TY - JOUR
T1 - Ebolavirus polymerase uses an unconventional genome replication mechanism
AU - Deflubé, Laure R.
AU - Cressey, Tessa N.
AU - Hume, Adam J.
AU - Olejnik, Judith
AU - Haddock, Elaine
AU - Feldmann, Friederike
AU - Ebihara, Hideki
AU - Fearns, Rachel
AU - Mühlberger, Elke
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank H. Feldmann (LV, NIAID, NIH) and members of the Laboratory of Virology for training L.R.D. in BSL-4 work and for help with BSL-4 experiments at the NIAID/NIH Rocky Mountain Laboratories, Hamilton, MT. We thank U.J. Buchholz, NIAID/NIH, Bethesda, MD for providing BSR T7/5 cells; J. Alonso, Texas Biomedical Research Institute, San Antonio, TX for sharing Huh7 cells; I. Jordan, ProBioGen AG, Berlin, Germany for providing R05T cells; T. Takimoto, St. Jude Children’s Research Hospital, Memphis, TN, and Y. Kawaoka, University of Wisconsin, Madison, WI for providing the pCAGGS-T7 plasmid; and J.R. Pacheco, Boston University for excellent technical assistance. This work was funded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Awards R03 AI114293 (to E.M.), R21 AI126457 (to E.M.), R01 AI133486 (to R.F. and E.M.), and R01 AI113321 (to R.F.). The work was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (to H.E.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.
PY - 2019
Y1 - 2019
N2 - Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3′ and 5′ terminal nucleotides because the promoters at the 3′ ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3′ and 5′ terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5′ ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5′, the 3′ termini are variable and are typically 3′-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3′ ends. The majority of 3′ terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3′ terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3′-CCUGU motif regardless of the identity of the 3′ terminal nucleotide(s) and of the position of this motif relative to the 3′ end. Deletion or mutation of the first residue of the 3′-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3′ overhangs. This could be a mechanism to avoid antiviral recognition.
AB - Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3′ and 5′ terminal nucleotides because the promoters at the 3′ ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3′ and 5′ terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5′ ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5′, the 3′ termini are variable and are typically 3′-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3′ ends. The majority of 3′ terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3′ terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3′-CCUGU motif regardless of the identity of the 3′ terminal nucleotide(s) and of the position of this motif relative to the 3′ end. Deletion or mutation of the first residue of the 3′-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3′ overhangs. This could be a mechanism to avoid antiviral recognition.
KW - Ebola virus genome ends
KW - Ebola virus replication
KW - Ebolavirus replication initiation
KW - Nonsegmented negative strand RNA virus replication
KW - Variable 3′ genome ends
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U2 - 10.1073/pnas.1815745116
DO - 10.1073/pnas.1815745116
M3 - Article
C2 - 30962389
AN - SCOPUS:85065214053
VL - 116
SP - 8535
EP - 8543
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
SN - 0027-8424
IS - 17
ER -