Biswas N, Weller S K
Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
J Biol Chem. 1999 Mar 19;274(12):8068-76. doi: 10.1074/jbc.274.12.8068.
Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex that is composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins retains all the enzymatic activities exhibited by the holoenzyme in vitro. The UL52 protein contains a putative zinc finger at its C terminus which is highly conserved among both prokaryotic and eukaryotic primases. We constructed a mutation in which two highly conserved cysteine residues in the zinc finger motif were replaced with alanine residues. A UL52 expression plasmid containing the mutation in the zinc finger region is unable to support the growth of a UL52 mutant virus in a transient complementation assay. Wild type and mutant UL5.UL52 subcomplexes were purified from insect cells infected with recombinant baculoviruses. Surprisingly, the mutant protein was severely affected in all biochemical activities tested; no helicase or primase activities could be detected, and the mutant protein retains only about 9% of wild type levels of single-stranded DNA-dependent ATPase activity. Gel mobility shift assays showed that DNA binding is severely affected as well; the mutant subcomplex only retains approximately 8% of wild type levels of binding to a forked substrate. On the other hand, the mutant protein retains its ability to interact with UL5 as indicated by copurification and with UL8 as indicated by a supershifted band in the gel mobility shift assay. In addition, the ability of individual subunits to bind single-stranded DNA was examined by photo cross-linking. In the wild type UL5.UL52 subcomplex, both subunits are able to bind an 18-mer of oligo(dT). The mutant subcomplex was severely compromised in the ability of both UL5 and UL52 to bind the oligonucleotide; total cross-linking was only 2% of wild type levels. These results are consistent with the proposal that the putative zinc binding motif of UL52 is required not only for binding of the UL52 subunit to DNA and for primase activity but also for optimal binding of UL5 to DNA and for the subsequent ATPase and helicase activities.
1型单纯疱疹病毒编码一种异源三聚体解旋酶-引发酶复合物，该复合物由UL5、UL52和UL8基因的产物组成。由UL5和UL52蛋白组成的亚复合物在体外保留了全酶所表现出的所有酶活性。UL52蛋白在其C末端含有一个假定的锌指，这在原核和真核引发酶中都高度保守。我们构建了一个突变体，其中锌指基序中的两个高度保守的半胱氨酸残基被丙氨酸残基取代。在瞬时互补试验中，含有锌指区域突变的UL52表达质粒无法支持UL52突变病毒的生长。野生型和突变型UL5.UL52亚复合物从感染重组杆状病毒的昆虫细胞中纯化得到。令人惊讶的是，突变蛋白在所有测试的生化活性中都受到严重影响；未检测到解旋酶或引发酶活性，并且突变蛋白仅保留约9%的野生型单链DNA依赖性ATP酶活性水平。凝胶迁移率变动分析表明，DNA结合也受到严重影响；突变亚复合物仅保留约8%的野生型与叉状底物结合水平。另一方面，如共纯化所示，突变蛋白保留了与UL5相互作用的能力，如凝胶迁移率变动分析中的超迁移带所示，也保留了与UL8相互作用的能力。此外，通过光交联检测了各个亚基结合单链DNA的能力。在野生型UL5.UL52亚复合物中，两个亚基都能够结合18聚体的寡聚(dT)。突变亚复合物中UL5和UL52结合寡核苷酸的能力都严重受损；总交联仅为野生型水平的2%。这些结果与以下提议一致，即UL52假定的锌结合基序不仅是UL52亚基与DNA结合以及引发酶活性所必需的，也是UL5与DNA最佳结合以及随后的ATP酶和解旋酶活性所必需的。
