Exploring the contribution of distal P4 promoter elements to the oncoselectivity of Minute Virus of Mice.
Exploring the contribution of distal P4 promoter elements to the oncoselectivity of Minute Virus of Mice.
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DOI：
10.1016/j.virol.2006.11.006
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发表时间：
2007-04
期刊：
影响因子：
3.7
通讯作者：
J. Paglino;E. Burnett;P. Tattersall
中科院分区：
文献类型：
--
作者：
J. Paglino;E. Burnett;P. Tattersall
小鼠微小病毒（MVM）与细小病毒属的其他成员具有固有的致瘤特性。两个元素负责，至少部分地，这种肿瘤选择性已被映射到一个Ets1结合位点附近的启动子，P4的P4 TATA盒，和一个更远的环AMP响应元件（CRE），位于端粒发夹茎。在这里，CRE重叠一个半位点的结合细小病毒起始因子（PIF），这是必不可少的病毒DNA复制。我们使用了一种简并寡核苷酸选择方法，以表明CRE结合蛋白（CREB）选择的序列ACGTCAC在这种情况下，而不是其更普遍接受的回文TGACGTCA识别位点。我们已经开发出直接在MVM感染性克隆的左端回文序列内操纵这些序列的策略，并使用它们来克隆其CRE与对称共有序列匹配或被打乱的突变体，或者其中PIF结合位点相对于CRE逐渐减弱的突变体。使用差异实时定量PCR测定，通过在共感染培养物中的多轮生长，在正常鼠成纤维细胞A9或转化的人成纤维细胞324 K中测试突变体组相对于野生型的适应性。我们证实，灭活CRE基本上废除了肿瘤选择性，但发现，提高其适合回文共识是有点衰弱的细胞类型。我们还证实，减少一个碱基对的PIF半位点间距增强肿瘤选择性，但发现进一步的碱基对删除显着降低这种效果。
Minute Virus of Mice (MVM) shares inherent oncotropic properties with other members of the genus Parvovirus. Two elements responsible, at least in part, for this oncoselectivity have been mapped to an Ets1 binding site adjacent to the P4 TATA box of the initiating promoter, P4, and to a more distal cyclic AMP responsive element (CRE), located within the telomeric hairpin stem. Here the CRE overlaps one half-site for the binding of parvoviral initiation factor (PIF), which is essential for viral DNA replication. We used a degenerate oligonucleotide selection approach to show that CRE binding protein (CREB) selects the sequence ACGTCAC within this context, rather than its more generally accepted palindromic TGACGTCA recognition site. We have developed strategies for manipulating these sequences directly within the left-end palindrome of the MVM infectious clone and used them to clone mutants whose CRE either matches the symmetric consensus sequence or is scrambled, or in which the PIF binding site is incrementally weakened with respect to the CRE. The panel of mutants were tested for fitness relative to wildtype in normal murine fibroblasts A9 or transformed human fibroblasts 324 K, through multiple rounds of growth in co-infected cultures, using a differential real-time quantitative PCR assay. We confirmed that inactivating the CRE substantially abrogates oncoselectivity, but found that improving its fit to the palindromic consensus is somewhat debilitating in either cell type. We also confirmed that reducing the PIF half-site spacing by one basepair enhances oncoselectivity, but found that a further basepair deletion significantly reduces this effect.