Infectious Viral DNA in Rous Sarcoma Virus-transformed Nonproducer and Producer Animal Cells

Hill, M.; Hillova, Jana; Dantchev, Dimitre; Mariage, Regine; Goubin, Gérard

doi:10.1101/sqb.1974.039.01.117

Cited by 21 publications

(6 citation statements)

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“…The infectious viral DNA which they characterized was almost certainly the integrated form of the provirus (18). The detection of unintegrated viral DNA by molecular hybridization shortly after infection (2,3) raised the possibility that this DNA, a probable precursor to the integrated form, was also infectious.…”

Section: Discussionmentioning

confidence: 99%

Infectious viral DNA of murine leukemia virus.

Smotkin¹,

Gianni²,

Rozenblatt³

et al. 1975

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

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A fraction of the unintegrated viral DNA that appears early after infection of mouse cells by Moloney leukemia virus is infectious. The infectivity could be demonstrated by an XC plaque assay of the cells exposed to DNA co-precipitated with calcium phosphate. The number of plaques deriving from closed-circular, supercoiled DNA was proportional to the concentration of added DNA, indicating that a single DNA molecule of about 5.5 X 106 daltons carries all the viral information. Nonsupercoiled viral DNA is also infectious; these molecules appear to be largely doublestranded and 5 to 6 X 106 daltons in mass.Following infection by RNA tumor viruses, various forms of unintegrated viral DNA appear and can be detected by molecular hybridization (1-3). Some of these forms, including the closed-circular, supercoiled form, are probable precursors to the integrated viral genome. In this report both the closed-circular, supercoiled DNA and a fraction of the nonsupercoiled DNA are shown to be infectious. The DNA infectivity assay we describe is simple and quantitative, and the number of infectious events, also referred to as "transfections," can be directly determined by counting the plaques produced in an XC assay (4). The demonstration of the biological activity of a fraction of the newly synthesized viral DNA proves that these forms contain the complete viral genetic information and thus makes stronger the hypothesis that these are intermediates in the synthesis of the integrated provirus. MATERIALS AND METHODSInfection. Roller bottle cultures of JLS-V9 cells, a murine line derived from Balb/c mice (5), containing about 108 cells per 257-mm bottle, were infected with 20 ml of a stock of cloned Moloney murine leukemia virus (M-MuLV) in the presence of 8 ,ug/ml Polybrene (6). The virus titers were between 1 and 2 X 107 plaque-forming units/ml as assayed by the XC plaque assay (4).Extraction of Viral DNA. Nine hours after the start of infection, cells were extracted by the Hirt procedure (7). The supernatant fraction was twice extracted with CHCl3-isoamyl alcohol followed by ethanol precipitation. This nucleic acid will be referred to as the Hirt-soluble nucleic acid. Unless otherwise noted, the nucleic acid was resuspended in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, and LiCl was added to a concentration of 2 M in order to precipitate single-stranded, high-molecular-weight RNA. The nucleic acid remaining in the supernatant was ethanol precipitated and treated with RNase A at 50 .ug/ml in 1 mM Tris-HCl, pH 7.5, 1 mM EDTA for 1 hr at 370. Closed-circular, supercoiled DNA and nonsupercoiled DNA were then separated by centrifugation in an ethidium bromide-cesium chloride (EtdBrCsCI) isopycnic gradient as described previously (3).Infectious DNA Assay. Transfection was performed by the method described by Graham and Van Der Eb (8,9) After 20 min at room temperature, 4.5 ml of Dulbecco's modified Eagle's medium containing 10% calf serum was added. In later experiments, the DNA-calcium phosphate precipitates were added directly to...

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Section: Discussionmentioning

confidence: 99%

Infectious viral DNA of murine leukemia virus.

Smotkin¹,

Gianni²,

Rozenblatt³

et al. 1975

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

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“…Despite the large number of proviruses, these cells contain less than 50 genome equivalents of viral RNA (Varmus et al, 1974a). Cell fusion and transfection experiments have clearly indicated the presence of at least some complete functional genomes (Svoboda et al, 1967(Svoboda et al, , 1973Hlo2~.nek & Svoboda, 1972;Hill et al, 1974;Levy et at., 1974). Recently, we have demonstrated modification of the majority of the proviral DNA copies in these cells and suggested an inverse relationship between methylation and gene activity .…”

Section: Introductionmentioning

confidence: 99%

Studies on the Structure and Organization of Avian Sarcoma Proviruses in the Rat XC Cell Line

Mitsialis

Svoboda

et al. 1983

Journal of General Virology

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SUMMARYThe structure and arrangement of the multiple provirus copies of avian sarcoma virus in a rat XC cell line were studied by restriction endonucleases. The following observations were made: (i) the majority of the proviruses integrated randomly with respect to cell DNA; (ii) no gross deletions or rearrangements in the proviruses were observed; (iii) two types of proviruses (type I and type II) could be distinguished on the basis of restriction endonuclease cleavage sites; (iv) the virus rescued from these cells was derived from type II provirus, which has a novel EcoRI site between the env andpol genes; (v) most of the provirus units contained the src gene.

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“…Until now the calcium method was used only in infectivity assays with double-stranded DNAs extracted from, for example, adenovirus types 1, 2, 5, 12 [7,10], herpes simplex virus [11,12], and SV40 [7,10,13], Earlier experi ments using DEAE-dextran have shown that the RSV can be recovered even when the DNA samples were previously denatured by alkali [14] or banded as single strands in alkaline CsCl gradients [15]. In the present study we have examined whether the calcium technique can also be used in transfections with single-stranded DNAs.…”

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confidence: 99%

Infectivity of Rous Sarcoma Cell DNA: Comparison of Two Techniques of Transfection Assay

Hillova¹,

Hill²,

Goubin³

et al. 1975

Intervirology

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A DNA extracted from a clone of chicken cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D (SR-RSV-D), was assayed for infectivity by means of DEAE-dextran and calcium techniques. The calcium technique like the previously described DEAE-dextran procedure gave rise to viruses in transfection assays with both native and denatured (SI nuclease susceptible) DNAs. The efficiency of these transfection techniques with native DNA was compared and found to be about the same provided that with the calcium technique carrier DNA was used to complement DNA concentrations lower than 2.5 µg/ml.

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Infectious Viral DNA in Rous Sarcoma Virus-transformed Nonproducer and Producer Animal Cells

Cited by 21 publications

References 0 publications

Infectious viral DNA of murine leukemia virus.

Infectious viral DNA of murine leukemia virus.

Studies on the Structure and Organization of Avian Sarcoma Proviruses in the Rat XC Cell Line

Infectivity of Rous Sarcoma Cell DNA: Comparison of Two Techniques of Transfection Assay

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