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. 2005 Jan;79(1):57-66.
doi: 10.1128/JVI.79.1.57-66.2005.

Substitution of feline leukemia virus long terminal repeat sequences into murine leukemia virus alters the pattern of insertional activation and identifies new common insertion sites

Chassidy Johnson  1 , Patricia A Lobelle-Rich, Adriane Puetter, Laura S Levy
Affiliations

Substitution of feline leukemia virus long terminal repeat sequences into murine leukemia virus alters the pattern of insertional activation and identifies new common insertion sites

Chassidy Johnson et al. J Virol. 2005 Jan.

Abstract

The recombinant retrovirus, MoFe2-MuLV (MoFe2), was constructed by replacing the U3 region of Moloney murine leukemia virus (M-MuLV) with homologous sequences from the FeLV-945 LTR. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. MoFe2 integrated infrequently (0 to 9%) near common insertion sites (CISs) previously identified for either parent virus. Using three different strategies, CISs in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parent virus in wild-type animals. Two of the newly identified CISs had not previously been implicated in lymphoma in any retrovirus model. One of these, designated 3-19, encodes the p101 regulatory subunit of phosphoinositide-3-kinase-gamma. The other, designated Rw1, is predicted to encode a protein that functions in the immune response to virus infection. Thus, substitution of FeLV-945 U3 sequences into the M-MuLV long terminal repeat (LTR) did not alter the target tissue for M-MuLV transformation but significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma. These observations support a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. The findings also demonstrate the oligoclonal nature of retrovirus-induced lymphomas by demonstrating proviral insertions at CISs in subdominant populations in the tumor mass. Finally, the findings demonstrate the utility of novel recombinant retroviruses such as MoFe2 to contribute new genes potentially relevant to the induction of lymphoid malignancy.

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Figures

FIG. 1.
FIG. 1.
Flow cytometric analysis of MoFe2-induced thymic lymphomas. Surface expression of CD4 and CD8 on three tumors was examined by flow cytometry. The results demonstrate CD4− CD8− (A), mixed tumor (CD4− CD8− and CD4+ CD8−) (B), and CD4+ CD8+ (C) surface phenotypes.
FIG. 2.
FIG. 2.
Southern blot analysis of DNA from MoFe2-induced lymphoma of animal MF8. (A) Genomic DNA (8 μg) from lymphoma in the spleen (S), in the thymus (T), and from uninvolved control tissue of the same animal (C) was digested with HpaI and hybridized to radiolabeled probe for the TCRβ locus. The closed circles indicate the germ line configuration of the TCRβ locus. (B) Genomic DNA samples (8 μg) from lymphomas in the spleen (S), thymus (T), and lymph node (LN) of animal MF8 were digested with KpnI and hybridized to rLTR1, a probe specific for the integrated provirus of MoFe2-MuLV. The open circle indicates a clonal host-virus junction fragment that distinguishes the thymic tumor from tumors in other tissue sites of the same animal.
FIG. 3.
FIG. 3.
Southern blot analysis of the MF8T locus in MoFe2-induced lymphomas. DNA was digested with HindIII and hybridized to the MF8T-HVJ probe. Shown are DNA samples from six tumors with insertions in the MF8T locus, indicated by the animal number and tissue origin of the tumor (T, thymus; S, spleen; K, kidney). The arrow indicates the germ line configuration of the locus.
FIG. 4.
FIG. 4.
Physical map of the MF8T locus. Depicted is the 3.9-kb domain of common proviral insertion. Vertical lines represent the positions of proviral integrations (indicated by animal number) with the transcriptional orientation of each provirus depicted by the direction of the arrow. Solid lines represent insertions detectable by Southern blot analysis. Checkered lines represent insertions undetectable by Southern blot analysis but identified by PCR amplification. Oligonucleotide primers used in PCR to amplify host-virus junctions are illustrated as horizontal arrowheads. The positions and transcriptional directions of the Spred-1 and Rasgrp1 genes are indicated.
FIG. 5.
FIG. 5.
Northern blot analysis of Rasgrp1 expression in tumors containing (MF8T+) or lacking (MF8T−) proviral insertion at MF8T. Total RNA (10 μg) was examined from lymphomas indicated by animal number and by the anatomic origin of the tumor (T, thymus) and also from normal tissues of uninfected NIH/Swiss mice (B, brain; K, kidney). Northern blots were hybridized to a probe specific for Rasgrp1 exons 9 to 16 and were subsequently hybridized to a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe as a loading control. Size of the observed transcript is indicated.
FIG. 6.
FIG. 6.
Southern blot analysis of the 3-19 locus in MoFe2-induced lymphomas. Shown are DNA samples from five lymphomas indicated by the animal number and anatomic origin of the tumor (T, thymus; S, spleen; LN, lymph node) and from an uninfected NIH/Swiss mouse (C). DNA was digested with EcoRI (477-4, -11, and -18) or KpnI (493-3 and 477-2) and hybridized to a radiolabeled probe representing the 3-19 locus. The arrow represents the germ line configuration of the locus.
FIG. 7.
FIG. 7.
Physical map of the 3-19 locus. Depicted is the 5.5-kb domain of common proviral insertion. Vertical lines represent the positions of proviral integrations (indicated by animal number) with the transcriptional orientation of each provirus depicted by the direction of the arrow. Solid lines represent insertions detectable by Southern blot analysis. Checkered lines represent insertions undetectable by Southern blot analysis but identified by PCR amplification. Oligonucleotide primers used in PCR to amplify host-virus junctions are illustrated as horizontal arrowheads. The positions and transcriptional directions of the Netrin-1, p101, and Q8K323 genes are indicated.
FIG. 8.
FIG. 8.
Northern blot analysis of p101 expression in tumors containing (3-19+) or lacking (3-19−) proviral insertion at 3-19. Total RNA (10 μg) was examined from lymphomas indicated by animal number and by the anatomic origin of the tumor (T, thymus; S, spleen; LN, lymph node). Northern blots were hybridized to a probe specific for p101 exon 10. The membranes were subsequently hybridized to a GAPDH probe as a loading control. The sizes of the observed transcripts are indicated.
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