A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA.
A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA.
复制标题
双发夹结构对于脾坏死病毒逆转录病毒RNA的有效包壳是必要的。
DOI：
10.1002/j.1460-2075.1994.tb06311.x
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Temin,HM
中科院分区：
文献类型：
--
作者：
Yang,S;Temin,HM
我们在先前定义的脾坏死病毒（SNV）（一种禽类逆转录病毒）的突变序列（E）内进行了突变分析。我们发现SNV的高效复制需要两个区域。第一个区域是双发夹结构，如Konings等（1992，J.Virol.，66，632 - 640）;第二区域位于发夹的下游。我们进一步表明，双发夹结构是有效的SNV RNA包埋所必需的。我们的工作是第一个证明，通过接头扫描和定点诱变，一个特定的RNA二级结构是需要逆转录病毒RNA的构象。对E区域内的一系列突变的分析表明：（i）保留两个发夹的二级结构对于有效的双糖苷化是重要的，以及（ii）发夹的茎区含有对双糖苷化至关重要的特异性序列。在发夹中，两个保守的GACG四残基环中至少有一个的存在，而不是第一个发夹的适度保守的凸起序列，对功能至关重要。发夹的功能与两个发夹的相对顺序无关。然而，这两个发夹不是多余的，并且在功能上不相同。用莫洛尼鼠白血病病毒（M-MLV）的双发夹序列替换SNV双发夹序列对基于SNV的逆转录病毒载体与网状内皮组织增生病病毒株A（REV-A）辅助病毒的复制没有可检测到的影响。此外，用莫洛尼鼠肉瘤病毒（M-MSV）和M-MLV的整个E序列替换SNV的整个E序列导致逆转录病毒载体与具有野生型SNV E的SNV载体一样复制。该结果表明，M-MSV/M-MLV和SNV的糖苷化序列不是病毒特异性的，并且在SNV和MLV RNA与来自REV-A的病毒蛋白包装期间，糖苷化序列主要通过其二级或三级结构识别。
We conducted a mutational analysis within the previously defined encapsidation sequence (E) for spleen necrosis virus (SNV), an avian retrovirus. We found that two regions are necessary for efficient SNV replication. The first region is a double hairpin structure as proposed by Konings et al. (1992, J. Virol., 66, 632‐640); the second region is located downstream of the hairpins. We showed further that the double hairpin structure is required for efficient SNV RNA encapsidation. Our work is the first to demonstrate, via linker‐scanning and site‐directed mutagenesis, that a specific RNA secondary structure is required for the encapsidation of retroviral RNA. Analysis of a series of mutations within the E region indicates (i) that preserving the secondary structure of the two hairpins is important for efficient encapsidation and (ii) that the stem regions of the hairpins contain specific sequences critical for encapsidation. Within the hairpins, the presence of at least one of the two conserved GACG four‐residue loops, but not the moderately conserved bulge sequence of the first hairpin, is crucial for function. The function of the hairpins is independent of the relative order of the two hairpins. However, the two hairpins are not redundant and are not functionally identical. Replacement of SNV double hairpin sequences with those of Moloney murine leukemia virus (M‐MLV) has no detectable effect on the replication of SNV‐based retrovirus vectors with reticuloendotheliosis virus strain A (REV‐A) helper virus. Furthermore, replacement of the entire E sequence of SNV with that of Moloney murine sarcoma virus (M‐MSV) and M‐MLV results in retroviral vectors that replicate as well as SNV vectors with wild type SNV E. This result indicates that the encapsidation sequences of M‐MSV/M‐MLV and SNV are not virus specific and that, during packaging of SNV and MLV RNA with viral proteins from REV‐A, the encapsidation sequences are recognized largely by their secondary or tertiary structures.