Targeting RNA decay with 2',5' oligoadenylate-antisense in respiratory syncytial virus-infected cells

Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1937-42. doi: 10.1073/pnas.94.5.1937.

Abstract

Treatment of human cells with 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine Nucleotides / genetics
  • Adenine Nucleotides / pharmacology*
  • Antiviral Agents / pharmacology*
  • Binding Sites
  • Cell Line
  • Electrophoresis, Agar Gel
  • Endoribonucleases / metabolism
  • Humans
  • Nucleic Acid Conformation
  • Oligonucleotides / genetics
  • Oligoribonucleotides / genetics
  • Oligoribonucleotides / pharmacology*
  • Polymerase Chain Reaction
  • RNA, Antisense / genetics
  • RNA, Antisense / pharmacology*
  • RNA, Messenger / metabolism*
  • RNA, Viral / metabolism*
  • Respiratory Syncytial Viruses / drug effects*
  • Respiratory Syncytial Viruses / metabolism
  • Respiratory Syncytial Viruses / physiology
  • Viral Proteins / metabolism
  • Virus Replication / drug effects

Substances

  • Adenine Nucleotides
  • Antiviral Agents
  • Oligonucleotides
  • Oligoribonucleotides
  • RNA, Antisense
  • RNA, Messenger
  • RNA, Viral
  • Viral Proteins
  • 2',5'-oligoadenylate
  • Endoribonucleases
  • 2-5A-dependent ribonuclease