Intracellular Cre-Mediated Deletion of the Unique Packaging Signal Carried by a Herpes Simplex Virus Type 1 Recombinant and Its Relationship to the Cleavage-Packaging Process

To gain further insight on the function of the herpes simplex virus type 1 (HSV-1) packaging signal (a sequence), we constructed a recombinant virus containing a unique a sequence, which was flanked by two loxP sites in parallel orientation. The phenotype of this recombinant, named HSV-1 LaL, was studied in cell lines which either express or do not express Cre recombinase. Although LaL virus multiplication was only slightly reduced in standard cell lines, its growth was strongly inhibited in Cre-expressing cells. In these cells, a sequences were detected mostly in low-molecular-weight DNA circles, indicating that they had been excised from virus DNA by site-specific recombination. Deletion of the a sequences from the viral genome resulted in the accumulation of uncleaved replication intermediates, as observed by pulsed-field gel electrophoresis. B-type capsids also accumulated in these cells, as shown both by electron microscopy and by sucrose gradient sedimentation. Further examination of the status of a sequences in Cre-expressing cells indicated that high-level amplification of this sequence can occur in the absence of the cleavage-packaging process. Moreover, the amplified a signals in small circular DNA molecules remained uncleaved, indicating that these molecules were not able to efficiently interact with the cleavage-packaging machinery. The cleavage-packaging machinery and the structural proteins required to assemble virions were, however, functional in HSV-1 LaL-infected Cre-expressing cells, since this system could be used to package plasmid DNA harboring an origin of virus replication and one normal a signal. This is the first study in which accumulation both of uncleaved replication intermediates and of B capsids has been obtained in the presence of the full set of proteins required to package virus DNA.

为进一步深入解析1型单纯疱疹病毒（herpes simplex virus type 1, HSV-1）包装信号（packaging signal）的功能，我们构建了一株携带唯一a序列的重组病毒，该序列两侧以同向平行取向连有两个loxP位点（loxP sites）。我们将该重组病毒命名为HSV-1 LaL，并在表达或不表达Cre重组酶（Cre recombinase）的细胞系中对其表型展开研究。尽管LaL病毒在标准细胞系中的增殖仅出现轻微下降，但在表达Cre重组酶的细胞中，其生长受到显著抑制。在这类细胞中，a序列主要以低分子量DNA环状分子的形式被检出，表明其已通过位点特异性重组从病毒DNA中被切除。脉冲场凝胶电泳（pulsed-field gel electrophoresis）结果显示，将a序列从病毒基因组中缺失会导致未切割的复制中间体积累。电子显微镜（electron microscopy）与蔗糖梯度沉降（sucrose gradient sedimentation）实验均证实，此类细胞中还出现了B型衣壳的积累。对表达Cre重组酶的细胞内a序列状态的进一步研究表明，在缺失切割-包装过程的情况下，该序列仍可发生高水平扩增。此外，小型环状DNA分子中的扩增a信号仍未被切割，提示这类分子无法与切割-包装系统有效结合。不过，在感染HSV-1 LaL的表达Cre重组酶的细胞中，切割-包装系统与组装毒粒（virions）所需的结构蛋白均保持功能正常——因为该系统可用于包装携带病毒复制起点（origin of virus replication）与一段正常a信号的质粒DNA。本研究首次实现了在具备完整病毒DNA包装所需蛋白的条件下，同时观察到未切割的复制中间体与B型衣壳的积累。