The activated Mlvi-4 locus in Moloney murine leukemia virus-induced rat T-cell lymphomas encodes an env/Mlvi-4 fusion protein

Abstract

A genomic DNA probe derived from the region immediately 3' of the clusters of integrated proviruses in the Mlvi-4 locus detects a 5.5-kb mRNA transcript which is specifically expressed in normal rat thymus and spleen. The same probe detects two tumor-specific mRNA transcripts 2.5 and 10 kb long, both of which are expressed only in tumors carrying a provirus in the Mlvi-4 locus. Sequence analysis of two cDNA clones (LE3a and B1.1) of the 2.5-kb tumor-specific mRNA, obtained from two independent tumors (6889 and B1), revealed that they are both derived from hybrid env/Mlvi-4 mRNA transcripts. The splicing of env to Mlvi-4 sequences linked a cryptic splice donor site at nucleotide position 6397 of the viral genome with a splice acceptor site in the region immediately 3' of the integrated provirus. The mRNA that gives rise to cDNA clone B1.1 terminates 1,005 bases 3' of the splice acceptor site without additional splicing. The mRNA that gives rise to cDNA clone LE3a terminates in the same site but undergoes differential splicing of an 81-base-long intron. The resulting mRNAs contain 247-amino-acid (clone B1.1) or 226-amino-acid (clone LE3a) open reading frames sharing 221 N-terminal amino acids, of which 207 are derived from the viral env gene and 14 are derived from Mlvi-4. RNase protection assays using 6889 tumor cell RNA and a probe derived from the cDNA clone LE3a detected both mRNA transcripts. More abundant of the two, however, was the one encoding the putative 247-amino-acid protein. Transient transfections of a construct expressing the RNA transcript defined by clone B1.1 into D17 cells led to the expression of an Env/Mlvi-4 fusion protein with an apparent molecular mass of 33 kDa. Given that cells with provirus insertions in the Mlvi-4 locus are selected and that retroviral env gene products may have profound effects in the biology of hematopoietic cells, we suggest that the detected fusion proteins may contribute to the growth of T-cell lymphomas.