Reciprocal Inhibition of Mouse Leukemia Virus Infection by Fv-1 Allele Cell Extracts

Kiggans

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

et al. 1980

Production of various forms of nonintegrated viral DNA was measured in cultured mouse cells carrying different Fv-1 alleles early after infection with N-tropic or B-tropic retroviruses. Quantitative analyses were performed by agarose gel electrophoresis, transfer to diazobenzyloxymethyl-paper, and molecular hybridization. In permissive infection of Fv-n cells (NIH Swiss and DBA mouse strains) with N-tropic virus and of Fv-1 b cells (BALB/c and C57BL/6 strains) with B-tropic virus, form III (double-stranded linear) DNA first appeared at 3-4 hr and reached a maximum at 8-10 hr; two form I (closed circle) DNAs appeared at 7-8 hr and reached a maximum at or beyond 12 hr. In the two Fv-1 b cells infected with N4ropic virus and in DBA (Fv-1 n) cells infected with B-tropic virus, formation of the two form I DNAs was quantitatively restricted but formation of form III DNA was unaltered. In Fv-1" NIH Swiss mouse embryo cells infected with B-tropic virus, the level of form III DNA was markedly depressed and hence the two form I DNAs were not detectable. In C57BL/6 cells as well as in DBA/2 cells 12 hr after infection, the quantity of form III DNA varied directly with the amount of restricted virus, whereas the quantity of form I DNA varied according to the square of the amount of restricted virus. The significance of these results for understanding the molecular basis of retrovirus replication and its restriction by the Fv-1 gene is discussed. The importance of the mouse Fv-1 gene locus in controlling infection by N-and B-tropic retroviruses has been well recognized in both animal studies and cell culture studies (1-5). This locus, mapped on chromosome 4 of the mouse (6), is presumably responsible for the production of specific inhibitor molecules in the cytoplasm of the cell (7-9). Inactivation of the restricted virus particles appears to be mediated by a virus protein, possibly the p30 core protein (10), which serves as a "target" for the Fv-1 gene product (11,12). However, the precise molecular mechanism of Fv-1 gene restriction is still unknown. Results of various previous investigations (13)(14)(15)(16)(17)(18)(19)(20) indicate that Fv-1 restriction occurs intracellularly at an early step of the virus replication cycle. Of particular significance are the observations (i) that, in Fv-1 restricted infection, the quantity of the cellgenome integrated viral DNA is markedly decreased whereas the formation of nonintegrated viral DNA appears to be unaffected (17-18), and (ii) that transfection by infectious integrated viral DNA is not restricted by the Fv-1 locus of the cell (19,20). These data imply that Fv-1 restriction may occur at or prior to the step of viral DNA integration.In the present study, we quantitatively measured the production of double-stranded linear and circular forms of viral DNA in Fv-1 permissive and restrictive cells early after infection with N-and B-tropic murine retroviruses. The results demonstrate that mouse cells may be assigned to either of two classes with regard to their ability to re...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Synthesis and circularization of N- and B-tropic retroviral DNA Fv-1 permissive and restrictive mouse cells.

Kiggans

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

et al. 1980

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

“…1A). Because, after first subcultivation, (27,28) and also by kinetic studies of resistance transfer with cell extracts (10) and by a cell fusion method (7). In contrast, two previous reports put forward the view that Fv-I gene action was at a step beyond viral gene integration (29,30 …”

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confidence: 99%

“…In a previous study (10) 10 passages of the original sources and were grown in Eagle's minimal essential medium containing 10% heat inactivated fetal calf serum, streptomycin (100;g/ml), and penicillin (100 units per ml).…”

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confidence: 99%

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses.

Hsu¹,

Yang²,

Tennant³

et al. 1978

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N-or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-1n (NIH-3T3) and two Fv-1 b (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an [N-tropic MuLVJSC- (Clinton, TN), respectively. All cells used were within 10 passages of the original sources and were grown in Eagle's minimal essential medium containing 10% heat inactivated fetal calf serum, streptomycin (100;g/ml), and penicillin (100 units per ml).DNA Preparations. SC-1 cells were inoculated with N-or B-tropic MuLV at a multiplicity of infection of 1. Forty-eight hours later they showed more than 90% infection by immunofluorescence measurement. After five serial passages in dishes, the chronically infected SC-1 cells were grown to confluency in medium containing hydrocortisone (1 ,uM) and insulin (0.1 international unit/ml) in 6 X 75 cm roller bottles. The cells were harvested by scraping, washed, and used for DNA isolation according to a modification of Marmur's method (12), which includes Pronase digestion, ribonuclease digestion, and extractions with chloroform/isoamyl alcohol, 25:1 (vol/vol), as described (13,11). The final DNA products were dissolved in 15 mM NaCI/1.5 mM Na citrate, pH 7.0, and stored in aliquots at -70°. DNA concentration was measured spectrophotometrically at 260 nm by assuming that 20 A20 units equal 1 mg of DNA. All DNA preparations showed A 2m/A28o ratios of 1.9-2.0.Infectious DNA Assay. The detailed procedure for DNA transfection in mouse cell cultures has been described (11). In this study, all cultures were plated at 2 X 105 cells per 35-mm dish 20 hr prior to DNA inoculation. The cells, with medium removed, were exposed to 0.3 ml of the freshly prepared calcium DNA suspension (14) which contained various doses of sample DNA and also an appropriate amount of calf thymus DNA to maintain a constant DNA concentration of 40 Ag/ml. After 15 min, 3 ml of medium was added and the dishes were placed in a 370 CO2 incubator for an additional 15 hr before DNA was removed by a change to fresh medium. Two to 3 days after DNA inoculation, when the cultures became confluent, the cells were trypsinized and used 1:5 for subculture. Serial Abbreviations: MuLV, murine leukemia virus; PFU, plaque-forming units.

“…We have previously reported the ability to transfer Fv-1 gene-specific resistance by means of crude cell lysates [1] and cytoplasmic RNA [2,3]. Although the specific gene product or cellular function involved in the transfer has not been determined, it appears to be mediated through a specific component of the virus [4,5].…”

mentioning

confidence: 99%

Characterization of Fv-1 Gene-Product-Mediated Resistance Transfer

et al. 1981

Self Cite

Fv-1 gene-specific resistance to eco tropic murine retro viruses can be transferred to permissive cells with RNA prepared from restrictive cells. The active RNA has a sedimentation coefficient of 20–22S at low ionic strength and multiple regions of activity (5–6S, 10–12S, 24–27S) at high ionic strength. The maximum level of resistance transfer cannot be enhanced with noninhibitory carrier RNA or facilitators of RNA uptake, and the maximum inhibition (40–60%) is independent of the MOI. The low efficiency of resistance transfer is, therefore, probably the result of a fraction of cells resistant to RNA uptake.