Schenck Sabine, Kehm Elke, Epstein Alberto L, Zentgraf Hanswalter, Müller Martin, Knopf Charles W
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, DKFZ-ATV, INF 242, 69120, Heidelberg, Germany.
Virus Genes. 2008 Oct;37(2):131-43. doi: 10.1007/s11262-008-0247-8. Epub 2008 Jun 12.
Expression of human papilloma virus type 16 (HPV16) antigens by herpes simplex virus type 1 (HSV-1)-based amplicon vectors was investigated. Amplicons were packaged using HSV-1 LaL, a virus with a floxed 'a' sequence, and the CRE recombinase-expressing cell line TE-CRE30. In amplicon-infected BHK-21 cells, a 60-mer E7 peptide was weakly expressed, but its expression was significantly improved using a transcription unit with humanized codons. In contrast, the HPV16 structural proteins L1 and L2, as well as L1 fusion proteins with E7, were expressed at high levels. L1 displayed nuclear and cytoplasmic localization, and L1-E7 fusion proteins with a synthetic nuclear localization signal (NLS) from simian virus 40 (SV40) accumulated in the nuclei. The self-assembly of L1 into virus-like particles (VLPs) in HSV-1 amplicon-infected cells was confirmed by electron microscopy and a specific antigen-capture ELISA. For targeting of herpes virions with HPV16 antigens, a 60-mer E7 peptide was fused to the herpesviral tegument protein VP22 and envelope glycoprotein C (gC). Both fusion proteins, VP22E7 and gCE7, were demonstrated to be associated with sucrose gradient-purified infectious HSV-1 virion particles.
研究了基于1型单纯疱疹病毒（HSV-1）的扩增载体对16型人乳头瘤病毒（HPV16）抗原的表达。扩增子用HSV-1 LaL包装，HSV-1 LaL是一种具有floxed 'a'序列的病毒，以及表达CRE重组酶的细胞系TE-CRE30。在扩增子感染的BHK-21细胞中，一种60聚体E7肽表达较弱，但使用人源化密码子的转录单元可显著提高其表达。相比之下，HPV16结构蛋白L1和L2，以及与E7的L1融合蛋白均高水平表达。L1显示出核定位和胞质定位，带有来自猴病毒40（SV40）的合成核定位信号（NLS）的L1-E7融合蛋白在细胞核中积累。通过电子显微镜和特异性抗原捕获ELISA证实了HSV-1扩增子感染细胞中L1自组装成病毒样颗粒（VLP）。为了用HPV16抗原靶向疱疹病毒粒子，将一种60聚体E7肽与疱疹病毒被膜蛋白VP22和包膜糖蛋白C（gC）融合。两种融合蛋白VP22E7和gCE7均被证明与蔗糖梯度纯化的感染性HSV-1病毒粒子相关。
