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. 1999 Dec;73(12):10472-9.
doi: 10.1128/JVI.73.12.10472-10479.1999.

Introduction of a cis-acting mutation in the capsid-coding gene of moloney murine leukemia virus extends its leukemogenic properties

M Audit  1 , J Déjardin, B Hohl, C Sidobre, T J Hope, M Mougel, M Sitbon
Affiliations

Introduction of a cis-acting mutation in the capsid-coding gene of moloney murine leukemia virus extends its leukemogenic properties

M Audit et al. J Virol. 1999 Dec.

Abstract

Inoculation of newborn mice with the retrovirus Moloney murine leukemia virus (MuLV) results in the exclusive development of T lymphomas with gross thymic enlargement. The T-cell leukemogenic property of Moloney MuLV has been mapped to the U3 enhancer region of the viral promoter. However, we now describe a mutant Moloney MuLV which can induce the rapid development of a uniquely broad panel of leukemic cell types. This mutant Moloney MuLV with synonymous differences (MSD1) was obtained by introduction of nucleotide substitutions at positions 1598, 1599, and 1601 in the capsid gene which maintained the wild-type (WT) coding potential. Leukemias were observed in all MSD1-inoculated animals after a latency period that was shorter than or similar to that of WT Moloney MuLV. Importantly, though, only 56% of MSD1-induced leukemias demonstrated the characteristic thymoma phenotype observed in all WT Moloney MuLV leukemias. The remainder of MSD1-inoculated animals presented either with bona fide clonal erythroid or myelomonocytic leukemias or, alternatively, with other severe erythroid and unidentified disorders. Amplification and sequencing of U3 and capsid-coding regions showed that the inoculated parental MSD1 sequences were conserved in the leukemic spleens. This is the first report of a replication-competent MuLV lacking oncogenes which can rapidly lead to the development of such a broad range of leukemic cell types. Moreover, the ability of MSD1 to transform erythroid and myelomonocytic lineages is not due to changes in the U3 viral enhancer region but rather is the result of a cis-acting effect of the capsid-coding gag sequence.

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Figures

FIG. 1
FIG. 1
A replication-competent mutant of Moloney MuLV (MSD1) with synonymous differences in the capsid-coding gene. (A) Schematic organization of the MuLV genome and DNA sequence of the mutated capsid region. The potential amino acid coding ability, common to WT Moloney MuLV and MSD1, is indicated in italics below the nucleotide sequences. Nucleotides are numbered starting from the first nucleotide of R in the 5′ LTR. (B) NIH 3T3 cell extracts producing WT Moloney MuLV or the MSD1 mutant were immunoblotted with the anticapsid R187 monoclonal antibody (7). Also shown are mock-infected cell extracts, and positions of molecular weight markers are indicated. The band corresponding to the cleaved capsid p30 as well as those corresponding to the Pr65 Gag and Pr75 glyco-Gag precursors was detected at the same position and at similar levels in cells infected with either of the two viruses.
FIG. 1
FIG. 1
A replication-competent mutant of Moloney MuLV (MSD1) with synonymous differences in the capsid-coding gene. (A) Schematic organization of the MuLV genome and DNA sequence of the mutated capsid region. The potential amino acid coding ability, common to WT Moloney MuLV and MSD1, is indicated in italics below the nucleotide sequences. Nucleotides are numbered starting from the first nucleotide of R in the 5′ LTR. (B) NIH 3T3 cell extracts producing WT Moloney MuLV or the MSD1 mutant were immunoblotted with the anticapsid R187 monoclonal antibody (7). Also shown are mock-infected cell extracts, and positions of molecular weight markers are indicated. The band corresponding to the cleaved capsid p30 as well as those corresponding to the Pr65 Gag and Pr75 glyco-Gag precursors was detected at the same position and at similar levels in cells infected with either of the two viruses.
FIG. 2
FIG. 2
Latency of leukemia in mice inoculated as newborns with either WT Moloney MuLV or the MSD1 mutant. Newborn Swiss mice were inoculated intraperitoneally with 50 μl of either WT Moloney MuLV, MSD1 mutant, or Friend MuLV viral stocks, containing between 50 and 500 FFU each. Animals were regularly monitored for gross organ enlargement and hematocrit changes. The number of animals used in each series is indicated in parentheses. Latency is defined as the time at which 50% of the animals were either dead or presented with spleen, thymus, or lymph nodes grossly enlarged.
FIG. 3
FIG. 3
Staining for erythroid and myelomonocytic cells in smears from leukemic spleens. Suspensions of splenocytes were stained for nonspecific esterase activity (red cells in the upper panels) and Sudan black-positive cytosolic grains (examples shown with white arrowheads in the lower panels). These colorations allowed the identification of abnormal erythroid and myelomonocytic proliferations, respectively. (A) Prototypic WT Moloney MuLV-induced T lymphoma; (B) MSD1-induced pure T lymphoma; (C) MSD1-induced erythroleukemia; (D) MSD1-induced mixed erythroid and myelomonocytic leukemia; and (E) prototypic Friend MuLV-induced erythroleukemia.
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