TY - JOUR
T1 - Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses
AU - Li, Yize
AU - Banerjee, Shuvojit
AU - Wang, Yuyan
AU - Goldstein, Stephen A.
AU - Dong, Beihua
AU - Gaughan, Christina
AU - Silverman, Robert H.
AU - Weiss, Susan R.
N1 - Funding Information:
We thank Dr. Paul Bates and Stephen Bart for plasmids psPAX2 and pCMV-VSV-G and for advice on CRISPR-Cas9 technology; Dr. Babal Kant Jha for the 2-5A and RNase L used in the FRET assays; and Danika Baskar for help with the Western blots. This work was supported by NIH Grants R01-AI104887 (to S.R.W. and R.H.S.), R01-NS081008 (to S.R.W.), and R01-CA044059 (to R.H.S.) and by the Mal and Lea Bank Chair Fund (R.H.S.). Y.W. was supported in part by the China Scholarship Council.
PY - 2016/2/23
Y1 - 2016/2/23
N2 - 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.
AB - 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.
KW - 2-5A
KW - Antiviral response
KW - Oligoadenylate synthetase 3
KW - Ribonuclease L
KW - Type I interferon
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U2 - 10.1073/pnas.1519657113
DO - 10.1073/pnas.1519657113
M3 - Article
C2 - 26858407
AN - SCOPUS:84959253492
SN - 0027-8424
VL - 113
SP - 2241
EP - 2246
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 8
ER -