Baumann Richard G, Mullaney Julienne, Black Lindsay W
USNA, Department of Chemistry, Mailstop 9B, 572 Holloway Road, Annapolis, MD 21402-5070, USA.
Mol Microbiol. 2006 Jul;61(1):16-32. doi: 10.1111/j.1365-2958.2006.05203.x.
Architecturally conserved viral portal dodecamers are central to capsid assembly and DNA packaging. To examine bacteriophage T4 portal functions, we constructed, expressed and assembled portal gene 20 fusion proteins. C-terminally fused (gp20-GFP, gp20-HOC) and N-terminally fused (GFP-gp20 and HOC-gp20) portal fusion proteins assembled in vivo into active phage. Phage assembled C-terminal fusion proteins were inaccessible to trypsin whereas assembled N-terminal fusions were accessible to trypsin, consistent with locations inside and outside the capsid respectively. Both N- and C-terminal fusions required coassembly into portals with approximately 50% wild-type (WT) or near WT-sized 20am truncated portal proteins to yield active phage. Trypsin digestion of HOC-gp20 portal fusion phage showed comparable protection of the HOC and gp20 portions of the proteolysed HOC-gp20 fusion, suggesting both proteins occupy protected capsid positions, at both the portal and the proximal HOC capsid-binding sites. The external portal location of the HOC portion of the HOC-gp20 fusion phage was confirmed by anti-HOC immuno-gold labelling studies that showed a gold 'necklace' around the phage capsid portal. Analysis of HOC-gp20-containing proheads showed increased HOC protein protection from trypsin degradation only after prohead expansion, indicating incorporation of HOC-gp20 portal fusion protein to protective proximal HOC-binding sites following this maturation. These proheads also showed no DNA packaging defect in vitro as compared with WT. Retention of function of phage and prohead portals with bulky internal (C-terminal) and external (N-terminal) fusion protein extensions, particularly of apparently capsid tethered portals, challenges the portal rotation requirement of some hypothetical DNA packaging mechanisms.
结构保守的病毒门户十二聚体对于衣壳组装和DNA包装至关重要。为了研究噬菌体T4的门户功能，我们构建、表达并组装了门户基因20融合蛋白。C末端融合（gp20-GFP、gp20-HOC）和N末端融合（GFP-gp20和HOC-gp20）的门户融合蛋白在体内组装成活性噬菌体。噬菌体组装的C末端融合蛋白对胰蛋白酶不可接近，而组装的N末端融合蛋白对胰蛋白酶可接近，这分别与衣壳内部和外部的位置一致。N末端和C末端融合都需要与大约50%野生型（WT）或接近WT大小的20am截短门户蛋白共同组装成门户，以产生活性噬菌体。对HOC-gp20门户融合噬菌体进行胰蛋白酶消化显示，对被蛋白酶水解的HOC-gp20融合蛋白的HOC和gp20部分有类似的保护作用，这表明这两种蛋白在门户和近端HOC衣壳结合位点都占据受保护的衣壳位置。通过抗HOC免疫金标记研究证实了HOC-gp20融合噬菌体中HOC部分的外部门户位置，该研究显示噬菌体衣壳门户周围有一条金“项链”。对含有HOC-gp20的原头部的分析表明，只有在原头部扩张后，HOC蛋白对胰蛋白酶降解的保护作用才增强，这表明在这种成熟后，HOC-gp20门户融合蛋白被整合到保护性近端HOC结合位点。与WT相比，这些原头部在体外也没有显示出DNA包装缺陷。具有庞大内部（C末端）和外部（N末端）融合蛋白延伸的噬菌体和原头部门户功能的保留，特别是明显与衣壳相连的门户，对一些假设的DNA包装机制的门户旋转要求提出了挑战。
