Fate of viral RNA of murine leukemia virus after infection.

Abstract

ABSTRACT['H]Uridine-labeled Rauscher leukemia virus was used to infect mouse embryo fibroblasts. After the infected cells were separated into nuclear and cytoplasmic fractions nucleic acid was extracted by sodium dodecyl sulfate-phenol-chloroform treatment and analyzed by C82SO4 and sucrose density gradient centrifugation. Between 45 and 70 min after infection a transient and synchronized shift of the acid-insoluble radioactive peak toward the RNA-DNA hybrid region occurred in both the nuclear and cytoplasmic fractions. The density of the cytoplasmic hybrid shifted to 1.56 g/ml (RNA = about 50%), while the sedimentation rate decreased from 36 S to 14 S; however, the density of the nuclear hybrid shifted to 1.58-1.48 g/ml (RNA = 57-17%, respectively), while its sedimentation rate remained about 65 S. The hybrids in both the nuclear and the cytoplasmic fractions still showed hybrid density after heat denaturation. The processes of the early stages of RNA tumor virus infection are discussed with regard to the functions of viral RNA-dependent DNA polymerase (reverse transcriptase) and a possible integration of viral genetic information into the host chromosome.DNA synthesis in cells in the early stages of infection by an RNA tumor virus appears to play an important role in both the expression of transformation (1, 2) and the production of the progeny viruses (3). DNA copies homologous to the viral RNA have been found in virus-producing (4, 20) as well as virally transformed cell lines (5). These results suggest that reverse transcription of the viral information into DNA and the integration of this DNA into the host chromosome may be necessary for the maintenance and the expression of the viral information (6).The process of the integration of the viral genetic information, however, is entirely obscure. In order to study these processes, we followed the fate of ['H] mmol, New England Nuclear). After 10-to 14-hr incubation at 370, the radioactive medium was discarded and an equal volume of fresh MEM containing 3% FBS was added to each bottle. The virus was collected for 90 min after the medium change. DNase I (Worthington Biochemical Corp.; RNasefree) was added to give a final concentration of 10 ,g/ml for the last 30 min of each collection period. This collection procedure was repeated after another feeding with nonradioactive medium. The combined medium containing radioactive virus was purified by sucrose gradient centrifugation. Virus was pelleted onto a 60% sucrose cushion and then banded in a 15-50% sucrose gradient. The peak fraction of radioactivity in the final sucrose gradient (density = 1.16 g/ml) was used as a preparation of purified virus. The purified virus was diluted with 5 volumes of Earles' balanced salt solution (BSS, Flow Laboratories) and used for infection on the same day the purification was done.Infection with 3H-Labeled Viruses. Monolayers of MEF were plated 8-12 hr before infection at a density of 1.5 X 106 cells per 60-mm plastic petri dish with 5 ml of MEM supplemented with 10% FBS, and ...

Kinetics of murine type C virus-specific DNA synthesis newly infected cells

“…The inhibitor of DNA synthesis, ara-C, was found to be most inhibitory for RLV progeny formation during a similar interval: 30 to 90 min postinfection. This timing is in substantial agreement with the results of Takano and Hatanaka (29), demonstrating that the [3H ]RNA of RLV showed a shift under nondenaturing conditions toward the RNA-DNA hybrid region in Cs2SO, gradients between 45 and 70 min after infection. Since (-) strand viral DNA could not be detected in the presence of ara-C, the drug most likely inhibited the DNA synthetic step catalyzed by the viral RNA-directed DNA polymerase.…”

“…This has been supported by considerable indirect evidence involving the effect of inhibitors on DNA synthesis of infected cells (4,30), detection of RNA-directed DNA polymerase in virions (7,33), and isolation of biologically active DNA from transformed cells (16). More direct evidence for the synthesis of viral DNA beginning within 1 to 3 h after infection has been obtained by autoradiography (14), isopycnic Cs2SO4 centrifugation of incoming viral RNA (19,28,29), and nucleic acid hybridization of unintegrated DNA (1,17,21,36).…”

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

“…6; 0. Richards, P. Shank, and authors, unpublished data), although covalent hybrids have been reported by others (9,13,25,26). Chen and Temin (2) have recently described infrequent ribonucleotides in strands of spleen necrosis virus DNA; however, the presence of the ribonucleotides in both plus and minus strands of spleen necrosis virus DNA and their absence from ASV DNA ( Fig.…”

Thermolabile reverse transcriptase of a mammalian leukemia virus mutant temperature sensitive in its replication and sarcoma virus helper functions

“…Recent work with avian and muriine type C viruses has begun to elucidate the early intracellular steps involved in the synthesis of viral DNA and its subsequent integration into the host cell genome. Thus, covalent viral RNA-DNA hybrids (15,26) and closed circular viral DNA duplexes (8,30) have been demonstrated in the cytoplasm of newly infected cells. Subsequently, the viral DNA becomes integrated into the genome of the cell (30).…”