Adelman K, Salmon B, Baines J D
Department of Microbiology and Immunology, Cornell University, VMC C5-164, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3086-91. doi: 10.1073/pnas.061555698.
The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of U(L)28 in this process, purified U(L)28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for U(L)28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by U(L)28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by U(L)28 protein. To determine the relevance of the observed U(L)28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of U(L)28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the U(L)28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U(L)28-encoded component of the herpesvirus cleavage and packaging machinery.
单纯疱疹病毒1型U(L)28基因的产物对于将串联体病毒DNA切割成基因组长度单位并将该DNA包装到病毒原衣壳中至关重要。为了研究U(L)28在此过程中的作用，对纯化的U(L)28蛋白识别保守疱疹病毒DNA包装序列的能力进行了检测。我们报告称，含有pac1 DNA包装基序的DNA片段可通过热处理诱导形成新的DNA构象，在非变性凝胶中其迁移速度比相应的双链DNA更快。令人惊讶的是，这些新的DNA结构是U(L)28蛋白结合的高亲和力底物，而相同序列组成的双链DNA则不被U(L)28蛋白识别。我们证明，pac1基序中只有一条链负责形成能被U(L)28蛋白紧密且特异性结合的新DNA结构。为了确定观察到的U(L)28蛋白与pac1的相互作用与切割和包装过程的相关性，我们分析了U(L)28蛋白对先前已证明在体内切割和包装存在缺陷的pac1突变体的结合亲和力。每个pac1突变体均表现出U(L)28蛋白与DNA结合能力的下降，这与报道的切割和包装效率降低直接相关，从而支持了U(L)28蛋白与pac1的相互作用在体内的作用。因此，这些数据表明，pac1基序形成的新DNA结构赋予了疱疹病毒切割和包装机制中由U(L)28编码的组分识别DNA包装序列额外的特异性。
