Isolation of a replication-defective murine leukaemia virus from cultured AKR leukaemia cells

Rein, Alan; Athan, E; Benjers, B M; Bassin, Robert H.; Gerwin, Brenda I.; Slocum, Donald R.

doi:10.1038/282753a0

Cited by 16 publications

(16 citation statements)

References 21 publications

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“…Viral RNAs were separated on denaturing agarose gels (27), transferred to nitrocellulose, and hybridized with 32P-labeled nick-translated probes (27) as described (28). Hybridized filters were washed three times in 2x SSC/0.01% NaDodSO4 at 42°C for 5 min, two times in 1 x SSC/0.01% NaDodSO4 at 54°C for 20 min, and two times in 0.5x SSC at 54°C for 5 min (lx SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

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Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence.

Gorelick

Henderson

Hanser

et al. 1988

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

305

290

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All retroviruses encode a nucleic acid-binding or nucleocapsid protein that is believed to be associated with RNA in the virion. Further, all retroviral nucleocapsid proteins contain either one or two copies of the sequence Cys-Xaa2-CysXaa4-HisXaa4-Cys. The conservation of this sequence suggested that it is important for virus replication, and its resemblance to the "zinc-finger" sequences found in eukaryotic transcription factors raised the possibility that it recognizes specific sequences in viral RNA during retrovirus assembly. We used oligonucleotide-directed mutagenesis to generate a series of mutations in the nucleocapsid protein-coding region of Moloney murine leukemia virus. These mutations changed single amino acids, including each of the cysteines, to serine. The mutant viral genomes direct the synthesis of virus particles; these particles lack detectable viral RNA but do contain significant levels of cellular RNAs. Thus it appears that the mutations have destroyed the ability of the viral proteins to specifically package viral RNA during virus assembly. We propose that the conserved sequence in retroviral nucleocapsid proteins functions in RNA sequence recognition and suggest that it is evolutionarily related to the zinc fingers that recognize specific sequences in double-stranded DNA.

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Section: Methodsmentioning

confidence: 99%

“…Infectivity and reverse transcriptase assays were as described (19)(20)(21)(22) Protein Immunoblotting. Viral proteins were fractionated by NaDodSO4/PAGE in 10-20% acrylamide gradient gels and transferred to diazotized paper as described (24).…”

Section: Methodsmentioning

confidence: 99%

Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence.

Gorelick

Henderson

Hanser

et al. 1988

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

305

290

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“…Infectivity assays were performed as described (20,21). Virion-associated reverse transcriptase activity was assayed as described (22).…”

Section: Methodsmentioning

confidence: 99%

Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.

Rein¹,

McClure²,

Rice³

et al. 1986

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

270

211

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It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.

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“…The XC 'complementation-plaque assay' of Rein et al (1979) was performed on F7 and A8 cells. This assay depends on the ability of a replication-competent XC-virus to complement an XC ÷ defective virus, which can then be detected as an XC + pseudotype.…”

Section: Replication-competent Viruses Produced By Sl3 Cellsmentioning

confidence: 99%

“…The XC complementation test was a modification of the procedure described by Rein et al (1979). Cells were infected with XC-virus and passaged ten times until a high level of RT was detectable.…”

Section: Cellsmentioning

confidence: 99%

Several Classes of Retroviruses Are Produced by an AKR Mouse T Lymphoma Cell Line

Hays¹,

Swanson²,

Silva

1984

Journal of General Virology

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SUMMARYCharacterization of the viruses produced by the spontaneous T lymphoma cell line SL3 is presented. Using supernatant fluids or direct co-cultivation of cells, the SL3 cell line was found to produce replication-defective viruses in excess of replicationcompetent viruses. The replication-competent viruses released were predominantly those negative in the XC plaque assay (XC-); XC + viruses represented a minor population. However, when the SL3-derived viruses were passed in mouse embryo fibroblasts, XC-viruses were rarely recovered, and XC + viruses were readily isolated. These viruses were all ecotropic and lymphomagenic. Viruses with dual host range and nononcogenic ecotropic viruses were not isolated from the lymphoma cells. Two replication-defective viruses from SL3 cells were studied. Both could be rescued by non-oncogenic retroviruses and were then lymphomagenic. One defective virus appeared related to XC + viruses. In these studies, the XC ÷ and XC-viruses appeared to represent two different interference classes using separate cell receptors. Taken together, these experiments show that the SL3 T lymphoma cells replicate a variety of viruses most of which are lymphomagenic. Virus replication and/or virus integration may be the means of maintaining the malignant phenotype of these T lymphoma cells.

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Isolation of a replication-defective murine leukaemia virus from cultured AKR leukaemia cells

Cited by 16 publications

References 21 publications

Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence.

Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence.

Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.

Several Classes of Retroviruses Are Produced by an AKR Mouse T Lymphoma Cell Line

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