Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs.
The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex mRNA compared to pX-tax-orfII and pX-rex-orfI mRNAs, but did not affect exon skipping. Ectopic expression of hnRNP A1 did not affect pX splice site utilization, but increased exon skipping, as the level of pX-p21rex mRNA was increased by almost 10-fold.
Princler
Gerald L
GL
Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.
Julias
John G
JG
Hughes
Stephen H
SH
Derse
David
D
D017398:Alternative Splicing; D002460:Cell Line; D002461:Cell Line, Transformed; D018076:DNA, Complementary / Q000235:genetics; D016370:Gene Products, rex / Q000235:genetics / Q000378:metabolism; D016356:Gene Products, tax / Q000235:genetics / Q000378:metabolism; D016355:Genes, pX; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011533:Proviruses; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D013601:T-Lymphocytes / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D016370:Gene Products, rex; D016356:Gene Products, tax; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D000068103:Serine-Arginine Splicing Factors