Raymond W. Tennant

Requirement of cellular synthesis for Kilham rat virus replication

Abstract X-irradiation of rat embryo cells before infection with Kilham rat virus (KRV) impaired the capacity of the cells to synthesize the virus. Dose-response assays indicated that the cell population was composed of a radiosensitive fraction with an approximate mean lethal dose (D 0 ) of 82 R and a radioresistant fraction with a D 0 of about 1990 R. Irradiation with ultraviolet light likewise abolished cellular capacity for virus synthesis; and, as with the X-rays, the dose-response assay could be resolved into a two-component curve. A dose of approximately 8 ergs/mm 2 was the D 0 for the radiosensitive fraction of cells and 129 ergs/mm 2 the D 0 for the resistant fraction. Finally, treatment of cells with 5-fluorouracil prior to infection also impaired capacity for replication of KRV, but neither X radiation, ultraviolet light, nor 5-fluorouracil decreased the capacity of rat embryo cells to support pseudorabies virus replication. Therefore, KRV requires cell function(s) not required by other DNA viruses which replicate in the nucleus, and evidence suggests that the function(s) involves DNA synthesis.

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

Fv-1 gene-specific resistance to ecotropic murine retroviruses 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.

IN VITRO QUANTITATION OF LETHAL AND PHYSIOLOGIC EFFECTS OF TOTAL BODY IRRADIATION ON STROMAL AND HEMATOPOIETIC STEM CELLS IN CONTINUOUS BONE MARROW CULTURES FROM Rf MICE

The role of stromal-supportive cells in hematopoietic stem cell responses to irradiation is poorly understood. The effects of in vivo total body irradiation (TBI) and interval from TBI to explant of marrow on: stromal cell proliferation in vitro; stromal cell support of hematopoiesis in continuous bone marrow culture; and generation of WEHI-3 growth factor (GF)-dependent lines of hematopoietic progenitor cells were evaluated. Continuous marrow cultures from non-irradiated control RfM/UN, C57BL/6J, C3H/HeJ, and N:NIH (Swiss) mice generated pluripotential hematopoietic stem cells (CFUs) and committed granulocyte-macrophage progenitor cells (GM-CFUc) for over 20 weeks. Explant of marrow at 2, 4, 5, or 6 months after single fraction TBI (300-800 rad) was associated with decreased longevity of hemopoiesis (2-12 weeks), and a decrease in the proliferative capacity of fibroblastic adherent-stromal colony forming cells (CFUf) as measured by colony size at 14 days and number of colonies per 10(6) cells plated. In contrast, explant of marrow 8 to 24 months after TBI produced cultures with longevity that was indistinguishable from age-matched control cultures (19-24 weeks). Marrow from irradiated first and second generation recipients of serially transferred marrow demonstrated a similar 7-month in vivo recovery period; however, the plateau maximum duration of hemopoiesis did not return to control levels. Purified stromal cell cultures were prepared by corticosteroid-deprivation of explanted marrow for 28 days and were then engrafted in vitro with marrow from C57BL/6J or RfM/UN mice that had been irradiated 1 month previously. Hemopoiesis in these cultures was restored, and they produced GM-CFUc and granulocytes for 15-24 weeks. Thus, healthy stroma supported growth of recently irradiated hemopoietic cells in vitro. Nonadherent cells removed from the above continuous marrow cultures generated clonal non-leukemogenic WEHI-3 GF-dependent hemopoietic progenitor cell lines with a frequency concordant with radiation effects on culture longevity, and this was increased by the presence of purified healthy stromal cultures. Indirect effects of x-irradiation on hemopoietic stem cells through damage and repair in the stromal cell compartment can be effectively studied with the present bone marrow culture system.

Characterization of Fv-1 host range strains of murine retroviruses by titration and p30 protein characteristics☆☆☆

Abstract A standardized, direct XC plaque assay was used to determine the titration hitness patterns of Fv-1 host range murine retroviruses obtained from various laboratories. The N- or B-tropic viruses were tested on a variety of cells with Fv-1 nn , Fv-1 bb , or Fv-1 nb genotypes, and, with one exception, a discrete two-hit pattern was obtained on cells with the restrictive genotype (i.e., N-tropic virus on BALB/c, SIM·R, and B6C3F 1 cells, and B-tropic virus on SME, SIM, and B6C3F 1 cells). The single exception to the two-hit titration effect was a strain (TOR-B) which is poorly infectious for all cells, and which does not show a clear N- or B-tropism. In addition, it was possible to convert the two-hit pattern of another B-tropic virus (OR-B) to a one-hit curve by coinfection with an XC-negative N-tropic virus. Relative to SC-1 (Fv-1 −− ) cells, it was possible to define three components of restriction of ecotropic virus infection of mouse cells: The first two components, the two-hit kinetics and 10 2- to 10 3 -fold reduction in titer on restrictive cells, are Fv-1 determined; the third a decreased infectivity on all cells relative to SC-1 cells, is independent of Fv-1 genotype.

Induction of endogenous murine retrovirus by hydroxyurea and related compounds

Abstract Hydroxyurea and two related compounds, carbamoyloxyurea and formamidoxime, induce endogenous retrovirus expression in AKR mouse embryo cells. Only the induction of ecotropic (N-tropic) virus was detected. The ability of each compound to induce shows a linear dose-response over a limited range of concentrations which correlate with the cytotoxicity of each of the compounds. The data suggest that these compounds may induce virus expression via damage to cellular DNA.

Murine leukemia virus: restriction in fused permissive and nonpermissive cells.

Cultures of human cells nonpermissive for mouse leukemia virus replication could not be induced to support virus replication by homologous fusion in the presence of Moloney leukemia virus. Human cells were also fused with permissive mouse cells, and the fate of the virus in heterokaryons was determined by a simultaneous autoradiography and fluorescent antibody technique. Heterokaryons containing the full chromosome complement of both cells were likewise nonpermissive for virus synthesis, but hybrids of human and mouse cells, which lacked up to half of the human chromosome complement, were permissive for virus synthesis. The results suggest that human cell genes can direct a repressive control over mouse leukemia virus replication.

Involvement of DNA damage in hydroxyurea-mediated induction of endogenous murine retrovirus☆

Hydroxyurea (HU) induces AKR cells to produce endogenous murine retrovirus at a low frequency (approx. 1 x 10/sup -5/), and DNA synthesis is required soon after treatment with HU for induction to be observed (i.e., no stable induction intermediate is formed). Induction by HU can be enhanced by simultaneous treatment with halogenated pyrimidines, with the concomitant appearance of a stable induction intermediate state. The effects of the two compounds are synergistic, indicating an actual stimulation of HU-mediated induction by iodode-oxyuridine. Since HU inhibits semiconservative replication, and since (/sup 3/H)bromodeoxyuridine is incorporated into the cellular genome predominantly by unscheduled DNA synthesis (repair replication) under these conditions, this stimulation appears to be the result of insertion into DNA of the thymidine analogs during the repair of HU-induced alterations in the DNA. The nature of HU damage to DNA is not defined; if single-strand breaks are involved, they may occur at a frequency <10/sup -8/ and escape detection, but induction could also be due to other alterations in DNA. The characteristics of induction by HU, therefore, are similar to those of induction by other DNA-damaging treatments such as ..gamma.. or x irradiation or methylcholanthrene. This suggests that these agents may induce by similar,morexa0» if not identical, mechanisms. Further, the ability of halogenated pyrimidines to form a stable induction intermediate when incorporated by repair synthesis, similar to the intermediate formed when the analogs are incorporated during semiconservative replication, suggests that the same sites are involved for induction by damaging agents or by halogenated pyrimidine incorporation.«xa0less

Immunofluorescent analysis of murine leukemia virus-infected cells by flow microfluorometry.

Flow microfluorometric assay with a Cytofluorograf model 4801 in combination with immunofluorescence was applied to the quantitative assay of cells exogenously infected with mouse leukemia viruses and to the chemical induction of virus in AKR cells. The Cytofluorograf provides sensitivity equal to the visual method and is capable of assaying up to 5000 cells/sec with specificity equivalent to that of the direct visual immunofluorescence assay, thereby providing a large, statistically valid sampling in a fraction of the time required by visual counting. Flow microfluorometry also provides a method of quantitatively resolving fluorescent cell populations on the basis of relative size and degree of fluorescence.

Assay of mouse-cell clones for retrovirus p30 protein by use of an automated solid-state radioimmunoassay

A solid-state radioimmunoassay system has been developed that is useful for automated analysis of samples in microtiter plates. Assays for interspecies and type-specific antigenic determinants of the C-type retrovirus protein, p30, have been used to identify clones of cells producing this protein. This method allows testing of at least 1000 clones a day, making it useful for studies of frequencies of virus protein induction, defective virus production, and formation of recombinant viruses.

Fv-1 Locus restriction of mouse retroviruses in glucocorticoid-treated cells

Abstract Treatment of mouse embryo cells with hydrocortisone (10 −6 M ) or dexamethasone (10 4 −10 −6 M ) increases virus synthesis whether the cells are permissive or restrictive at the Fv-1 locus. However, the number of cells infected was not increased in either permissive or restrictive cells by treatment with either glucocorticoid, and the two-hit titration pattern in restrictive cells remained unaltered. Therefore, the enhancement of virus replication by the glucocorticoids is independent of Fv-1 restriction and appears to occur after the Fv-I locus-sensitive step in virus synthesis.