TY - JOUR
T1 - Using SHAPE-MaP to Model RNA Secondary Structure and Identify 3=UTR Variation in Chikungunya Virus
AU - Madden, Emily A.
AU - Plante, Kenneth S.
AU - Morrison, Clayton R.
AU - Kutchko, Katrina M.
AU - Sanders, Wes
AU - Long, Kristin M.
AU - Taft-Benz, Sharon
AU - Cruz Cisneros, Marta C.
AU - White, Ashlyn Morgan
AU - Sarkar, Sanjay
AU - Reynolds, Grace
AU - Vincent, Heather A.
AU - Laederach, Alain
AU - Moorman, Nathanial J.
AU - Heisea, Mark T.
N1 - Funding Information:
We thank members of the Heise, Moorman, and Laederach laboratories for reviews of the manuscript. We thank Kevin Weeks for his advice and thoughtful conversations on SHAPE-MaP analysis. We thank Michael Diamond of Washington University and Robert Tesh of the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch for kindly sharing the CHIKV Caribbean isolate with us. We also thank the reviewers of our paper for their thoughtful and constructive comments in the midst of a pandemic. Their attention to detail and commitment to improving our paper was greatly appreciated. This study was supported by the National Institutes of Health (U19 AI142759 to M.T.H., U19 AI 107810 to M.T.H., U19 AI 109761 to M.T.H., R21 AI 138056 to M.T.H. and N.J.M., AI 123811 to N.J.M., T32 AI 007151-36A1 to E.A.M., and F32 AI 126730 to K.S.P.). Funding for the open access charge was provided by the National Institutes of Health.
Funding Information:
This study was supported by the National Institutes of Health (U19 AI142759 to M.T.H., U19 AI 107810 to M.T.H., U19 AI 109761 to M.T.H., R21 AI 138056 to M.T.H. and N.J.M., AI 123811 to N.J.M., T32 AI 007151-36A1 to E.A.M., and F32 AI 126730 to K.S.P.). Funding for the open access charge was provided by the National Institutes of Health. We report no conflicts of interest.
Publisher Copyright:
© 2020 American Society for Microbiology. All Rights Reserved.
PY - 2020/12
Y1 - 2020/12
N2 - Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3=UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3=UTR variants. A duplication in the 3=UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3=UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3=UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.
AB - Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3=UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3=UTR variants. A duplication in the 3=UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3=UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3=UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.
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U2 - 10.1128/JVI.00701-20
DO - 10.1128/JVI.00701-20
M3 - Article
C2 - 32999019
AN - SCOPUS:85096815705
SN - 0022-538X
VL - 94
JO - Journal of Virology
JF - Journal of Virology
IS - 24
M1 - e00701-20
ER -