Sørensen Annette Balle, Lund Anders H, Kunder Sandra, Quintanilla-Martinez Leticia, Schmidt Jörg, Wang Bruce, Wabl Matthias, Pedersen Finn Skou
Department of Molecular Biology, University of Aarhus, Denmark.
Retrovirology. 2007 Jul 6;4:46. doi: 10.1186/1742-4690-4-46.
Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus.
By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.
We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.
先前已表明，位于gag区域内的一个可变剪接受体位点的突变会扩大致T淋巴细胞瘤的γ逆转录病毒莫洛尼鼠白血病病毒的致病潜力，而在致红白血病的弗氏鼠白血病病毒中的等效突变似乎对该病毒的致病潜力没有影响。在本研究中，我们研究了可变剪接模式以及突变可变剪接位点对致B淋巴细胞瘤的Akv鼠白血病病毒致癌特性的可能影响。
通过外显子捕获程序，我们鉴定出一个新的γ逆转录病毒外显子，它是由位于B细胞淋巴瘤致瘤性Akv鼠白血病病毒gag衣壳区域的可变剪接受体（SA'）和剪接供体（SD'）位点的使用产生的。为了分析这个新外显子在体内的可能影响，构建了三种不同的可变剪接位点突变病毒，分别在SA'、SD'或两个位点发生突变，并将其注射到新生近交NMRI小鼠体内。大多数受感染的小鼠（约90%）在250天内发生造血肿瘤，对肿瘤的组织学检查表明，引入的同义gag突变对诱导肿瘤的表型有显著影响，改变了不同类型肿瘤的分布，并产生了额外特异性的肿瘤，如新发弥漫性大B细胞淋巴瘤（DLBCL）和组织细胞肉瘤。有趣的是，与野生型和双剪接位点突变体相比，两个单剪接位点突变体的诊断范围更广。单剪接和双剪接转录本在体内均使用SA'和/或SD'位点产生，但观察到的对肿瘤发生影响的潜在机制仍有待阐明。同样，对肿瘤组织中前病毒整合位点的分析，鉴定出111个新的逆转录病毒整合位点（RIS）和35个新的共同整合位点（CIS），但并未明确指出与特定肿瘤诊断或单个病毒突变体相关的特定靶基因或途径。
我们在此展示了γ逆转录病毒组中双剪接转录本的首个实例，并表明定义这种新RNA产物的可变剪接位点的突变改变了Akv鼠白血病病毒的致癌潜力。
