Inter-RNA Interaction of Phage φ29 pRNA to Form a Hexameric Complex for Viral DNA Transportation

Abstract

Ds-DNA viruses package their DNA into a preformed protein shell (procapsid) during maturation. Bacteriophage phi29 requires an RNA (pRNA) to package its genomic DNA into the procapsid. We report here that the pRNA upper and lower loops are involved in RNA/RNA interactions. Mutation in only one loop results in inactive pRNAs. However, mixing of two, three and six inactive mutant pRNAs restores DNA packaging activity as long as an interlocking hexameric ring can be predicted to form by base pairing of the mutated loops in separate RNA molecules. The stoichiometry of pRNA for the packaging of one viral DNA genome is six. Homogeneous pRNA purified from a single band in denaturing gels showed six bands when rerun in native gels. These results suggest that six pRNAs form a hexameric ring by the intermolecular interaction of two RNA loops to serve as part of the DNA transportation machinery.

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

“…10 pRNA forms dimers, trimers, and, ultimately, hexamers through hand-in-hand interaction of the right and left interlocking loops. 11,12,31 The structural features of pRNA, which have been studied extensively, allow for easy manipulation and permit the conversion of pRNA into a gene targeting and delivery vehicle. The pRNA molecule contains two independent folding domains with distinct functions.…”

Structure of the bacteriophage φ29 DNA packaging motor

“…The ϕ29 connector, a cone-shaped dodecamer of gene product 10 (gp10), occupies the pentagonal vertex at the base of the prohead 5 and is the portal for DNA entry during packaging and DNA ejection during infection 6 . The connector, in association with the oligomeric, ϕ29-encoded prohead RNA (pRNA) and a viral ATPase (gp16), is required for DNA packaging [7][8][9] . However, only the first 120 bases of the 174-base pRNA are essential for packaging 7 the genomic dsDNA with gp3 (DNA-gp3) can be packaged into proheads in about three minutes in vitro (P.J.J., unpublished results).…”

“…1d) was based on reconstructions in which the five-fold symmetry had been imposed along the long axis of the particles. If the pRNA were six-fold symmetric as suggested by genetic data 8,9 , the resultant averaged density would be weak and smeared. However, we observe that the pRNA has excellent density, higher by a factor of ~1.5 than the density of the head, thus demonstrating that the pRNA is consistent with the imposed five-fold symmetry.…”

“…Although genetic studies 8,9 had shown that the pRNA forms hexamers by intermolecular base pairing, only a pentameric model could be readily fitted into the cryo-EM density, with the five A helices fitting into the radial spokes (Fig. 3b).…”

“…A hexameric ring would have had too large a radius. It is possible that a hexameric pRNA might be required to initiate pRNA binding, but that at some stage one pRNA molecule 8,9 is shifted out of the ring or eliminated. As the handedness of the pRNA density is not known, the direction of the pRNA helices within the ring is uncertain.…”