Zaia John A, Li Xiuli, Franck Anne E, Wu Xiwei, Thao Lia, Gallez-Hawkins Ghislaine
Beckman Research Institute of the City of Hope, Department of Virology, 1500 E. Duarte Rd., Duarte, CA 91010, USA.
Clin Vaccine Immunol. 2009 Jun;16(6):935-43. doi: 10.1128/CVI.00011-09. Epub 2009 Apr 15.
The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A0201 target cell line. In HHDII mice expressing HLA A0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.
人巨细胞病毒（CMV）pp65蛋白在CMV pp65羧基末端附近的氨基酸（aa）415至438以及aa 537至561处含有两个二分核定位信号（NLS），并且在赖氨酸436处有一个与激酶活性相关的磷酸结合位点。将pp65的K436突变为N（CMV pp65mII）并进一步缺失aa 537至561，产生了一种新蛋白（pp65mIINLSKO，其中NLSKO表示NLS敲除），该蛋白无激酶活性且核定位明显降低。本研究的目的是对该蛋白进行生物学特性分析，并将其免疫原性与天然pp65的免疫原性进行比较。与天然CMV pp65不同，在体外将CMV pp65mIINLSKO通过基于DNA或重组腺相关病毒的转导导入细胞后，首次观察到pp65mIINLSKO的表达位于细胞质中，并且pp65mIINLSKO的表达水平高于天然蛋白。CMV pp65mIINLSKO mRNA比CMV pp65 mRNA更早丰度更高（分别在4小时和8小时），但蛋白质的半衰期相同。通过在pp65mIINLSKO转导的人HLA A0201靶细胞系中细胞毒性杀伤效率的提高来衡量，这种修饰改变了体外CMV pp65的抗原加工过程。在表达HLA A0201的HHDII小鼠中，pp65mIINLSKO与CMV pp65具有相同的免疫原性。通过RNA微阵列分析，与天然CMV pp65相比，CMV pp65mIINLSKO的表达对细胞周期途径的影响较小，而对涉及免疫活性的细胞表面信号通路的影响较大。得出的结论是，从pp65中去除主要的NLS基序不会损害其免疫原性，在疫苗设计中应予以考虑。
