Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus

Abstract

Background: Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus.

Results: By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.

Conclusion: We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.

Figure 1

Location of the trapped exon. Upper panel shows the structure of proviral Akv MLV DNA with the positions of the splice sites indicated (SD; splice donor, SA; splice acceptor). Arrows signify the PCR primers used to verify the stability of the introduced mutations. Lower panel shows the positions and types of the introduced mutations, marked by asterisks and underlined. The SA'/SD'-delineated exon is indicated by the box. The boldfaced A in the sequence indicates the presumed branch point.

Figure 2

Pathogenicity of Akv and derived splice site mutants in inbred NMRI mice. Shown are the cumulative incidences of tumor development related to age of injected mice (in days).

Figure 3

Histopathology of tumors induced by Akv and derived splice site mutants. Representative examples are shown. (A to D) de novo diffuse large B-cell lymphoma. (A) Low magnification of a spleen infiltrated by a vaguely nodular lymphoid neoplasia (H&E staining). Magnification, ×25. (B) Higher magnification demonstrates that the neoplasia is composed of a monotonous population of large cells with blastic chromatin, one to three nucleoli and abundant eosinophilic cytoplasm characteristic of centroblasts (H&E staining). Magnification, ×640. (C) Anti-B220 highlights the large neoplastic cells, which are strongly positive (immunohistochemistry). Magnification, ×400. (D) Anti-CD3 shows that only few residual reactive T-cells are present (immunohistochemistry). Magnification, ×400. (E to H) Follicular lymphoma. (E) Low magnification of a spleen infiltrated by a clear nodular lymphoid proliferation (H&E staining). Magnification, ×25 (F) Higher magnification shows a combination of large centroblasts intermingled with small- to medium-sized lymphocytes or centrocytes (H&E staining). Magnification, ×640. (G) Anti-B220 highlights the expansion of the follicles, mainly of the germinal center lymphoid cells (light brown) (immunohistochemistry). Magnification, ×25. (H) Anti-CD3 reveals the presence of abundant reactive T-cells intermingled with the neoplastic B-cells (immunohistochemistry). Magnification, ×400. (I to L) Marginal zone cell lymphoma. (I) Low magnification of a spleen infiltrated by a marginal zone lymphoma. Note that the follicles (F) are small and the cells surrounding these follicles expand and infiltrate the red pulp in a marginal zone pattern (H&E staining). Magnification, ×100. (J) Higher magnification showing that the neoplasia is composed of a monotonous population of small- to medium-sized cells with open fine chromatin, inconspicuous nucleoli and abundant light eosinophilic cytoplasm (H&E staining). Magnification, ×400. (K) Anti-CD79a reveals that the tumor cells in the marginal zone area are strongly positive, whereas the cells in the germinal centers (F) are weakly positive. The opposite staining pattern is seen with anti-B220 (data not shown) (immunohistochemistry). Magnification, ×200. (L) Higher magnification with anti-CD79a shows a uniform membranous positivity of the tumor cells (immunohistochemistry). Magnification, ×400. (M to O) Histiocytic sarcoma. (M) Low magnification of a spleen diffusely infiltrated by a histiocytic sarcoma (H&E staining). Magnification, ×25. (N) Higher magnification shows the presence of large cells with abundant eosinophilic cytoplasm and bland nuclei characteristic of histiocytes (H&E staining). Magnification, ×400. (O) Anti-Mac 3 shows that all tumor cells are positive for this histiocytic marker, both in the cytoplasm and in the cell membrane (immunohistochemistry). Magnification, ×4 Histopathological and immunohistological analyses of tumor tissues.

Figure 4

RT-PCR analyses of splice products generated in vivo. (A) The structures of the potential splice products A to D are illustrated at the top, with the positions and orientations of the PCR primers (see Materials and Methods) from the four primer sets depicted below. The predicted origins and sizes of the amplified fragments are given at the right. (B) Shown are examples from each series of amplified RT-PCR products visualized on ethidium bromide-stained agarose gels. The employed primer sets (#1 to #4) are listed above the lanes. Size markers are indicated at the left.

Figure 5

Northern blot hybridizations with an ecotropic specific env probe and a gag probe of RNA isolated from NIH 3T3 cells chronically infected with the viruses listed above each lane. The sizes of the full-length transcript (unspliced) and the single-spliced env transcript are indicated at the left. The arrow indicates splice product C. For verification of integrity and concentration of the loaded RNA, the original ethidium bromide stained agarose gel exposing 18S and 28S rRNAs is shown below.

Figure 6

DNA sequence alignment around the Akv MLV SA' site in the capsid-coding region of a series of different ecotropic MLVs. The 3' splice acceptor site consensus sequences are shown on top, with the border of the novel gag exon indicated by a vertical line. The boldfaced A in the sequence indicates the presumed branch point.