Open Access

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

  • Annette Balle Sørensen1, 6,
  • Anders H Lund1, 7,
  • Sandra Kunder2,
  • Leticia Quintanilla-Martinez2,
  • Jörg Schmidt3,
  • Bruce Wang4,
  • Matthias Wabl5 and
  • Finn Skou Pedersen1Email author
Retrovirology20074:46

DOI: 10.1186/1742-4690-4-46

Received: 07 March 2007

Accepted: 06 July 2007

Published: 06 July 2007

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.

Background

Many murine leukemia viruses (MLVs) belonging to the genus gammaretroviruses induce cancer when injected into susceptible newborn mice [1, 2]. These simple retroviruses do not themselves harbor transduced oncogenes, and their ability to cause cancer relies on the host cellular genes that are transcriptionally activated or otherwise mutated as a result of the integrated provirus [36].

Regarding the virus itself, it is well documented that the LTR region plays a crucial role for both the strength and cell type specificity of disease induction [7, 8]. Within the LTR the specificity has been located mainly to the enhancer region in U3, and further narrowed down to the sequences defining different transcription factor binding sites [912]. In spite of this predominant role of the LTR in MLV pathogenesis, also sequences outside this region have been shown to be important for the ability and potency of a particular virus to induce cancer. Infection is mediated by interaction between the viral envelope protein (Env) and a specific host cell receptor, and for the ecotropic MLVs such as Moloney, Akv, and SL3-3, this receptor has been identified as the mouse cationic amino acid transporter 1 (mCAT1) [13, 14]. A significant role of env in MLV pathogenesis is the involvement in the generation of recombinant polytropic viruses that takes place during T-cell lymphoma development. These MCF (mink cell focus-forming) viruses have the ability to superinfect cells, an aspect which is thought to contribute to tumor formation [15, 16]. In addition to the env gene, and perhaps somewhat surprisingly, the viral gag gene sequences have also proven to play a role in MLV pathogenesis. Thus, Audit et al. (1999) [17] showed that the introduction of only three synonymous nucleotide mutations in the capsid-coding gene of Moloney MLV (Mo-MLV) changed the oncogenic properties of this virus. The mutations were located at an alternative splice donor site (SD'), which together with the canonical env splice acceptor site was shown to produce a subgenomic transcript of 4.4 kb [18]. The equivalent transcript, produced by Friend MLV, was subsequently shown to be packaged into virions, reversely transcribed and integrated in the host genome by normal viral mechanisms [19]. While wild-type Mo-MLV induces T-cell lymphomas in 100% of the inoculated mice, the SD' mutant virus exhibited a much broader specificity, thus inducing – besides the expected T-cell tumors – erythroid or myelomonocytic leukemias. In contrast, the corresponding mutations in a Friend MLV background did not seem to influence the pathogenic potential of this virus at all. Both wild-type and mutant Friend MLVs induced exclusively the characteristic erythroleukemia [17]. So it seems that the importance for the disease-inducing potential of the SD' site, although conserved among many species, is strongly dependent on the virus type.

The SD' site has also been found to be used for production of the oncogenic gag-myb fusion RNAs in promonocytic leukemias induced by Mo-MLV in pristane-treated BALB/c mice [20]. When the SD' site was mutated in this model, the overall disease incidence was not affected; however the proportion of myeloid leukemia decreased significantly, while the proportion of lymphoid leukemia increased. Moreover, no 5' insertional activation of c-myb (using alternative splice donor sites) could be found, thereby signifying a specific requirement of the SD' site for this mechanism [21].

Here we report of the identification of an alternative splice acceptor site, SA', located in the capsid region of gag, which together with the gag splice donor site, SD' (corresponding to the one reported for Moloney and Friend MLV), or together with a second alternative gag splice donor site, SD*, defines a novel exon within the genus gammaretroviruses. We show that RNA splicing by use of the alternative splice sites does indeed take place in tumor tissue, and that both double- and single-spliced transcripts are produced. When mutating the SD', the SA', or both sites simultaneously, the splicing pattern is affected in a predictable way. Moreover, we demonstrate that the SA' and SD' mutations alter the oncogenic specificity of the Akv MLV, displayed by a change in the distribution of the diagnoses of the resulting tumors as well as by an induction of tumors of altered specificity such as histiocytic sarcoma and de novo diffuse large B cell lymphoma (DLBCL).

Results

Identification of a novel exon residing within the gagregion of Akv MLV

In order to identify potential alternative splice donor and splice acceptor sites in Akv MLV, exon-trapping was performed using the exon-trapping vector pSPL3 (see Materials and Methods). In short, an exon resulting from usage of the alternative splice acceptor (SA') and either one of two alternative splice donor (SD' or SD*) sites located in the capsid region of gag (Fig. 1), was isolated and verified by RT-PCR analyses of RNA isolated from Akv MLV infected cells (data not shown). The size of the exon is 235 bp or 180 bp, depending on the splice donor site used.
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.

Mutations of the alternative splice sites affect the specificity of the induced tumors

To analyze a possible effect in vivo of the novel exon, defined by SA' and SD', three different alternative splice site mutant viruses, Akv-CD, Akv-EH, and Akv-CDH, mutated in either the SA' or SD' site, or in both sites simultaneously, were constructed and injected into newborn mice of the inbred NMRI strain. Fig. 1 shows the precise locations of the synonymous mutations around the trapped exon. Without altering the coding potential of the capsid gene, the mutations affect the branch point site, the pyrimidine region, the conserved splice junction AG and GT dinucleotides, and the fairly well-conserved exonal A at the SD' junction site. The positions of the three intron mutations at the SD' junction site are identical to those in Moloney and Friend MLV described by Audit et al. (1999) [17].

As can be seen from Fig. 2 and Table 1 the majority of the infected mice (about 90%) developed tumors within 250 days with similar average latency periods of about 200 days for the four types of virus. Histological examination (examples shown in Fig. 3) and diagnosis according to the Bethesda classification [22] revealed that a large proportion (approx. 70%) of the total numbers of tumors could be classified as either follicular B-cell lymphoma (FBL) (13%), diffuse large B-cell lymphoma (DLBCL) progressed from FBL (33%), or plasmacytoma (PCT) (25%) (Table 2). However, the distribution was quite different within the different virus series; thus, almost one quarter of the Akv-wt induced tumors were diagnosed as FBL, while no tumor of the Akv-CD group (p < 0.05) or one tumor each of the Akv-EH or Akv-CDH groups fell into this group. In contrast, within the DLBCL tumors progressed from FBL the frequencies are similar (ranging from 24% to 39%) no matter if the causative virus contained mutated SA' and/or SD' sites or not. In the PCT group it appears that mutating the SA' site significantly impaired the ability of the virus to induce PCT (p < 0.05). On the other hand, this effect was not statistically significant if the SD' site was mutated, and curiously if both sites were mutated, wild-type level for PCT induction was restored.
Table 1

Disease latency and frequency

Virus

Average latency period (days)

Frequency of mice developing hematopoitic tumors

Akv-wt

184 ± 26

40/40

Akv-CD

201 ± 30

17/19

Akv-EH

184 ± 34

17/18

Akv-CDH

190 ± 46

14/16

Table 2

Frequency and latency of induced tumors

Virus

FBL

DLBCL (progression from FBL)

De novoDLBCL#

PCT

SMZL

DLBCL (progression from SMZL)

SBL

PTLL

STL

Histiocytic sarcoma

Akv-wt

9/40 (23%)

13/40 (33%)

0/40 (0%)

13/40 (33%)

3/40 (8%)

0/40 (0%)

2/40 (5%)

0/40 (0%)

0/40 (0%)

0/40 (0%)

Akv-CD*

0/18 (0%)

7/18 (39%)

0/18 (0%)

1/18 (6%)

5/18 (28%)

1/18 (6%)

0/18 (0%)

0/18 (0%)

0/18 (0%)

4/18 (22%)

Akv-EH

1/17 (6%)

4/17 (24%)

6/17 (35%)

3/17 (18%)

0/17 (0%)

1/17 (6%)

0/17 (0%)

0/17 (0%)

1/17 (6%)

0/17 (0%)

Akv-CDH

1/14 (7%)

5/14 (36%)

0/14 (0%)

5/14 (36%)

0/14 (0%)

0/14 (0%)

0/14 (0%)

1/14 (7%)

0/14 (0%)

0/14 (0%)

Total

11/88 (13%)

29/88 (33%)

6/88 (7%)

22/88 (25%)

8/88 (9%)

2/88 (2%)

2/88 (2%)

1/88 (1%)

1/88 (1%)

4/88 (5%)

Av. latency period (days)

188 ± 30

198 ± 31

187 ± 43

180 ± 27

207 ± 20

174 ± 18

153 ± 12

107

146

211 ± 36

Abbreviations: FBL, follicular B cell lymphoma; DLBCL, diffuse large B cell lymphoma; PCT, plasmacytoma; SMZL, splenic marginal zone lymphoma; SBL, small B cell lymphoma; PTLL, precursor T cell lymphoblastic lymphoma; STL, small T-cell lymphoma.

# De novo DLBCL refers to Bethesda classification "DLBCL centroblastic"; however, to stress the parallel to human de novo lymphomas we use this term.

*In this group one of the 17 mice that developed tumors had two tumors, hence a total number of 18 tumors.

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.

In line with this, the most dramatic effect in general was seen when only the SA' site was mutated as shown for Akv-CD; the tumor incidence of this mutant with respect to splenic marginal zone lymphoma (SMZL) increased from 8% to 28% (p < 0.1) and decreased to 0% as shown for Akv-EH (p < 0.05) and for Akv-CDH (p = 0.5). Moreover, the Akv-CD mutant virus was the only one that displayed a capability for inducing histiocytic sarcoma, a tumor type which has not been observed in any of our previous studies using NMRI mice (inbred or random-bred) infected with Akv, SL3-3, or different derived mutants of these. So in brief, synonymous mutations at the SA' site of Akv MLV markedly altered the oncogenic potential of the virus by significantly impairing the ability to induce both FBL and PCT. Besides, while the development of SMZL was increased by Akv-CD, it was abolished in Akv-EH and Akv-CDH, and most notably, a novel potential for inducing histiocytic sarcoma was established.

The most pronounced effect of mutating the SD' site (Akv-EH) is the frequent occurrence (35%) of diffuse tumors, which according to the Bethesda classification represent DLBCL centroblastic (more than 50% of the infiltrating population is centroblasts). These tumors, where progression is not from either a follicular or a marginal lymphoma, are comparable to the de novo lymphomas in humans, and to emphasize this association we have used the term de novo DLBCL (Table 2). Strikingly, de novo DLBCLs were never observed among the wild-type induced tumors or among the other mutant induced tumors (p < 0.05). The finding of such tumors in mice is rare and could be exploited to understand the molecular changes in de novo DLBCL of mice, and eventually a useful mouse model of human de novo DLBCL might be generated from this set-up.

Quite unexpectedly, the effect of mutating the SA' and SD' sites simultaneously (Akv-CDH) was the less manifested one. FBL incidence dropped from 23% to 7%; otherwise this mutant in our experimental setting displayed similar tumorigenic potential as the wild-type Akv MLV.

Conservation of the introduced splice site mutations in the tumors

To determine the stability of the introduced mutations, the regions containing the mutations were PCR amplified from genomic DNA prepared from the induced tumors, using the primers depicted in Fig. 1. The sequences of the amplified fragments confirmed in all cases the integrity of the introduced mutations (data not shown).

Both single- and double-spliced transcripts are generated in vivo

The observed effect of the mutated splice sites on the oncogenic properties advocates that RNA splicing by means of the alternative SA' and SD' sites does indeed take place in vivo. To clarify and confirm the identity of the produced transcripts, the splice pattern in tumor tissues (and for comparison in NIH 3T3 cells infected with the same four viruses) was analyzed. RNA from the individual end-stage tumors (or from virally infected cells) was isolated, and conventional RT-PCRs were performed with primers designed in such a way that it should be possible to identify all four potential splice products using 4 different primer sets as shown in Fig. 4A.
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.

With a few exceptions, all tumors were analyzed, and sequences of the amplified RT-PCR products determined to validate the specificity of the fragments (data not shown). Representative results from each virus series are shown in Fig. 4B. In all cases, PCR products representing splice product A (the regular env transcript; primer set #4) was observed, which implies that damage of the alternative splice sites, SA' and SD', does not impair the production of the regular single-spliced env RNA. Concerning splice product D (primer set #1) it was never amplified, neither from tumor tissues nor from cell culture studies, strongly indicating that this is not a bona fide transcript. The lack of detection of product D is unlikely to result from a technical PCR-problem since the two primers have been validated in other PCRs.

For the Akv wild-type induced tumors, RT-PCR products representing the double-spliced product B (primer set #2), and fragments of expected size amplified by primer set #3, indicative of splice product B or C, were observed in all cases. As would be expected primer set #2, which is dependent on an intact SA' site, did not result in any amplification products using RNA from Akv-CD or Akv-CDH tumors. Surprisingly however, in five out of 14 analyzed Akv-EH tumor samples (represented by Akv-EH tumor no. 14 in Fig. 4B), a product slightly smaller than that of transcript version B was amplified. The subsequent sequence analyses revealed that the alternative splice donor site SD* (depicted in Fig. 1) in these cases consistently had been used, resulting in the generation of a splice product equivalent in structure to product B, however 54 nucleotides shorter. No correlation between tumor cell specificity and usage of the SD* site could be observed, since the five tumor samples originated from FBL, DLBCL progressed from FBL, de novo DLBCL, and the single case of STL (small T-cell lymphoma). The presence of the same splice product from the SD* site was verified by sequence analysis of RT-PCR products derived from tumors induced by the wild-type virus in some cases, although the product was consistently less prominent than product B.

Transcript C corresponds to the single-spliced transcript of 4.4 kb, which previously has been reported to be produced by both Friend and Moloney MLV using the SD' together with the canonical env SA' site [18, 19]. Our RT-PCR results confirm the existence of this single-spliced transcript, since products of the expected size were always amplified with primer set #3 using RNA from Akv-CD tumors (Fig. 4B), whereas product B (primer set #2) was never amplified in this material.

In summary, by means of the alternative splice sites that define the novel gag exon, both a single-spliced transcript C as well as a novel double-spliced transcript B is produced in vivo, and when these alternative splice sites are destroyed, the splicing pattern is changed concordantly.

The same RT-PCR analyses were performed for NIH 3T3 cells infected with the four viruses, which led to the same splice pattern (data not shown). In addition, Northern blot hybridizations with an ecotropic env-probe and with a probe covering the novel SA'-SD' defined exon in gag were performed with RNA isolated from these cells (Fig. 5). Besides the expected hybridization patterns of prominent bands of full-length (env and gag probe) and env mRNA (only env probe) sizes, a weaker band of a size corresponding to splice product C (4.4 kb) was detected with both probes. No distinct band corresponding to spliced RNA B was observed, suggesting a very low level of production and/or significant messenger instability.
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.

Provirus integration site analyses

In order to identify a possible connection between specific retroviral integration sites (RIS) and specific diagnostic tumor types, provirus integration sites from the majority of the induced tumors were isolated and sequenced. We have then by subsequent homology searches of the mouse genome databases identified 240 unambiguous integration sites (Table 3). These integration site sequences represent tumors from 30 out of 40 (104 sequences), 14 out of 19 (46 sequences), 14 out of 18 (51 sequences), and 11 out of 16 (39 sequences) mice infected by Akv-wt, Akv-CD, Akv-EH, and Akv-CDH, respectively. This corresponds to an average of 3.6 integrations per analyzed tumor. Based on the searches in the UCSC database [23], and the Mouse Retrovirus Tagged Cancer Gene Database, RTCGD [24, 25], both version mm8, 111 novel RISs were identified. In an attempt to pick up candidate cancer genes that might be associated with specific tumor diagnoses, we looked for common integration sites (CISs), which would infer such genes [25, 26]. Hence, we compared the integration sites with each other as well as with previously defined RISs in RTCGD. In principle, using the recommendations from RTCGD with a window size of 100 kb, 50 kb, and 30 kb for CISs with 4 (or more), 3, or 2 insertions, respectively, we were hereby able to define 35 novel common integration sites (CISs) (Table 3). Just a single one of these could be correlated with a specific diagnosis and with a specific virus, since in two independently Akv-wt induced plasmacytomas a definite region of chromosome 15 was targeted. However, this region does not contain genes/RefSeqs within a 100 kb distance from the integrated proviruses, so for the present we cannot predict what – if any – candidate gene(s) that might have been influenced by the integrated proviruses. For the remaining 34 CISs more than one virus and more than one tumor diagnosis were implicated, which implies that no straightforward association between target gene (and/or causative virus) and tumor type can be deduced.
Table 3

Positions of integrated proviruses in tumor DNA

#

Virus

Diagnosis

Chromosome

Position (mm8)

Gene/RefSeqa

No. of hits in RTCGD (mm8)

Novel RISsb

Novel CISsc

1

Akv-EH

DLBCL (from FBL)

1

24641886

Lmbrd1

0

1

-

2

Akv-EH

DLBCL (from FBL)

1

36406157

Cnnm4

0

1

-

3

Akv wt

DLBCL (from FBL)

1

78743292

Kcne4

0

1

-

4

Akv-EH

PCT

1

82855932

Slc19a3

0

1

-

5

Akv wt

FBL

1

93014894

Ramp1

5

-

-

6

Akv-CD

n.d.

1

93022552

    

7

Akv-EH

Lymphoma, NOS

1

120226476

AK080782

0

1

-

8

Akv-CD

DLBCL (from FBL)

1

130341056

Cxcr4

3

-

-

9

Akv wt

PCT

1

135878316

Fmod/Btg2

8

-

-

10

Akv wt

DLBCL (from FBL)

1

135882183

    

11

Akv-CD

Abscess

1

139604557

---

1

-

1

12

Akv wt

PCT

1

144940508

---

0

1

-

13

Akv wt

PCT

1

163725782

AK029097

0

1

-

14

Akv wt

FBL

1

173476364

Slamf7

0

1

-

15

Akv wt

PCT

1

174350588

Tagln2/AK006449

2

-

-

16

Akv wt

DLBCL (from FBL)

1

182219328

MGC68323/AK038867

2

-

-

17

Akv-CD

SMZL

2

11542293

Il2ra

4

-

-

18

Akv-CDH

DLBCL (from FBL)

2

13133178

Rsu1

0

1

-

19

Akv-CDH

PCT

2

35270244

Ggta1

5

-

-

20

Akv wt

DLBCL (from FBL)

2

44741201

Gtdc1

0

1

-

21

Akv wt

DLBCL (from FBL)

2

46263959

---

0

1

-

22

Akv wt

DLBCL (from FBL)

2

71667822

Itga6/Pdk1

0

1

-

23

Akv wt

DLBCL (from FBL)

2

90883313

Slc39a13/Sfpi1

6

-

-

24

Akv wt

SMZL

2

90883476

    

25

Akv wt

PCT

2

102668507

Cd44

0

1

-

26

Akv wt

DLBCL (from FBL)

2

102782324

Pdhx

0

1

-

27

Akv wt

FBL

2

118352393

Pak6

0

1

-

28

Akv wt

FBL

2

119028536

Spint1

0

1

-

29

Akv-CDH

Lymphoma, NOS

2

120301032

Zfp106

1

-

1*

30

Akv-CDH

PTLL

2

128875013

Slc20a1

0

1

-

31

Akv-CDH

Lymphoma, NOS

2

129283153

Ptpns1

1

-

1

32

Akv-EH

PCT

2

131711284

Rassf2

0

1

-

33

Akv-CDH

DLBCL (from FBL)

2

158379688

Ppp1r16b

4

-

-

34

Akv wt

SMZL

2

164051325

Slpi

0

1

-

35

Akv wt

DLBCL (from FBL)

2

169860192

Zfp217

5

-

-

36

Akv wt

FBL

3

22265638

Tbl1xr1

0

1

-

37

Akv-CDH

PCT

3

27464311

Aadacl1

0

1

-

38

Akv-CDH

DLBCL (from FBL)

3

30203814

Evi1

5

-

-

39

Akv-CDH

PCT

3

30203870

    

40

Akv-CDH

Lymphoma, NOS

3

76043446

Golph4

0

1

-

41

Akv-EH

DLBCL (from FBL)

3

79339620

---

0

1

-

42

Akv wt

FBL

3

90334704

Slc39a1

0

1

-

43

Akv-CD

DLBCL (from FBL)

3

96900321

Cd160

0

1

-

44

Akv wt

SMZL

3

98031475

LOC433632

13

-

-

45

Akv-EH

PCT

3

98041399

    

46

Akv-CD

SMZL

3

98043109

    

47

Akv-CDH

DLBCL (from FBL)

3

98043150

    

48

Akv wt

PCT

3

98043377

    

49

Akv wt

DLBCL (from FBL)

3

98043659

    

50

Akv-CD

SMZL

3

98064421

    

51

Akv-CDH

DLBCL (from FBL)

3

98127957

Notch2

8

-

-

52

Akv-EH

PCT

3

108214198

Ampd2

0

1

-

53

Akv-CD

SMZL

3

115828351

Dph5

1

-

1

54

Akv wt

DLBCL (from FBL)

3

131582947

Papss1

1

-

-

55

Akv wt

SBL

3

145870070

Bcl10

3

-

-

56

Akv wt

DLBCL (from FBL)

3

146091393

Mcoln2

0

1

-

57

Akv-EH

DLBCL (from FBL)

3

157996860

Lrrc40

0

1

-

58

Akv wt

SBL

4

8842182

BC034239

1

-

1

59

Akv-CDH

PCT

4

11915327

AK132816

0

1

-

60

Akv-CD

DLBCL (from FBL)

4

32560128

Bach2

14

-

-

61

Akv wt

FBL

4

32611866

    

62

Akv-CD

n.d.

4

32619341

    

63

Akv wt

PCT

4

32702311

    

64

Akv-CD

Histiocytic sarcoma

4

44734542

Pax5

4

-

-

65

Akv wt

SMZL

4

55369934

Rad23b

1

-

1

66

Akv-CDH

Plasma cell prolif.

4

57933461

Akap2

0

1

-

67

Akv-CD

DLBCL (from FBL)

4

97386196

Nfia/D90173

2

-

-

68

Akv-EH

DLBCL (from FBL)

4

132220004

Fgr

3

-

-

69

Akv wt

SMZL

4

134699599

Dscr1l2

0

1

-

70

Akv-CDH

PCT

4

138050107

Pla2g2d

0

1

-

71

Akv wt

FBL

5

39921672

Hs3st1

0

1

-

72

Akv-CDH

Lymphoma, NOS

5

65179064

Tlr1

1

-

1

73

Akv-CD

Histiocytic sarcoma

5

75074642

---

0

1

-

74

Akv-CDH

PTLL

5

107966364

Gfi1

78

-

-

75

Akv-CDH

PCT

5

121838640

Aldh2

0

1

-

76

Akv-CDH

DLBCL (from FBL)

5

141077075

Gna12

4

-

-

77

Akv wt

DLBCL (from FBL)

6

29717975

4631427C17Rik

0

1

-

78

Akv-EH

de novo DLBCL

6

40821642

BC048599

0

1

-

79

Akv-CDH

Lymphoma, NOS

6

40955151

2210010C04Rik

0

1

-

80

Akv-CDH

PCT

6

54425558

Scrn1

0

1

-

81

Akv wt

FBL

6

72441620

BC100525

2

-

-

82

Akv wt

PCT

6

84016089

Dysf

0

1

-

83

Akv-CDH

Lymphoma, NOS

6

88923549

Gpr175

0

1

-

84

Akv-EH

DLBCL (from SMZL)

6

99153396

Foxp1

1

-

1*

85

Akv-CDH

Lymphoma, NOS

6

113010477

Thumpd3

1

-

1

86

Akv-CD

DLBCL (from FBL)

6

120535110

Cecr5

3

-

-

87

Akv-CD

Histiocytic sarcoma

6

136905161

Arhgdib

0

1

-

88

Akv-CD

PCT

6

145079282

Lrmp

4

-

-

89

Akv-CDH

Plasma cell prolif.

7

18841894

Apoc4

0

1

-

90

Akv wt

FBL

7

24263292

Xrcc1

0

1

-

91

Akv wt

PCT

7

28498093

5830482F20Rik

0

1

-

92

Akv-EH

de novo DLBCL

7

28691963

Map4k1

1

-

1

93

Akv-EH

PCT

7

29760852

Zfp14

0

1

-

94

Akv-CD

Histiocytic sarcoma

7

30746023

Fxyd5

0

1

-

95

Akv wt

PCT

7

45003648

Flt3l

0

1

-

96

Akv wt

DLBCL (from FBL)

7

66812108

Adamts17

0

1

-

97

Akv wt

DLBCL (from FBL)

7

73481277

---

7

-

-

98

Akv-CD

SMZL

7

76335733

D430020F16

0

1

-

99

Akv-EH

DLBCL (from FBL)

7

79083435

Mfge8

0

1

-

100

Akv-EH

PCT

7

80764333

AK034740

0

1

-

101

Akv wt

PCT

7

82574727

Eftud1

1

-

1*

102

Akv-EH

de novo DLBCL

7

99422958

Arrb1/mmu-mir-326

0

1

-

103

Akv wt

SBL

7

113972740

Rras2/Copb1

32

-

-

104

Akv-CDH

DLBCL (from FBL)

7

121248057

AK043969

0

1

-

105

Akv-EH

de novo DLBCL

7

126957227

Cd2bp2

2

-

-

106

Akv wt

DLBCL (from FBL)

7

132147451

4631426J05Rik

0

1

-

107

Akv-CD

DLBCL (from FBL)

7

144741729

Ccnd1

21

-

-

108

Akv-CD

SMZL

8

8401964

---

0

1

-

109

Akv-EH

DLBCL (from FBL)

8

10980425

---

4

-

-

110

Akv wt

FBL

8

35575516

Dctn6

0

1

-

111

Akv wt

PCT

8

37098099

---

0

1

-

112

Akv-CD

SMZL

8

74527100

Pgls

3

-

-

113

Akv wt

DLBCL (from FBL)

8

98522228

Gins3

0

1

-

114

Akv wt

SBL

8

112753504

BC027816

0

1

-

115

Akv-EH

de novo DLBCL

8

118119184

Wwox

1

-

1

116

Akv-EH

Lymphoma, NOS

8

123611854

Irf8

1

-

-

117

Akv-EH

DLBCL (from FBL)

8

126312417

Tubb3/Mela

1

-

1

118

Akv wt

DLBCL (from FBL)

8

126312418

    

119

Akv wt

PCT

9

46235263

---

0

1

-

120

Akv-CD

Abscess

9

86514607

A330041J22Rik

0

1

-

121

Akv-CDH

PCT

9

104103363

Acpp

1

-

1

122

Akv-EH

FBL

9

115327386

---

0

1

-

123

Akv wt

PCT

9

117143474

Rbms3

0

1

-

124

Akv-EH

PCT

10

5056902

Syne1

1

-

-

125

Akv wt

SBL

10

7626524

Map3k7ip2

1

-

1

126

Akv wt

FBL

10

19681745

Map3k5

2

-

-

127

Akv wt

PCT

10

43100419

Pdss2

0

1

-

128

Akv-EH

PCT

10

59295561

Dnajb12

2

-

-

129

Akv-CD

Histiocytic sarcoma

10

75678805

Prmt2

0

1

-

130

Akv wt

FBL

10

77412375

Pfkl

0

1

-

131

Akv wt

DLBCL (from FBL)

10

8009951

BC058238

2

-

-

132

Akv wt

DLBCL (from FBL)

10

84366577

Ric8b

0

1

-

133

Akv-EH

de novo DLBCL

10

87574719

BC070476

0

1

-

134

Akv-EH

de novo DLBCL

10

92501677

Pctk2

1

-

-

135

Akv-CD

n.d.

10

123752010

---

0

1

-

136

Akv-CDH

PCT

11

3236433

1500004A08Rik

0

1

-

137

Akv wt

PCT

11

5331124

AK133342

0

1

-

138

Akv wt

DLBCL (from FBL)

11

23587218

4933435A13Rik

4

-

-

139

Akv-CD

DLBCL (from FBL)

11

23587651

    

140

Akv wt

PCT

11

32443618

Stk10

1

-

1

141

Akv wt

PCT

11

46693128

Timd4

0

1

-

142

Akv-CD

PCT

11

51687729

Phf15

0

1

-

143

Akv-EH

de novo DLBCL

11

62648918

Trim16

0

1

-

144

Akv wt

PCT

11

67380781

Gas7

0

1

-

145

Akv-EH

de novo DLBCL

11

74962635

Smg6

9

-

-

146

Akv-EH

de novo DLBCL

11

78821537

Ksr1

0

1

-

147

Akv-EH

PCT

11

86862765

Gdpd1

0

1

-

148

Akv wt

DLBCL (from FBL)

11

95031687

Tac4

0

1

-

149

Akv-EH

de novo DLBCL

11

102249514

Grn

0

1

-

150

Akv wt

DLBCL (from FBL)

11

102990732

Fmnl1

2

-

-

151

Akv wt

DLBCL (from FBL)

11

106946907

Nol11

0

1

-

152

Akv-CD

Abscess

11

107232889

Pitpnc1

1

-

1

153

Akv-EH

PCT

11

116126666

Exoc7

0

1

-

154

Akv-CDH

Lymphoma, NOS

11

118058083

Pscd1

1

-

1*

155

Akv-CD

SMZL

12

3288080

Rab10

0

1

-

156

Akv wt

PCT

12

13172238

Ddx1

0

1

-

157

Akv-CD

Abscess

12

56600484

Garnl1

0

1

-

158

Akv-EH

de novo DLBCL

12

77286036

Zbtb25

1

-

1

159

Akv wt

DLBCL (from FBL)

12

80214408

AK132344

1

-

1

160

Akv wt

DLBCL (from FBL)

12

86569587

Batf

2

-

-

161

Akv-CD

DLBCL (from FBL)

12

113688885

BC004786

5

-

-

162

Akv wt

PCT

13

24453563

Cmah

1

-

1*

163

Akv-CDH

DLBCL (from FBL)

13

28624333

---

0

1

-

164

Akv wt

PCT

13

28727388

---

3

-

-

165

Akv-CDH

DLBCL (from FBL)

13

28764182

    

166

Akv-CDH

DLBCL (from FBL)

13

28950798

Sox4

79

-

-

167

Akv-CD

DLBCL (from FBL)

13

28950905

    

168

Akv-EH

de novo DLBCL

13

28955972

    

169

Akv wt

DLBCL (from FBL)

13

28958981

    

170

Akv-EH

DLBCL (from FBL)

13

30695323

Dusp22

4

-

-

171

Akv-CD

Histiocytic sarcoma

13

30727958

    

172

Akv wt

PCT

13

31914670

Gmds

1

-

1

173

Akv-CD

SMZL

13

36214883

Fars2

0

1

-

174

Akv wt

PCT

13

37804150

Rreb1

8

-

-

175

Akv-CD

Histiocytic sarcoma

13

38701503

Eef1e1

1

-

-

176

Akv-CD

DLBCL (from FBL)

13

43205444

Gfod1

1

-

1*

177

Akv-CD

SMZL

13

63488458

Fancc

4

-

-

178

Akv wt

DLBCL (from FBL)

13

84050271

Mef2c

11

-

-

179

Akv-CDH

DLBCL (from FBL)

14

6779744

Dnase1l3

1

-

1

180

Akv-CDH

Lymphoma, NOS

14

24439077

Rai17

10

-

-

181

Akv-CDH

PCT

14

25348097

Slmap

1

-

1

182

Akv-EH

Lymphoma, NOS

14

29013598

Cacna1d

0

1

-

183

Akv wt

SBL

14

30491074

Btd

0

1

-

184

Akv wt

DLBCL (from FBL)

14

59481891

---

0

1

-

185

Akv wt

FBL

14

59637396

AK151394

3

-

-

186

Akv-EH

de novo DLBCL

14

63115780

Msra

1

-

1*

187

Akv wt

PCT

14

72420139

Sucla2

0

1

-

188

Akv wt

SMZL

14

113921088

microRNA cluster

2

-

-

189

Akv wt

FBL

15

57752356

BC066830

0

1

-

190

Akv-CD

SMZL

15

58238320

15Ertd621e

0

1

-

191

Akv wt

PCT

15

61240727

---

0

1

1

192

Akv wt

PCT

15

61240729

    

193

Akv wt

FBL

15

73548838

Gpr20/Ptp4a3

6

-

-

194

Akv-EH

DLBCL (from FBL)

15

79567673

Unc84b

4

-

-

195

Akv wt

DLBCL (from FBL)

15

79728346

Apobec3

0

1

-

196

Akv-EH

Plasma cell prolif.

15

90445122

Cpne8

1

-

-

197

Akv wt

FBL

16

23906462

Bcl6

5

-

-

198

Akv-EH

de novo DLBCL

16

24049008

---

5

-

-

199

Akv-CD

DLBCL (from FBL)

16

24086635

    

200

Akv-EH

DLBCL (from FBL)

16

24101470

    

201

Akv-EH

Lymphoma, NOS

16

24152772

    

202

Akv wt

FBL

16

24178252

    

203

Akv wt

DLBCL (from FBL)

16

32049420

Lrrc33

3

-

-

204

Akv-CD

n.d.

16

52388367

Alcam

1

-

1

205

Akv wt

PCT

17

6624877

Vil2

3

-

-

206

Akv wt

PCT

17

11630998

Park2

1

-

1*

207

Akv wt

DLBCL (from FBL)

17

36597927

2410137M14Rik

0

1

-

208

Akv wt

FBL

17

49537999

2310015N21Rik

1

-

1*

209

Akv-EH

DLBCL (from FBL)

17

63705275

Fert2

0

1

-

210

Akv-CD

SMZL

17

71389894

Kntc2

0

1

-

211

Akv-CDH

PTLL

17

74614066

Birc6

1

-

-

212

Akv wt

PCT

18

11242422

---

0

1

-

213

Akv-CD

SMZL

18

12368360

Npc1

1

-

1

214

Akv-CD

SMZL

18

36016553

Cxxc5

3

-

-

215

Akv-CDH

DLBCL (from FBL)

18

39790170

---

0

1

-

216

Akv wt

FBL

18

42916219

Ppp2r2b

0

1

-

217

Akv-EH

de novo DLBCL

18

60930595

Ii/Cd74

0

1

1

218

Akv-EH

DLBCL (from FBL)

18

60930793

    

219

Akv-CD

DLBCL (from FBL)

18

60930833

    

220

Akv wt

PCT

18

60931233

    

221

Akv-CDH

PCT

18

60934151

    

222

Akv wt

DLBCL (from FBL)

18

61103722

Camk2a

0

1

-

223

Akv-EH

PCT

18

65601468

Malt1

1

-

-

224

Akv wt

DLBCL (from FBL)

18

67824697

Ptpn2

0

1

-

225

Akv wt

FBL

19

11607493

Ms4a4d

0

1

-

226

Akv wt

DLBCL (from FBL)

19

34362400

Fas

2

-

-

227

Akv-EH

de novo DLBCL

19

37505125

Hhex/Exoc6

44

-

-

228

Akv-CD

DLBCL (from FBL)

19

37532944

    

229

Akv-EH

de novo DLBCL

19

37537038

    

230

Akv-CD

SMZL

19

37560104

    

231

Akv-CD

Histiocytic sarcoma

19

37560104

    

232

Akv-EH

PCT

19

43474871

Cnnm1

0

1

-

233

Akv-CDH

PCT

19

47583866

Obfc1

0

1

-

234

Akv wt

DLBCL (from FBL)

19

47963460

AK014581

0

1

-

235

Akv wt

PCT

19

53994934

Shoc2

0

1

-

236

Akv wt

FBL

19

55633727

Vti1a

1

-

1*

237

Akv wt

FBL

X

103293531

P2ry10

0

1

-

238

Akv-CDH

Lymphoma, NOS

X

109985132

Dach2

1

-

1

239

Akv-EH

DLBCL (from FBL)

X

129928954

Btk

0

1

-

240

Akv wt

DLBCL (from FBL)

X

162452646

Tmsb4x

1

-

1

aThe gene (or RefSeq) closest to the integrated provirus is given (UCSC, mouse mm8 assembly). --- indicates that the distance to the closest gene/RefSeq is more than 100 kb.

bFor each insertion, it is indicated, based on RTCGD (mm8), whether a novel RIS has been defined.

cFor each insertion, it is indicated, based on RTCGD (mm8), whether a novel CIS has been defined. The definition follows the recommendations from RTCGD with a window size of 100 kb, 50 kb, and 30 kb for CISs with 4 (or more), 3, or 2 insertions, respectively. * indicates an exception from this rule, if two integration sites are found within the same gene/RefSeq.

n.d., not determined.

In six cases, the same chromosomal locus was targeted several times. These cases include Bach2 (hit 4 times), Sox4 (hit 4 times), Hhex (hit 5 times), Ii (hit 5 times), a region of chr. 16 not containing any genes/RefSeqs within a distance of 100 kb from the integration sites (hit 5 times), and LOC433632 (hit 7 times). Five of these integration sites were already registered in RTCGD, only the Ii locus define a completely novel RIS/CIS. This latter finding may suggest that Ii targeting is strongly associated with the applied model system (virus/mouse strain)[27]. We also note that an integration has taken place within the first intron of Stk10 in a plasmacytoma induced by Akv-wt (Table 3, #140), which appears to be in conflict with the work of Shin et al., 2004 [28] where Stk10 was described as a SMZL specific candidate gene.

Finally, we examined if specific regions of the targeted gene/RefSeq have been favored with respect to orientation and position of the integrated provirus. We have recently reported of differences between Akv MLV and an enhancer mutant hereof, Akv1-99, in their patterns of proviral insertions around host transcription units in the induced tumors [29]. In line with this, it might be envisioned that destroying the alternative splice sites of the virus could lead to a different pattern of integration site selection during tumorigenesis; e.g. it might be speculated if particular positions relative to the target gene somehow would facilitate gene deregulation dependent on the presence or absence of intact SA' and/or SD' sites. Accordingly, we allocated each individual integration site position and orientation to a defined region, i.e. either upstream, within the promoter, within 1. intron, within last intron, within all other introns, within exons, or downstream of the target gene (Table 4). Eleven cases in total were excluded as they were positioned – almost with the same distance – in between two target genes, upstream of one and downstream of the other. As seen in table 4, no clear differences with respect to the four viruses were observed, signifying that mutations of the alternative splice sites do not have major effect on the ability of the Akv MLV to affect the target gene/RefSeq from certain positions and/or orientations. It might be worth to notice that the overall picture shows that about half of all integrations are found within introns, and among these there appear to be a tendency for a provirus orientation opposite to that of the target gene. In these cases the formation of chimeric RNA species by promoter insertion and/or splicing would not be predicted.
Table 4

Frequency of proviral insertions within defined genomic regions

Virus

Upstreama

 

Promoterb

 

1. intron

 

Internal intron

 

Last intron

 

Downstreamc

 

Exon

 

Outsided

 

+

-

+

-

+

-

+

-

+

-

+

-

+

-

 

Akv-wt

2/104 2%

7/104 7%

5/104 5%

3/104 3%

9/104 9%

16/104 15%

6/104 6%

17/104 16%

2/104 2%

1/104 1%

5/104 5%

8/104 8%

2/104 2%

1/104 1%

11/104 10%

Akv-CD

3/46 7%

6/46 13%

1/46 2%

0/46 0%

3/46 7%

5/46 11%

2/46 4%

8/46 17%

0/46 0%

0/46 0%

4/46 9%

6/46 13%

1/46 2%

0/46 0%

5/46 11%

Akv-EH

3/51 6%

4/51 8%

1/51 2%

0/51 0%

4/51 8%

6/51 12%

2/51 4%

11/51 22%

2/51 4%

0/51 0%

7/51 14%

1/51 2%

2/51 4%

2/51 4%

6/51 12%

Akv-CDH

1/39 3%

2/39 5%

2/39 5%

3/39 8%

7/39 18%

3/39 8%

3/39 8%

5/39 13%

0/39 0%

1/39 3%

5/39 13%

4/39 10%

0/39 0%

0/39 0%

3/39 8%

Total

9/240 4%

19/240 8%

9/240 4%

6/240 3%

23/240 10%

30/240 13%

13/240 5%

41/240 17%

4/240 2%

2/240 1%

21/240 9%

19/240 8%

5/240 2%

3/240 1%

25/240 10%

a Within 3–100 kb upstream of target gene/RefSeq

b Within 0–3 kb upstream of exon1 of target gene/RefSeq

c Within 100 kb downstream of target gene/RefSeq

d Proviral integrations > 100 kb away from gene/RefSeq

+ or - denotes the orientation of the integrated provirus relative to the target gene/RefSeq.

Discussion

We have in the B-lymphomagenic Akv MLV identified a novel exon, which is defined by the alternative splice acceptor (SA') and the splice donor (SD') sites located in the capsid encoding region. While previous studies of Moloney and Friend MLV have demonstrated production of a 4.4 kb transcript using the same SD' site together with the canonical env SA site, this is the first report demonstrating the existence of an alternative SA' site and production of a double-spliced transcript during the life cycle of a replication-competent simple retrovirus. Yet it remains to be investigated how widespread this competence is. An alignment between six murine retroviruses shows that the conserved splice junction dinucleotide AG is present neither in Cas-Br-E nor in Moloney MLV, although the region in general is well-conserved (Fig. 6).
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.

We did not perform detailed analyses of the influence of the splice site mutations on the viral replication. However, since the same number (105 to 106) of infectious virus particles, as measured by infectious center assays, were injected from each virus series, and since the mutant viruses induced tumors with comparable incidences and latencies as the wild-type virus, it is not likely that the mutations had imposed severe weakening on the in vivo spreading capability. Hence the observed shifts in specificity of the induced tumors most likely are a direct result of the introduced mutations. However, we note that Houzet et al. [19] observed a reduction in titer of SD'-mutants of Friend virus.

The in vivo significance of the alternative splice sites was exposed by a change of the oncogenic properties of Akv MLV, when synonymous mutations destroying the SA' site, the SD' site, or both sites simultaneously were introduced. First and foremost, the obvious capability of Akv MLV to induce follicular B cell lymphoma was seriously weakened, when one or both of the alternative splice sites were mutated, suggesting that this competence relies on intact SA' and SD' sites and a proper balance between all the produced transcripts (one full-length, two single-spliced, and one double-spliced). The integrity of Akv MLV seems fundamental for its capability to induce FBL; thus we just reported that Akv MLVs with mutated enhancer sequences retained the ability to induce tumors of B-cell type, but altered specificities were observed, including an impaired ability of FBL induction [11]

A more complex picture was observed regarding the strong predisposition of Akv MLV for plasmacytoma induction. This predisposition was affected significantly only if the SA' alone was mutated. Thus, if the SD' site was mutated along with SA', wild-type potential was restored. This may indicate that the ability to induce plasmacytoma is dependent on a fine-tuned balance between the alternative single-spliced and double-spliced transcripts. If no double-spliced transcript is produced, while the single-spliced 4.4 kb transcript still is, as is the case for the SA' mutant, the single-spliced transcript somehow seems to be related with a barrier for plasmacytoma induction. On the other hand, if both transcripts are produced (Akv-wt) or none of them are produced (the SA'/SD' double mutant) the virus will hold a potential for inducing plasmacytomas. This is in line with the overall observation that the most pronounced effects were observed when the SA' or SD' splice sites were mutated individually, while the outcome of infection with the SA'/SD' double mutant in essence, except for the capability of FBL induction, was comparable to that of the wild-type virus. It may thus be speculated if a delicate balance between the alternatively single-spliced and double-spliced transcripts is a key determinant for the shift in oncogenic specificity, as demonstrated by the SA' and SD' splice site Akv mutants.

The most striking shifts in specificity observed were the increased tendency to stimulate development of splenic marginal zone lymphoma and the exposure of a novel ability for inducing histiocytic sarcoma for the SA' site mutant. Although we have shown that mutation of the SA' site results in inhibition of generation of the double-spliced product, we are at this point not able to explain or point to any detailed mechanisms underlying the observed changes in specificity. The other remarkable shift in specificity was detected with the SD' mutant, which was the only virus capable of inducing centroblastic DLBCL, i.e. tumors for which an origin from the follicle or marginal zone could not be inferred and comparable to de novo DLBCL in humans. Moreover, since the exposed potential appeared quite strong (35% of the SD'-mutant induced tumors fell within this diagnosis), and since such tumors are in general rare in mice, this mutant virus may be a helpful starting point tool to create a solid mouse model of human de novo DLBCL.

Obviously, the proposed significance of proper balances between the four different transcripts for the observed shifts in tumor specificity may reflect a need for a well-regulated balance between resulting translational products. We did not investigate if novel proteins were produced, but the open reading frames (ORFs) of both the alternatively singly and doubly spliced transcripts clearly reveal a potential for additional proteins to be produced. The gene products of the single-spliced 4.4 kb transcript most probably correspond to the p50 and p60 proteins made from the equivalent Friend MLV transcript [19]. These proteins were produced with translation initiations at two initiation codons (AUG gag and CUGglyco-gag) in the same ORF and were shown to harbor the N-terminal Gag domain including matrix, p12, and the first 110 amino acids of the capsid in frame with the last 116 amino acids of integrase [19]. Also the smaller double-spliced transcript harbors smaller ORFs providing a scene for even more MLV proteins.

Intragenic elements such as gag enhancers have been known for many years in avian retroviruses [30, 31]. However, it seems unlikely that a similar element is involved here, since the mutant virus with SA' and SD' sites mutated together was clearly the less affected one. This observation, in concert with the observed consequence on generation of different splice products, more likely suggests that the effect on disease specificity is related to an RNA processing phenomenon rather than an intragenic gag determinant with an effect on transcription.

Lymphoma-induction by non-acute murine retroviruses is associated with multiple proviral insertions that affect critical host genes. To achieve such multiple insertions the superinfection resistance caused by Env-expression must be by-passed. One possibility could be that reduced Env-expression caused by mutation of the gag splice sites as reported here might favor superinfection and thereby multiple proviral insertions. While we cannot exclude this possibility, our finding of the same number of sequence tags for proviral insertions for the wild-type and mutated viruses gives no immediately support to such a mechanism.

Retroviral insertional mutagenesis has been established as a solid strategy for the identification of candidate cancer-causing genes [6, 26, 3234]. Accordingly, in an effort to relate specific genes or pathways with specific diagnoses, splice pattern, or causative virus, we identified a pool of 240 integration sites from which 111 novel RISs and 35 novel CISs were defined. Our analyses did not immediately point to any clear correlations; nevertheless the collection of candidate genes may prove to be a central input in future attempts to understand the exact roles of the different splice transcripts and/or their resulting translational products in hematopoietic differentiation and tumorigenesis.

Conclusion

We have in the B-lymphomagenic Akv MLV in the gag region identified a novel exon, which represents the first example of a doubly spliced gammaretroviral transcript. Mutations of the alternative splice sites that define this novel transcript change the distribution of the different induced tumor phenotypes as well as generate tumors of additional specificities such as de novo diffuse large B cell lymphoma and histiocytic sarcoma. Provirus integration site analyses revealing 111 novel RISs and 35 novel CISs did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants. However, the list of potential target genes will be useful for future studies of hematopoietic differentiation and tumorigenesis.

Methods

Exon trapping

Exon trapping was performed by using an Exon Trapping System kit (GibcoBRL) in essence according to supplied protocol. In brief, Akv DNA was digested with BamHI or BglII and all restriction fragments were subcloned into the pSPL3 plasmid, which in addition to sequences necessary for replication and growth in Escherichia coli contains SV40 sequences that provide for replication and transcription in COS-7 cells, splicing signals, and a multiple cloning site. Following transformation into E. coli, plasmid DNA was isolated and transfected into COS-7 cells. Total RNA was isolated from cultured cells and used for first-strand cDNA synthesis. The cDNA was PCR amplified in two rounds with primers located in the vector exons. The outcome of the PCR amplifications was several different fragments, which were all sequenced. Two of trapped sequences could be verified as exons by RT-PCR analyses of RNA isolated from Akv MLV infected cells. The two trapped exons were defined by the same splice acceptor site (SA', located in the gag region, Fig. 1), but by different splice donor sites (SD' and SD*, Fig. 1), and the sizes were 235 bp and 180 bp, respectively.

Generation of viruses

The mutations of Akv MLV at splice acceptor (SA') and/or splice donor site (SD') sites were introduced by PCR-based oligonucleotide directed mutagenesis using the following primers harboring the wanted mutations (underlined): Mut-C: 5'-CTATATAACTGGAAAAATAATAATCCATCATTCAGTGAAGATCCAGGTAAACT-3', Mut-D: 5'-GGATTATTATTTTTCCAGTTATATAGATCGCTGGAGGAAAACG-3', and Mut-H: 5'-TTGGGATTACACCACCCAAAGGGGACGAAACCACCT-3'. A 720 bp Bsu36I – Bsu36I fragment harboring the mutations was cloned into the full length parental provirus. The correct sequence of the introduced Bsu 36I fragment was verified by sequence analysis.

Pathogenicity experiments

Akv wild-type virus λ623 and the three different alternative splice site mutant viruses, Akv-CD, Akv-EH, and Akv-CDH, mutated in either the SA' or SD' site, or in both sites simultaneously, were injected into newborn mice of the inbred NMRI strain, as described in details [35]. Control mice of the same colony were mock injected with 0.1 mL complete medium. The animals were monitored 5 days per week. Mice were sacrificed and autopsied when showing signs of illness or tumor development. Tumor development was diagnosed on the basis of grossly enlarged lymphoid organs after having reached the size described earlier, which is compatible with lymphoma [36]. Lymphoid tumor tissues and the liver were dissected, stored frozen (-80°C) and/or fixed in formalin for further analysis. Statistical analysis was carried out using the two-tailed Fisher's exact test.

Histopathological examination and immunohistochemical analysis

Formalin-fixed, paraffin-embedded sections from lymph nodes, thymus, spleen and liver were analyzed. Three-to-five micrometer-thick sections were cut and stained with hematoxylin and eosin (H&E), and when indicated with Giemsa, PAS or chloroacetate esterase. Tumors were classified according to the Bethesda proposals for classification of murine hematopoietic neoplasms [22, 37]. Immunohistochemistry was performed on an automated immunostainer (Ventana Medical System, Inc.; AZ, USA), according to the protocol provided by the company with minor modifications. After deparaffinization and rehydration, the slides were placed in a microwave pressure cooker in 0.01 M citrate buffer (pH 6.0), containing 0.1% Tween-20 and heated in a microwave oven at maximum power for 30 min. After cooling in Tris-buffered saline, the sections were incubated with 3% goat or rabbit serum for 20 min. The antibody panel used included CD3, CD79acy, TdT, myeloperoxidase (Dako, Hamburg, Germany), B220/CD45R and MAC3 (BD Pharmingen, NJ, USA). Appropriate positive controls were used to confirm the adequacy of the staining.

Northern blot analysis

Total cellular RNA was extracted from chronically infected NIH 3T3 cells by Trizol Reagent (Invitrogen), following the manufacturer's recommendations. Approximately 25 μg of RNA from each series (Akv-wt, Akv-CD, Akv-EH, Akv-CDH, and mock-infected cells) was size-fractionated on a 1.2% formaldehyde/agarose gel, and transferred to a nylon filter membrane (Zeta-Probe GT; Bio-Rad) under alkaline conditions (50 mM NaOH). Prehybridzation, hybridization, and washing procedures were according to standard protocol described in the instruction manual from Zeta-Probe GT [(pre)hybridization buffer: 0.25 M sodium phosphate, pH 7.2, 7% SDS, and washing buffers: 20 mM sodium phosphate, ph 7.2, 5% (1%) SDS). The hybridization probes were a 32P random priming labeled envelope specific probe (a 330 bp SmaI fragment of Akv MLV (positions 6240to 6570) [38]]) and a 32P random priming labeled gag specific probe covering the novel exon. The gag probe was a 380 bp PCR-fragment amplified by the following primers: gag-forward: 5'-ATGGTCAGTTGCAGTACTGGCCGT-3' and gag-reverse: 5'-TGGGGCTTCGGCCCGCGTTTTGGA-3'. The integrity and concentration of the RNA were confirmed by visual inspection of ethidium bromide-stained 18S and 28S rRNAs.

PCR and RT-PCR analyses

Genomic DNA was purified from frozen tumor tissues by DNeasy Tissue Kit (Qiagen). Conservation of the introduced mutations was examined by PCR amplifying the region enclosing the mutations and by using the primers depicted in Fig. 1 (primer sequences and positions in Akv provirus: Forward primer, 5'-CCTATGAACCCCCTCCGTGGGTCA-3', nucleotides 1387–1410, and Reverse primer, 5'-TATTAAAGATCCTTTCGGCTTC-3', nucleotides 2412–2390). The resulting PCR products were analyzed by agarose gel electrophoresis, purified, and sequenced with nested sequencing primers (numberings refer to positions in Akv provirus). Forward primer, 5'-CGGGGAGGAGAAGCAGCGGGTGCT-3', nucleotides 1952–1976, and Reverse primer, 5'-GTCCCTAATAATTGCTGGCAAT-3', nucleotides 1942-1921.

For the RT-PCR analyses, total cellular RNA was extracted from tumor tissues or chronically infected NIH 3T3 cells by Trizol Reagent (Invitrogen), following the manufacturer's recommendations. 1–5 μg total RNA was used to make first-strand cDNA by First-Strand cDNA Synthesis Kit (Amersham Biosciences) with an oligo-dT primer. This was followed by standard PCR amplification using four different primer sets, #1 to #4. Primer set #1: Forward primer, 5'-CCGACCCACCGTCGGGAGGAT-3', and reverse primer, 5'-CCTCATCAAACAGGGTGGGACT-3'. Primer set #2: Forward primer, 5'-CCGACCCACCGTCGGGAGGAT-3', and reverse primer, 5'-CACCCACACGGAGTCTCCAAT-3'. Primer set #3: Forward primer, 5'-GATTACACCACCCAAAGAGCTC-3', and reverse primer, 5'-CACCCACACGGAGTCTCCAAT-3'. Primer set #4 (env transcript): Forward primer, 5'-TTGGAGACCCCCGCCCAGGGACCACC-3', and reverse primer, 5'-CACCCACACGGAGTCTCCAAT-3'. The resulting RT-PCR products were analyzed by agarose gel electrophoresis, and in most cases purified and sequenced.

Provirus tagging and analyses

Genomic DNA isolated from the induced tumors was analyzed for provirus integration sites by a splinkerette-based PCR method [26], described in details in [39]. The resulting host/virus junction fragments were sequenced, and the cellular flanking sequences were compared (BLAT search) to the UCSC Genome Browser, version mm8, to determine the chromosomal position of the integrated provirus. To identify possible novel retrovirus integration sites (RISs) and common integration sites (CISs), the individual integration sites were concomitantly matched up to the Retroviral Tagged Cancer Gene Database (RTCGD), version mm8 [24, 25]. The definition of a CIS follows the recommendations from RTCGD with a window size of 100 kb, 50 kb, and 30 kb for CISs with 4 (or more), 3, or 2 insertions, respectively. Exception from the recommended window sizes was allowed in a few cases when two (or more) integrations were found within the same gene/RefSeq (Table 3).

DNA sequencing analysis

Amplified PCR products or purified plasmid preparations were sequenced with the DYEnamic ET terminator cycle sequencing kit (Amersham Pharmacia Biotech), following the manufacturer's recommendations, and reaction products were analyzed on an automated DNA sequencer (Applied Biosystems Inc.).

Declarations

Acknowledgements

We thank Astrid van der Aa Kühle, Angelika Appold, Katrin Reindl, Jaqueline Müller, Claudia Kloß, Nadine Kink, and Elenore Samson for excellent technical assistance. This work was supported by the Danish Cancer Society, the Novo Nordic Foundation, the Karen Elise Jensen Foundation, the Danish Natural Sciences and Medical Research Councils, NIH grant CA100266, Synergenics LLC, and the National Danish Research Foundation through the Centre for mRNP Biogenesis and Metabolism at the University of Aarhus.

Authors’ Affiliations

(1)
Department of Molecular Biology, University of Aarhus
(2)
Institute of Pathology, GSF-National Research Center for Environment and Health
(3)
Department of Comparative Medicine GSF-National Research Center for Environment and Health
(4)
Picobella
(5)
Department of Microbiology and Immunology, University of California-San Francisco
(6)
The State and University Library, Universitetsparken
(7)
Biotech Research and Innovation Centre (BRIC), University of Copenhagen

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© Sørensen et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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