Doorbar J, Foo C, Coleman N, Medcalf L, Hartley O, Prospero T, Napthine S, Sterling J, Winter G, Griffin H
National Institute for Medical Research, London, United Kingdom.
Virology. 1997 Nov 10;238(1):40-52. doi: 10.1006/viro.1997.8768.
HPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 x 10(10) phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infection in vitro.
随着受感染的基底细胞穿过表皮的分化层，人乳头瘤病毒（HPV）晚期基因开始表达，导致病毒DNA进行增殖性复制，并表达病毒晚期蛋白。我们利用展示在噬菌体上的大型合成免疫球蛋白文库（多样性为6.5×10¹⁰噬菌体）分离出三种能识别HPV16 E4晚期蛋白上不同表位的Fab片段（TVG405、406和407）。通过杂交瘤技术制备了C端单克隆抗体（TVG404），并通过肽免疫制备了N端多克隆抗血清（α N端）。最有效的抗体（TVG405）对E4的亲和力约为1.0 nM。所有抗体均可识别石蜡包埋存档材料中的该蛋白，这使我们能够确定病毒感染后期的事件。E4在体内的表达与主要病毒衣壳蛋白L1的合成不一致，但在由HPV16引起的癌前病变中比L1的合成早1或2个细胞层，而在HPV63诱导的疣中则早多达20个细胞层。在与HPV16相关的高级别病变中，E4在没有L1的情况下产生。相比之下，增殖性病毒DNA复制与E4表达完全相关，并且在某些病变中，随着受感染的上皮细胞离开基底层而开始。在E4阳性细胞中无法检测到诸如丝聚合蛋白、兜甲蛋白和某些角蛋白等分化标志物，并且核变性延迟。HPV16 E4在下皮层呈丝状分布，但在分化程度更高的细胞中与单个核周结构相关。针对该蛋白N端的抗体对这些结构的染色效果不佳。我们的发现与HPV16 E4蛋白在增殖性DNA复制中或在改变受感染细胞的表型以促进病毒合成或病毒释放方面所起的作用相符。这些Fab片段在评估体外模拟HPV感染的模型系统中具有价值。
