Nonconditional replication mutants of type C and type D retroviruses defective in gag gene-coded polyprotein post-translational processing

Sacks, Thomas L.; Devare, S G; Blennerhassett, Gail T.; Stephenson, John

doi:10.1016/0042-6822(78)90383-5

Cited by 21 publications

(14 citation statements)

References 24 publications

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“…This antigen is expressed in the thymus, ascites fluid, serum, and genital tract secretions in several strains of mice (38)(39)(40)(41)(42)(43)(44), but BALB/c mice express only low amounts of xenotropic murine leukemia virus-induced cell surface antigen (45). 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).…”

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

Proc. Natl. Acad. Sci. U.S.A.

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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...

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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

Proc. Natl. Acad. Sci. U.S.A.

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“…for 10 min in a Beckman JA20 rotor at 4'C, then equivalent amounts of RT-containing medium were used to infect HeLa cells in the presence of 2.0 ag/mI of polybrene (hexadimerthrine bromide; Sigma) at 37°C for 1.5 h. On days 1, 4, 6, 8, 10 and 12 postinfection, culture fluid was harvested from the cells, and assays for RT were carried out (Rhee and Hunter, 1987;Sacks et al, 1978). They were clarified by centrifigation 10 000 r.p.m.…”

Section: Cells Immunoprecipitation and Gel Electrophoresismentioning

confidence: 99%

“…They were clarified by centrifigation 10 000 r.p.m. for 10 min in a Beckman JA20 rotor at 4'C, then equivalent amounts of RT-containing medium were used to infect HeLa cells in the presence of 2.0 ag/mI of polybrene (hexadimerthrine bromide; Sigma) at 37°C for 1.5 h. On days 1, 4, 6, 8, 10 and 12 postinfection, culture fluid was harvested from the cells, and assays for RT were carried out (Rhee and Hunter, 1987;Sacks et al, 1978). A 7.5 A1 portion of the disrupted virus was incubated at 37°C for 1 h with 30 M1 RT mixture containing poly(rA)-oligo(dT) as synthetic RNA template-DNA primer (Boehringer Mannheim Biochemicals) and [3H]TTP (57 Ci/mmol; Amersham Corp.).…”

Section: Fractionation Of Gag Polyproteinmentioning

confidence: 99%

Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid.

Rhee

Hunter

1991

The EMBO Journal

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The functional roles of the matrix (MA) protein in the assembly and maturation of retroviruses was investigated with a series of MA mutants of Mason‐Pfizer monkey virus (M‐PMV), an immunosuppressive type D retrovirus. The mutants we describe here were generated by the introduction of random point mutations within the MA coding domain by use of sodium bisulphite mutagenesis. Studies of these mutants show that the MA protein plays a critical role in three different, sequential events in the final stages of type D retrovirus replication: (i) folding of the gag gene‐encoded precursor poly‐proteins into a stable conformation for capsid assembly in the cytoplasm of infected cells; (ii) capsid transport from the site of assembly to the plasma membrane; and (iii) capsid association with, and extrusion of the membrane during virus budding. The mutants described here interfere with or block M‐PMV replication at each of these stages. Large numbers of preassembled capsids accumulate within the cytoplasm of transport‐defective mutant‐infected cells, suggesting that transport of M‐PMV capsids to the plasma membrane is an active and specific intracellular targeting process. The initial association of the capsid with the membrane may depend upon this intracytoplasmic transport process but additional protein‐lipid interactions that involve the MA protein are required for membrane extrusion around the preformed capsids; in cells infected with the budding‐defective mutant, assembled capsids accumulate under the inner surface of the cell plasma membrane, and are retarded in their release from the infected cell.

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“…Nonconditional mutants of mammalian RNA tumor viruses will provide valuable tools for the analysis of steps in viral infection and for the study of cell-virus interactions such as Fv-1 restriction (1) which are unique to mammalian cells. Reports from several laboratories indicate that the generation of replication-defective variants of mammalian RNA tumor viruses occurs with high frequency (16,35,38,39). To date, the number of extensively characterized mutants is quite small.…”

mentioning

confidence: 99%

Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule

Gerwin¹,

Rein²,

Levin³

et al. 1979

J Virol

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A nonconditional mutant of B-tropic murine leukemia virus (MuLV), defective in polymerase, has been isolated by cloning chronically infected cells. The cell clone containing the mutant produced virus particles which were noninfectious. However, superinfection of the cells by replication-competent XC-negative viruses resulted in the rescue of virus capable of forming plaques in a modified XC test, termed the "complementation plaque assay" (A. Rein and R. H. Bassin, J. Virol. 28:656-660, 1978). Analysis of the noninfectious virions produced without superinfection demonstrated that they contained only 2 to 5% of the wild-type level of reverse transcriptase activity. Purification of this activity indicated that it was associated with a smaller molecule than that produced by wild-type virus. Cells producing the mutant virions did not contain the gag-pol precursor, Pr1809"'-1°'; however the cells contained proteins of 147K and 114K daltons precipitable with anti-pol serum. All of the normal structural proteins as well as 70S genomic RNA could be detected in the mutant particles. An interference test indicated that a functional ecotropic glycoprotein was synthesized by the mutant. These results indicate that the mutant has a unique defect in the pol gene.

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Nonconditional replication mutants of type C and type D retroviruses defective in gag gene-coded polyprotein post-translational processing

Cited by 21 publications

References 24 publications

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

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

Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid.

Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule

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