INHIBITION OF PHAGE PHI-29 ASSEMBLY BY ANTISENSE OLIGONUCLEOTIDES TARGETING VIRAL PRNA ESSENTIAL FOR DNA PACKAGING

A sensitive and efficient system for the functional assay of antisense oligonucleotides (oligos) was developed based on an in vitro viral as sembly system. A 120-base RNA (pRNA), which indispensably participates in bacteriophage phi 29 DNA packaging, was the target for antisense a ction. Antisense oligos bound to pRNA, as revealed by a slower electro phoretic mobility of pRNA/oligo complexes in comparison with native pR NA. Infectious viruses were assembled in vitro with synthetic pRNA and DNA, as well as with viral proteins produced from cloned genes. Up to 10(7) plaque-forming units per milliliter were obtained in the absenc e of antisense oligos, while as few as zero plaques were detected in t he presence of certain antisense oligos. A 1-base mismatch greatly inf luenced the inhibitory effect of the antisense oligos, but this 1-base d mismatch was not important when the mismatch was placed at the end o f the oligo. Five oligos did not bind pRNA or inhibit the assembly of the virion, suggesting that the RNA sequences complementary to these o ligos are nonessential or buried internally in the RNA. Viral assembly was strongly inhibited by antisense oligos P15 and P10, targeting eit her the 5'- or the 3'-end of the pRNA, respectively. Viral assembly wa s also strongly inhibited by oligo P6, targeting an internal region, r esidues 75-91, of pRNA. Oligo P6 inhibited DNA packaging activity by b locking the binding of pRNA to the procapsid, while P10 and P15 inhibi ted DNA packaging activity but did not block the binding of pRNA to th e procapsid, suggesting that in addition to the reported internal doma in for procapsid binding, pRNA contains another domain at the paired 5 '/3'-ends with a yet to be defined role in DNA translocation. (C) 1995 Academic Press, Inc.