukemia virus-and avian sarcoma virus-infected cells (56)(57)(58)(59)(60), and a Mr 250,000 precursor bearing gp7O sequences has been reported in Rauscher murine leukemia virus-producing cells (61). Abelson antigen (Mr 100,000-130,000) (62) may be present on the surface of these cells, either in native form or as a precursor containing murine leukemia virus gag proteins (63,64).…”

Section: Methodsmentioning

confidence: 99%

Malignancies of metastatic murine lymphosarcoma cell lines and clones correlate with decreased cell surface display of RNA tumor virus envelope glycoprotein gp70.

Reading¹,

Brunson²,

Torrianni³

et al. 1980

Metastasis is dependent on both host and tumor cell characteristics (1-3). In order to study tumor cell properties associated with metastasis, a number of animal tumor models have been developed by sequential selection methods in vivo (4-9) and in vitro (10-13). Of particular interest are cell surface properties of these tumor models because in several systems the cell surface appears to be important in determining the degree and location of experimental metastasis (3,6,(12)(13)(14)(15)(16).A tumor model for lymphosarcoma metastasis has been developed from the murine RAW117 lymphosarcoma line which is capable of forming some solid tumor nodules in livers, lungs, and spleen of syngeneic BALB/c mice (5). By selecting 10 times for liver colonization, a more malignant lymphosarcoma subline RAW117-HlO was obtained which rapidly killed its host (5) and formed t200-250 times more gross liver tumor nodules than did the parental line RAW117-P after intravenous injection (5, 13). Utilizing the low-malignancy RAW117-P parental line, we selected in vitro for nonadherence to immobilized lectins such as concanavalin A (Con A) to obtain variant sublines that show increased potential for experimental metastasis in vivo (13,17). Similarly, selecting in vitro of the highly malignant subline RAW117-H1O on wheat germ agglutinin (WGA) yielded variant sublines with decreased metastatic potential (13). The cell surface properties of these lymphosarcoma sublines and several clones derived from them have been analyzed, and we find that there is an inverse relationship between the metastatic potentials of these sublines and clones and the expression of cell surface RNA tumor virus envelope glycoprotein gp7O. MATERIALS AND METHODSCell Lines. RAW117 lymphosarcoma cells were grown as described (5). The methods of sequential in vivo selection for malignant variant sublines (RAW117H1 through RAW117-H10) with enhanced ability to form solid tumor colonies in the livers of syngeneic BALB/c mice (5) and the methods for repeated in vitro selections for lectin-binding variant sublines (RAW117-P Con Aa10 and RAW117-H10 WGAal1) on lectincoated petri dishes have been reported (13,17).Cell Clones. Clones were obtained from the established parental cell line RAW117-P, from the in vivo-selected subline RAW117-H10, and from the in vitro-selected sublines RAW117-P Con Aa10 and RAW117-H10 WGAal1 by the limiting-dilution technique. Briefly, cells were diluted to one cell per ml in Dulbecco's modified Eagle's medium (GIBCO) containing 10% heat-inactivated (560C, 30 min) fetal bovine serum (Flow Laboratories, Inglewood, CA), and 0.2 ml was distributed to each of 96 wells in a Microtest II plate (Falcon). After 2 weeks, clones were visible as a small white spot on the bottom of the well; infrequent wells with two clones were discarded. Cloning efficiencies of the RAW117 lines and sublines were approximately 80%In Vivo Assays. RAW1 17 lines were assayed for organ colonization (experimental metastasis) after intravenous injection of 5000 viable lymphosarc...

“…These two pathways begin with two independent translation products, gPr80gag and Pr65ag. Pr65gag is processed by proteolytic cleavage to yield the internal capsid proteins of the virus particle and is analogous to the gag polyprotein precursors of other retroviruses (3)(4)(5). gPr8Ogg contains the amino acid sequences of Pr65gag as well as [4][5][6] kilodaltons (kDa) of amino-terminal protein (6,7).…”

mentioning

confidence: 99%

Construction and characterization of Moloney murine leukemia virus mutants unable to synthesize glycosylated gag polyprotein.

Fan¹,

Chute²,

Chao³

et al. 1983

Retroviruses contain three genes for replication: gag, pol, and env, which encode polyprotein precursors for the internal capsid proteins, reverse transcriptase, and envelope glycoprotein, respectively (1). Murine leukemia virus (MuLV) differs from most other retroviruses in that it encodes two different pathways for gag gene expression (2). These two pathways begin with two independent translation products, gPr80gag and Pr65ag. Pr65gag is processed by proteolytic cleavage to yield the internal capsid proteins of the virus particle and is analogous to the gag polyprotein precursors of other retroviruses (3-5). gPr8Ogg contains the amino acid sequences of Pr65gag as well as 4-6 kilodaltons (kDa) of amino-terminal protein (6, 7). The additional amino-terminal peptides result in glycosylation of gPr8O'ag during translation (8, 9). gPr8 gag is processed by the further addition of carbohydrate and exported to the cell surface where it appears as a glycoprotein of 95 kDa (8-11). It may be also be released into the extracellular medium as cleavage products of 55 and 40 kDa (8,12). Glycosylated gag products are not incorporated into MuLV virions, but they associate with the extracellular matrix (13).While the function of Pr65gag is known, the role of gPr809ae in MuLV infection is not known. Glycosylated gag might provide some function required for viral replication, or it might play an accessory role. We previously isolated mutants of Moloney MuLV (M-MuLV)-infected mouse fibroblasts that did not express gag proteins at the cell surface, and they were deficient for virus production. However, these mutants were cellular, not viral, in nature and they produced normal amounts of gPr8g'ag intracellularly (14). To obtain a more definitive answer, we constructed two mutants of M-MuLV at the recombinant DNA level and recovered virus by transfection. The constructions. and characterizations of the mutant viruses are described here.MATERIALS AND METHODS Cells and Viruses. All cells were grown in Dulbecco-modified Eagle's medium/10% calf serum; Mouse NIH-3T3 cells were described previously (15), as were M-MuLV-infected NIH-3T3 cells (clone A9) (16).The UV-XC assay for MuLV was carried out as described (17). Assay of viral reverse transcriptase by the addition of exogenous poly(rA):oligo(dT) template-primer (16) and banding of virus in sucrose density gradients (18) was carried out as described.Recombinant DNA Cloning. A phage recombinant DNA clones (A-MLV clones 48, 61, and 63) carrying integrated copies of M-MuLV provirus were described previously (19). pMSV-1 is a plasmid clone of unintegrated Moloney murine sarcoma virus (M-MSV) DNA permuted about the unique HindIII site (20). pSLT is a subclone of pMSV-1 deleted from the Sal I site in M-MSV DNA to the Sal I site in the pBR322.vector (see Fig. 2) and was kindly provided by Inder Verma. P90(Abl), a plasmid clone of unintegrated Abelson MuLV (Ab-MuLV) (P90 strain) DNA, was kindly provided by Owen Witte. Plasmid vector pBR328 has been described by Soberon et al. (21).Restriction ...