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. 1999 Sep;73(9):7175-84.
doi: 10.1128/JVI.73.9.7175-7184.1999.

Tandemization of a subregion of the enhancer sequences from SRS 19-6 murine leukemia virus associated with T-lymphoid but not other leukemias

S W Granger  1 , L M Bundy, H Fan
Affiliations

Tandemization of a subregion of the enhancer sequences from SRS 19-6 murine leukemia virus associated with T-lymphoid but not other leukemias

S W Granger et al. J Virol. 1999 Sep.

Abstract

Most simple retroviruses induce tumors of a single cell type when infected into susceptible hosts. The SRS 19-6 murine leukemia virus (MuLV), which originated in mainland China, induces leukemias of multiple cellular origins. Indeed, infected mice often harbor more than one tumor type. Since the enhancers of many MuLVs are major determinants of tumor specificity, we tested the role of the SRS 19-6 MuLV enhancers in its broad disease specificity. The enhancer elements of the Moloney MuLV (M-MuLV) were replaced by the 170-bp enhancers of SRS 19-6 MuLV, yielding the recombinants DeltaMo+SRS(+) and DeltaMo+SRS(-) M-MuLV. M-MuLV normally induces T-lymphoid tumors in all infected mice. Surprisingly, when neonatal mice were inoculated with DeltaMo+SRS(+) or DeltaMo+SRS(-) M-MuLV, all tumors were of T-lymphoid origin, typical of M-MuLV rather than SRS 19-6 MuLV. Thus, the SRS 19-6 MuLV enhancers did not confer the broad disease specificity of SRS 19-6 MuLV to M-MuLV. However, all tumors contained DeltaMo+SRS M-MuLV proviruses with common enhancer alterations. These alterations consisted of tandem multimerization of a subregion of the SRS 19-6 enhancers, encompassing the conserved LVb and core sites and adjacent sequences. Moreover, when tumors induced by the parental SRS 19-6 MuLV were analyzed, most of the T-lymphoid tumors had similar enhancer alterations in the same region whereas tumors of other lineages retained the parental SRS 19-6 MuLV enhancers. These results emphasize the importance of a subregion of the SRS 19-6 MuLV enhancer in induction of T-cell lymphoma. The relevant sequences were consistent with crucial sequences for T-cell lymphomagenesis identified for other MuLVs such as M-MuLV and SL3-3 MuLV. These results also suggest that other regions of the SRS 19-6 MuLV genome contribute to its broad leukemogenic spectrum.

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Figures

FIG. 1
FIG. 1
Generation of the ΔMo+SRS LTRs. (A) The DNA sequences deleted from positions −150 to −357 in the M-MuLV LTR to give the ΔMo LTR are shown (24). The 170-bp DNA fragment containing the SRS enhancer sequences (positions −298 to −125) was PCR amplified and cloned into the ΔMo LTR in either orientation to give the ΔMo+SRS+ and ΔMo+SRS− LTRs. The PstI restriction endonuclease sites used to discriminate enhancer orientation are shown. The downstream PstI site is in the 5′ M-MuLV sequences. wt, wild type. (B) The organization of the pΔMo+SRS+ M-MuLV plasmid is shown, with the chimeric LTR only in the downstream position. The internal viral sequences are all derived from M-MuLV.
FIG. 2
FIG. 2
Pathogenicity of ΔMo+SRS M-MuLVs. Neonatal NIH Swiss mice were inoculated intraperitoneally with ΔMo+SRS+ (14 animals) and ΔMo+SRS− M-MuLVs (7 animals) as described in Materials and Methods. The time course to death is shown. For comparison, mortality plots for animals inoculated with wild-type M-MuLV and SRS 19-6 MuLV are shown.
FIG. 3
FIG. 3
Southern blot analysis of ΔMo+SRS M-MuLV-induced tumors. High-molecular-weight DNAs from ΔMo+SRS M-MuLV-induced tumors were digested with PstI, which cleaves asymmetrically within the SRS enhancer fragment. (A) Hybridization with a labeled SRS enhancer-specific probe would yield a diagnostic 839-bp hybridizing fragment for ΔMo+SRS+ M-MuLV and a 757-bp fragment for ΔMo+SRS− M-MuLV. (B) Southern blots of the tumors all show fragments of the expected size when hybridized with an SRS 19-6 MuLV enhancer probe. Larger hybridizing fragments presumably represent endogenous MuLV sequences (also present in control spleen DNA) or host-virus junction fragments from the downstream LTR.
FIG. 4
FIG. 4
PCR analysis of proviral enhancers in ΔMo+SRS M-MuLV-induced tumors. (A) A diagram of the ΔMo+SRS+ or ΔMo+SRS− M-MuLV LTRs is shown, along with the locations of the oligonucleotide primers used to amplify the proviral LTRs from tumor DNAs. (B) PCR products from several different ΔMo+SRS M-MuLV-induced tumors were analyzed by agarose gel electrophoresis (2% agarose) and stained with ethidium bromide. Plasmids containing either wild-type (wt) M-MuLV or input ΔMo+SRS M-MuLV DNA provided size marker controls for the PCR amplification products, as shown to the left of the gel. Although some tumor DNAs yielded PCR fragments of the expected size, all tumors gave one or more enhancer-specific fragment of increased size. (C) Southern blot hybridization with an SRS enhancer-specific probe of a gel similar to the one in panel B is shown.
FIG. 5
FIG. 5
Southern blot analysis of ΔMo+SRS M-MuLV LTRs in tumor DNAs. Tumor DNAs were digested with NheI and SpeI and analyzed by Southern blot hybridization with an SRS enhancer-specific probe. NheI recognizes a site in the U3 region of M-MuLV 5′ of the inserted SRS sequences, and SpeI recognizes a site in the 5′ noncoding region 282 bp downstream from the U3-R junction (Fig. 4A). A diagnostic fragment of 680 bp was indicative of input ΔMo+SRS M-MuLV proviral DNA. The sizes of SRS-hybridizing fragments detected in this analysis corresponded to the fragment sizes detected by PCR amplification (Fig. 4). Fragments corresponding to duplications and triplications of the SRS enhancer sequences are indicated (2× and 3×, respectively). Hybridizing fragments migrating more slowly than the enhancer triplications might have represented virus-host junction fragments originating from the downstream LTRs.
FIG. 6
FIG. 6
Altered SRS enhancer regions amplified from ΔMo+SRS M-MuLV-induced tumors. PCR products of tumors similar to those shown in Fig. 4 were cloned and sequenced. The sequences were aligned with the input SRS enhancer sequences shown at the top of the figure. Binding sites for sequence-specific DNA binding proteins are included (6). The asterisk indicates a G residue that is not present in the standard core motif; it is an A residue in the M-MuLV enhancers. The sequences shown for the different PCR clones indicate sequences that were tandemly duplicated or triplicated. For 498-7S clone 4, the alteration consisted of a deletion (… …) in the NF-1 sequences. Two clones with different sequences were obtained for tumors 498-1S, 498-7S, and 501-2S. Otherwise, the sequences shown represent the predominant PCR products from the respective tumors. Tumors 498-1S, 498-3S, and 498-7S were induced by M-MuLV+ M-MuLV, and tumors 501-1S and 501-2S were induced by ΔMo+SRS+ M-MuLV. A specific region of the SRS enhancers was amplified in at least one of the proviruses in each tumor. The minimum size of the region of common amplification is indicated by the dotted box. This portion of the enhancers contains the highly conserved NF-1, LVb motifs, and core motif that differs from M-MuLV at the nucleotide indicated in the figure.
FIG. 7
FIG. 7
Proviral LTRs in tumors induced by parental SRS 19-6 MuLV. The same LTR analysis as in Fig. 5B was applied to tumor DNAs induced by wild-type (wt) SRS 19-6 MuLV. The location of the PCR primers is shown at the top of the figure. Abbreviations: BLL, B-cell lymphoblastic lymphoma; AML, acute myelogenous leukemia; TLL, T-cell lymphoblastic lymphoma; EL, erythroid leukemia; ∗, from the same animal. LTR alterations were detected in four of the six T-lymphoid tumors induced by wild-type SRS 19-6 MuLV, but in none of the tumors that did not involve TLL.
FIG. 8
FIG. 8
SRS enhancer regions amplified in wild-type SRS 19-6 MuLV-induced tumors. The novel sized enhancer-specific PCR products shown in Fig. 7 were cloned and sequenced and are displayed here. The analogous region to that in ΔMo+SRS M-MuLV-induced tumors was amplified in T-lymphoid tumors induced by wild-type SRS 19-6 MuLV.
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