Use of a weakly pathogenic mutant to ascertain the mechanisms of oncogenesis by SL3-3 MuLV.

摘要

The mechanisms by which SL3-3 MuLV (SL3) induces thymic lymphoma are incompletely understood. We performed a longitudinal analysis of SL3 infection in the thymus and bone marrow to assess and quantify the molecular and cellular alterations that occur during progression to lymphoma. Studies were performed in parallel using a weakly pathogenic enhancer mutant of SL3, termed SL3ΔMyb5, in order to discriminate between events that contribute to malignancy from those that occur as a result of viral infection alone. Semi-quantitative PCR reveals that proviral DNA is detectable in the bone marrow, thymus and spleen of SL3- or SL3ΔMyb5-infected mice during the premalignant period (1–8 weeks post-inoculation). Infection with either virus results in a significant alteration in normal intrathymic T-cell subpopulation distribution, as evidenced by an increase in the proportion of double-negative (CD4−CD8 −) T-cells and decrease in the proportion of double-positive (CD4+CD8+) T-cells. In addition, a marked thymic atrophy is observed at 6 weeks post-inoculation (p.i.) only in SL3-infected animals, suggesting that thymic atrophy may be a necessary step in the malignant process mediated by this virus. Immunocytochemical staining for viral coat protein (gp70) in the bone marrow revealed that SU and SL3ΔMyb5, show different patterns of infection in this tissue during the premalignant period. Colony forming assays and flow cytometric analysis of the bone marrow demonstrate a pattern of disrupted hematopoiesis in SL3- and SL3ΔMyb5, animals at 2, 3 and 4 weeks p.i. A PCR-based protocol was used to determine sites and timing of appearance of two types of polytropic envelope recombinant viruses in animals infected with SL3 or SL3ΔMyb5, Longitudinal analysis demonstrated that the initial site of recombinant virus formation in SL3-Infected mice is the thymus, which harbors both types of envelope recombinants as early as 2 weeks p.i. No recombinants were detectable in the bone marrow of SL3-infected mice until 6 weeks p.i, findings inconsistent with their role in the initial transforming event reported to occur in this tissue. Both types of recombinants were detectable in thymus, bone marrow and spleen of animals infected with the weakly pathogenic SL3ΔMyb5, mutant, although with reduced frequency and delayed kinetics of appearance.