Introduction of acis-Acting Mutation in the Capsid-Coding Gene of Moloney Murine Leukemia Virus Extends Its Leukemogenic Properties

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 acis-acting effect of the capsid-coding gagsequence.

A new retroelement constituted by a natural alternatively spliced RNA of murine replication-competent retroviruses

“…The spreading of such SD′ retroelement derived from alternative usage of splice donors is likely to bear consequences on dissemination and pathogenesis of MLV retroviruses. Indeed, we showed in a previous work that inactivation of the SD′ site leads to extensive modifications of MLV leukemogenic properties (Audit et al ., 1999). MLV and other simple oncoretroviruses are implicated in leukemogenic processes through insertional mutagenesis mechanisms, where provirus DNA integrates into the host genome and affects the transcription of the neighbouring genes.…”

“…Protein analyses were conducted from cells cultured on 10 cm plates. Extractions of cellular and viral proteins and western blot analysis were performed as previously described (Mougel et al ., 1996; Audit et al ., 1999). Proteins were detected by immunoblotting with a mouse anti‐capsid (p30) monoclonal antibody (H187) (a kind gift from B.Chesebro) used at a 1:300 dilution as the primary antibody and a peroxidase‐conjugated (HRP) goat anti‐mouse antibody (1:2000) as the secondary antibody (Sigma), or with a mouse anti‐matrix (p15) monoclonal antibody (H690) (a kind gift from M.Miyazawa) used at a 1:5 dilution as the primary antibody and an HRP goat anti‐mouse antibody (1:1000) as the secondary antibody (Sigma), or with a rabbit anti‐Pol polyclonal antibody (a kind gift from M.Roth) used at a 1:2000 dilution and an HRP goat anti‐rabbit used at a 1:4000 dilution, or with a rabbit anti‐integrase (p45) polyclonal antibody (a kind gift from F.D.Bushman) used at a 1:2000 dilution as the primary antibody and a peroxidase‐conjugated goat anti‐rabbit antibody (1:2000) as the secondary antibody, or with an anti‐envelope polyclonal antibody, anti‐gp70 of Raüscher (ViroMED) used at a 1:1000 and a peroxidase‐conjugated goat anti‐mouse antibody (1:2000) as the secondary antibody.…”

“…We also reported an SD′‐suppression mutation which extends Moloney‐MLV leukemogenic properties in vivo . Thus, while wt Moloney MLV induces exclusive development of T lymphomas when inoculated in newborn mice, SD′‐defective Moloney MLV leads to the rapid development of a broad panel of leukemic cell types, including cells from the thymic, erythroid and myelomonocytic lineages (Audit et al ., 1999).…”

Murine leukemia virus (MLV) P50 protein induces cell transformation via transcriptional regulatory function

“…Studies in vivo revealed that inoculation of a Mo-MLV mutant (MSD1/M1) harboring an inactive SD’ site in newborn Swiss mice changes the MLV leukemogenic properties [ 15 ]. Indeed, whereas WT MLV exclusively induces T lymphoma, the MSD1/M1 mutant can induce a broad panel of leukemia (T lymphoma, erythroleukemia, myelomonocytic leukemia and other leukemia with undetermined origin).…”

Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging

“…1). SD' mutant viruses replicated more slowly than wild type viruses and they induced altered tumorigenesis in mice (28) (30,31). SD' RNA is specifically incorporated into virions and two viral proteins, P50, which uses the bona fide Gag start codon and P60, which initiates from the CUG codon used by glycoGag and is expressed at lower levels than P50, are encoded by this alternatively spliced RNA (32) (Fig.…”