Functional interaction between gene expression and DNA replication of HPV lifecyle

人乳头瘤病毒(HPV)的基因表达和基因组扩增受到宿主细胞分化状态的严格调控。在基底层细胞中可检测到病毒早期基因的弱表达和病毒基因组的低拷贝数。在角质形成细胞分化过程中，病毒基因表达和DNA扩增水平上调，只有在完全分化的细胞中才能检测到病毒颗粒的产生。因此，有必要在反映角质形成细胞分化过程的系统中分析HPV基因的功能和复制机制。角质形成细胞的器官型培养是为了重建皮肤结构，其中HPV DNA可以表达病毒基因并以分化特异性的方式复制。我们采用了黑木博士等人开发的器官型培养系统。进行人乳头瘤病毒检测。用脂质体转染法将hpv18基因组dna导入人原代角质形成细胞，并将其用于…。更多的或器官类型的培养。在基底层细胞中，原位杂交难以检测到HPV DNA，Southern印迹分析可检测到HPV DNA。在分化标志之一转谷氨酰胺酶表达的分化细胞中可观察到DNA扩增。随着细胞分化程度的提高，mRNA表达水平也有所提高。结果表明，该器官型培养体系适合于HPV分化特异性功能的分析。在角质形成细胞中表达了两种主要的病毒癌蛋白E6和E7，并分析了它们对细胞增殖和分化的影响。正常情况下，细胞分裂仅限于基底层，但表达E6/E7的细胞即使在部分分化层也能增殖。然而，即使有E6/E7的表达，分化程序仍保持完整。我们现在正在扩大对其他病毒基因和低危型E6/E7基因的分析。我们在器官培养中观察到基因表达和DNA复制之间的联系。这两种功能主要受HPV基因组LCR调控，病毒E2蛋白参与其中。我们获得的证据表明，E2蛋白在这两种活动之间起到了中介作用。我们正在研究E2蛋白在分化特异性HPV复制机制中的作用。较少

Gene expression and genome amplification of human papillomavirus (HPV) are tightly regulated by the status of host cell differentiation. In basal layer cells, weak expression of viral early genes and low copy number of viral genome are detectable. In the course of keratinocyte differentiation, the levels of viral gene expression and DNA amplification are upregulated, and the viral particle production can be detected only in fully differentiated cells. It is necessary, therefore, to analyze the mechanisms of HPV gene function and replication in the system that reflects the keratinocyte differentiation program.The organotypic culture of keratinocytes is developed to reconstitute the skin structure, in which HPV DNA can express viral genes and replicate in differentiation specific manner. We adapted an organotypic culture system developed by Dr. Kuroki et al. for HPV analysis. HPV 18 genomic DNA was introduced into human primary keratinocytes by lipofection, and then the cells were used f … More or organotypic culture. In the basal layer cells, HPV DNA was difficult to detect by in situ hybridization, although it was detectable by Southern blot analysis. The DNA amplification could be found in the differentiated cells, in which transglutaminase, one of the differentiation markers, was expressing. mRNA expression level was also enhanced through cell differentiation. The result indicated that the organotypic culture system was suitable for the analysis of differentiation specific HPV function.The gene function of HPV was analyzed in the organotypic culture system. Two major viral oncoproteins, E6 and E7, were expressed in keratinocytes and the effects on cell proliferation and differentiation were analyzed. In normal condition, cell division was restricted only in the basal layer, but E6/E7 expressing cells could proliferated even in partially differentiated layer. However, differentiation program was maintained intact even with E6/E7 expression. We are now expanding the analysis for other viral genes and low-risk type E6/E7 genes.We observed a linkage between gene expression and DNA replication in the organotypic culture. Both functions are regulated mainly by LCR of HPV genome, in which viral E2 protein is involved. We obtained evidence that the E2 protein functioned as a mediator between both activities. We are now investigating the involvement of E2 protein in differentiation specific HPV replication mechanism. Less