Infectious viral DNA of murine leukemia virus.

Abstract

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...

A nucleoprotein complex mediates the integration of retroviral DNA.

“…Deproteinized unintegrated viral DNA, mixed with an extract of uninfected NIH-3T3 cells, sediments much more slowly, at about 20S (Fig. 2D), as anticipated from earlier analyses (Smotkin et al 1975;Varmus et al 1978). These results indicate that in an infected cell, unintegrated viral DNA resides within a large nucleoprotein complex of discrete size.…”

Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA.

“…We wished to determine whether we could impart metastatic ability to these Ha-ras transformants by introduction of genetic information from a metastatic tumor. These Ha-ras transformants were well-suited for such an experiment, in that they had a low background of spontaneous metastasis (see above) and, being of NIH 3T3 origin, were able to take up efficiently and express exogenous DNA (23). We applied DNAs from a variety of human metastatic tumor cell lines to cultures of these Ha-ras transformants, using the transfection procedure.…”

Infectious murine leukemia virus from DNA of virus-negative AKR mouse embryo cells.

“…The data are consistent with an IdUrd-sensitive restriction in the recipient NIH 3T3 cells that revents viral replication from endogenous AKR MuLV DNA Cut does not prevent viral replication from MuLV DNA of productively infected cells. Previous transfection studies have shown that retroviral DNA is infectious either as an unintegrated proviral DNA genome from acutely infected cells (1)(2)(3)(4) or as an integrated proviral DNA genome from chronically infected cells (5)(6)(7)(8)(9)(10). However, DNA from cells known to contain unexpressed but potentially infectious endogenous viral genomes is not infectious under the same transfection conditions (11).…”