Bacterial virus phi29 pRNA modeling and its use as a hammerhead ribozyme escort to destroy hepatitis B virus and other targets

Stephen Michael Hoeprich, Purdue University


A feature shared by all linear dsDNA viruses is that during their maturation, their genomes are translocated swiftly into the limited space of the procapsid and arranged into near crystal density. A DNA-translocating motor hydrolyzes ATP to perform this work. The phi29 bacteriophage uses a virus-encoded 120 base RNA, called pRNA, that forms a hexameric complex and is an integral and indispensible component of its DNA translocating motor. The six pRNAs work sequentially during the DNA translocation process. Phi29, including its functional DNA translocation motor, has been assembled and used to package its genome in vitro utilizing purified components synthesized by chemical or molecular biological approaches. To further the understanding of the phi29 DNA packaging motor, the nonstructural component gp16 was studied for its ATP hydrolysis activity. Three-dimensional computer models of pRNA structures were built using experimental data as limiting parameters. Using these constraints, the shapes of these computer models became very similar to the images of pRNA seen using cryo-AFM (atomic force microscopy). The pRNA hexamer docking with the connector crystal structure displays a remarkable match with the current data. pRNA's independent and tightly self-folded domains make it a great vector for therapeutic RNAs. The pRNA has two. pRNAs made using circularly permutation did not alter the folding of the independent pRNA domains, or hinder its function. The pRNA 5'/3' ends were linked using various sequences. When using therapeutic RNA, two common problems encountered are exonuclease degradation and misfolding of the RNA in the cell. A vector was made using pRNA to harbor hammerhead ribozymes that cleaves the Hepatitis B virus (HBV) polyA signal. The ribozyme targeting HBV was connected to the native pRNA 5'/3' ends to produce a circularly permuted pRNA chimera. In vitro studies demonstrated the nearly complete cleavage of HBV polyA signal mRNA by the chimeric ribozyme. Cell culture studies demonstrated the increased inhibition of HBV replication by the chimeric ribozyme compared to the original ribozyme. This was demonstrated by Northern blot and e-antigen assays. These findings imply that pRNA can also harbor (hold and protect) other ribozymes, antisense, siRNA, and other therapeutic RNA molecules. ^




Peixuan Guo, Purdue University.

Subject Area

Biology, Molecular|Biology, Virology

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