Lund A H, Schmidt J, Luz A, Sørensen A B, Duch M, Pedersen F S
Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
J Virol. 1999 Jul;73(7):6117-22. doi: 10.1128/JVI.73.7.6117-6122.1999.
Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta-chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant.
逆转录病毒的逆转录由一个与18碱基对引物结合位点序列退火的细胞tRNA分子引发。引物结合位点的序列与一个负向作用的顺式元件的序列一致，该元件介导未分化胚胎细胞中小鼠白血病病毒（MLV）的转录沉默。在本研究中，我们测试了SL3-3 MLV是否可以使用同源tRNAPro以外的tRNA引物稳定复制，并在小鼠致病性模型中分析改变引物结合位点序列以匹配tRNA1Gln、tRNA3Lys或tRNA1,2Arg的3'末端的效果。与对引物结合位点修饰的1型人类免疫缺陷病毒和禽逆转录病毒的细胞培养研究结果相反，我们的发现是SL3-3 MLV可能使用tRNAPro以外的tRNA引物稳定且高效地复制。尽管SL3-3 Lys3突变体诱导淋巴瘤的时间相对于野生型病毒显著延迟，但分子肿瘤分析表明，就T细胞受体β链、免疫球蛋白κ轻链和c-myc基因座内基因组重排的频率而言，所有引物结合位点修饰的病毒诱导的T细胞淋巴瘤与野生型病毒诱导的相似。虽然没有发现任何突变体恢复使用tRNAPro引物，但在注射SL3-3 Lys3突变体产生的两个肿瘤中，引物结合位点发生了改变，以匹配一种新的引物种类tRNA1,2Lys的位点。此外，在由SL3-3 Lys3突变体诱导的大多数肿瘤以及由野生型SL3-3和SL3-3 Arg1,2突变体诱导的两个肿瘤中，检测到与内源性病毒的重组，导致产生携带谷氨酰胺引物结合位点的重组病毒。
