MOLECULAR BIOLOGY OF THE DELTA AGENTS

我们建议再进行五年的研究

We propose to conduct an additional five years of research on the properties of the causative agent for human delta hepatitis and its circular genomic RNA molecular which make it unique among viral and cellular RNAs. Delta RNA remains the only non-coding genomic RNA to undergo efficient RNA-to-RNA copying using cellular components by a rolling circle mechanism so far identified in mammalian cells. The RNA-level events which this agent brings to bear on its replication and host cell interactions include RNA-catalyzed enzymatic events using built in ribozymes for RNA cleavage during replication; specific RNA ligation of progeny RNAs to form circles; specific RNA editing of a mRNA reading frame utilizing a host enzyme; extensive secondary and local tertiary structure, including characteristic UV- crosslinkable structures in both the genomic and complementary antigenomic strands which arise during replication; interaction(s) with a host protein kinase, PKR, at specific RNA sites; and binding sites (RNA promoters) for host RNA polymerase molecules. Many of these processes and interactions involve the conserved, non-coding region of delta RNA, some parts of which are involved in multiple specific interactions requiring conformational switching. It is our long-term goal to study the interactions of delta genomic and antigenomic RNAs with host-cell components; and to understand how these interactions and RNA-based activities combine to promote efficient replication of this small, circular highly structured single-stranded RNA agent which causes a serious human disease. Our specific aims are (i) to study delta viral pathogenesis by carrying out studies on specific host protein delta RNA interactions, with emphasis on the protein kinase and RNA editing activities; (ii) since delta's RNA:protein interactions employ specific protein and RNA motifs of great general biological interest, we aim to pursue the detailed structure of key elements in delta RNA which bind proteins and otherwise react to promote replication and pathogenesis, using physical as well as biochemical techniques; and (iii) to study, by RNase protection and other means, the RNA-level enzymes of delta responsible for cleavage and ligation of replicating strands and their regulation by conformational switching at the RNA level.