Increased induction of osteopetrosis, but unaltered lymphomagenicity, by murine leukemia virus SL3-3 after mutation of a nuclear factor 1 site in the enhancer

Abstract

SL3-3 is a murine leukemia virus which is only weakly bone pathogenic but highly T-cell lymphomagenic. A major pathogenic determinant is the transcriptional enhancer comprising several transcription factor binding sites, among which are three identical sites for nuclear factor 1 (NF1). We have investigated the pathogenic properties of NF1 site enhancer mutants of SL3-3. Two different mutants carrying a 3-bp mutation either in all three NF1 sites or in the central site alone were constructed and assayed in inbred NMRI mice. The wild type and both mutants induced lymphomas in all mice, with a mean latency period of 9 weeks. However, there was a considerable difference in osteopetrosis induction. Wild-type SL3-3 induced osteopetrosis in 11% of the mice (2 of 19), and the triple NF1 site mutant induced osteopetrosis in none of the mice (0 of 19), whereas the single NF1 site mutant induced osteopetrosis in 56% (10 of 18) of the mice, as determined by X-ray analysis. A detailed histological examination of the femurs of the mice was carried out and found to support this diagnosis. Thus, the NF1 sites of SL3-3 are major determinants of osteopetrosis induction, without determining lymphomagenesis. This conclusion was further supported by evaluation of the bone pathogenicity of other SL3-3 enhancer variants, the lymphomagenicity of which had been examined previously. This evaluation furthermore strongly indicated that the core sites, a second group of transcription factor binding sites in the viral enhancer, are necessary for the osteopetrosis induction potential of SL3-3.

FIG. 1

(A) Schematic outline of the SL3-3 LTR region. The transcriptional enhancer is found within a part of the U3 region that consists of 72 bp which are repeated one-and-a-half times. The three identical NF1 sites are indicated, and the CGG to ATT mutation used in this study is also shown. SL3(3mNF1) carries the mutation in all three NF1 sites, whereas SL3(1mNF1) carries the mutation in the central site only. (B) Lymphoma incidence in inbred NMRI mice infected with the wild type or the two different NF1 site mutants of SL3-3. Mock-infected control mice did not develop lymphomas within the 100-day observation period. Prolonged observation of the control mice showed one mouse with a lymphoma at the age of 321 days and five more mice with lymphomas between 12 and 20 months.

FIG. 2

X-ray analyses and histological appearance of skeletal lesions of SL3-3 NF1 mutant virus-infected mice presenting with (A to F) and without (G to I) osteopetrosis. The roentgenographs show the areas in the skeleton which are primarily affected by osteopetrosis-inducing MLVs (30) and include the os ilium (arrow), lumbar vertebrae (black arrowheads), distal femur (double arrows), and proximal tibia (white arrowhead). (A) Characteristic roentgenographic appearance of an SL3(1mNF1)-infected, 61-day-old mouse (no. 364-705) with increased cancellous and cortical bone mass, indicative of osteopetrosis, as diagnosed by X-ray analysis. (B) Histological appearance of the growth cartilage/metaphyseal junction and cancellous area of the distal femur of the SL3(1mNF1)-infected mouse shown in panel A. Abnormal endochondral ossification and abundant deposition of new bone are shown in the cancellous and cortical areas in the trabeculi and along the cortex. (C) Higher magnification of panel B, showing thickening of the trabeculi and replacement of the marrow cavity by newly formed bone. (D) X-ray analysis as in the legend to panel A, but of an SL3(3mNF1)-infected, 48-day-old mouse (no. 369-706). The increased, roentgen-dense bone structures, compared to those in panel A, are below the stage of unequivocal diagnosis of osteopetrosis by X-ray analysis. (E) Histological section as described in the legend to panel B, but of the femur from the mouse displayed in panel D, showing increased bone mass at the cortical region of the distal femur. (F) Higher magnification of panel E, showing a similar disorganized growth zone as in the section shown in panel C but with a smaller number of bone trabecules extending into the marrow cavity and containing nonresorbed, mineralized cartilage. Note the virtual absence of trabecular coalescence but similar abnormal endochondral ossification of this femur as that shown in panel C. (G) X-ray analysis as described in the legend to panel A but of an SL3(3mNF1)-infected, 69-day-old mouse (no. 369-843) without signs of osteopetrotic lesions. Note the clearly defined cancellous and cortical bone structures in the lumbar vertebrae, os ilium, femur, and tibia. (H) Histological section as shown in panel B but from the femur of the mouse displayed in panel G. (I) Higher magnification of panel H, showing mature bone trabeculi and a nearly occluded growth zone, indicating a normal pattern of endochondral ossification. Van Gieson-stained sections; bars in panels B, E, and H, 854 μm; bars in panels C, F, and I, 204 μm.

FIG. 3

Structure of the various enhancer variants of SL3-3. (A) Organization into 34- and 38-bp repeat elements is shown as hatched or filled boxes. The presence of 3-bp mutations is indicated below each structure, and deletions of either 18 or 28 bp are shown as gaps. The presence of intact binding sites is indicated above each of the structures: N, NF1 site; I, core site I; II, core site II. (B) The sequence of one 72-bp repeat element. The exact location of the various 3-bp mutations introduced into the enhancer and the 18-bp and 28-bp sequence deleted in some of the enhancer variants are shown.