Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

Proc Natl Acad Sci U S A. 2016 Feb 23;113(8):2241-6. doi: 10.1073/pnas.1519657113. Epub 2016 Feb 8.

Abstract

The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans--OAS1, OAS2, and OAS3--synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.

Keywords: 2-5A; antiviral response; oligoadenylate synthetase 3; ribonuclease L; type I interferon.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2',5'-Oligoadenylate Synthetase / antagonists & inhibitors
  • 2',5'-Oligoadenylate Synthetase / genetics
  • 2',5'-Oligoadenylate Synthetase / metabolism*
  • Alphavirus Infections / genetics
  • Alphavirus Infections / metabolism
  • CRISPR-Cas Systems
  • Cell Line
  • Endoribonucleases / antagonists & inhibitors
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism*
  • Enzyme Activation
  • Gene Knockout Techniques
  • Humans
  • Influenza, Human / genetics
  • Influenza, Human / metabolism
  • Models, Biological
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Sindbis Virus
  • Vaccinia / genetics
  • Vaccinia / metabolism
  • Virus Diseases / genetics
  • Virus Diseases / metabolism*
  • West Nile Fever / genetics
  • West Nile Fever / metabolism

Substances

  • RNA, Ribosomal
  • RNA, Viral
  • OAS1 protein, human
  • OAS2 protein, human
  • 2',5'-Oligoadenylate Synthetase
  • OAS3 protein, human
  • Endoribonucleases
  • 2-5A-dependent ribonuclease