A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

Joanna Sztuba-Solinska; Larissa Diaz; Mia R. Kumar; Gaëlle Kolb; Michael R. Wiley; Lucas Jozwick; Jens H. Kuhn; Gustavo Palacios; Sheli R. Radoshitzky; Stuart F.J. Le Grice; Reed F. Johnson

doi:10.1093/nar/gkw825

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

Joanna Sztuba-Solinska, Larissa Diaz, Mia R. Kumar, Gaëlle Kolb, Michael R. Wiley, Lucas Jozwick, Jens H. Kuhn, Gustavo Palacios, Sheli R. Radoshitzky, Stuart F.J. Le Grice, Reed F. Johnson

Environmental, Agricultural & Occupational Health Sciences

Research output: Contribution to journal › Article › peer-review

10 Scopus citations

Abstract

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.

Original language	English (US)
Pages (from-to)	9831-9846
Number of pages	16
Journal	Nucleic acids research
Volume	44
Issue number	20
DOIs	https://doi.org/10.1093/nar/gkw825
State	Published - 2016

ASJC Scopus subject areas

Genetics

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Sztuba-Solinska, J., Diaz, L., Kumar, M. R., Kolb, G., Wiley, M. R., Jozwick, L., Kuhn, J. H., Palacios, G., Radoshitzky, S. R., Le Grice, S. F. J., & Johnson, R. F. (2016). A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8. Nucleic acids research, 44(20), 9831-9846. https://doi.org/10.1093/nar/gkw825

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8. / Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R.; Kolb, Gaëlle; Wiley, Michael R.; Jozwick, Lucas; Kuhn, Jens H.; Palacios, Gustavo; Radoshitzky, Sheli R.; Le Grice, Stuart F.J.; Johnson, Reed F.

In: Nucleic acids research, Vol. 44, No. 20, 2016, p. 9831-9846.

Research output: Contribution to journal › Article › peer-review

Sztuba-Solinska, Joanna ; Diaz, Larissa ; Kumar, Mia R. ; Kolb, Gaëlle ; Wiley, Michael R. ; Jozwick, Lucas ; Kuhn, Jens H. ; Palacios, Gustavo ; Radoshitzky, Sheli R. ; Le Grice, Stuart F.J. ; Johnson, Reed F. / A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8. In: Nucleic acids research. 2016 ; Vol. 44, No. 20. pp. 9831-9846.

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title = "A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8",

abstract = "Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.",

author = "Joanna Sztuba-Solinska and Larissa Diaz and Kumar, {Mia R.} and Ga{\"e}lle Kolb and Wiley, {Michael R.} and Lucas Jozwick and Kuhn, {Jens H.} and Gustavo Palacios and Radoshitzky, {Sheli R.} and {Le Grice}, {Stuart F.J.} and Johnson, {Reed F.}",

note = "Funding Information: This work was supported by the NIAID Division of Intramural Research. S.L.G. and J.S.-S. were supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. A subcontractor to Battelle Memorial Institute who performed this work is: J.H.K. an employee of Tunnell Government Services, Inc. S.R.R was supported by The Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD) of the Defense Threat Reduction Agency (DTRA) (proposal # 1323839 and CCAR# CB3849). Portions of this work performed by S.R.R. and L.J. were funded by the Defense Threat Reduction Agency, CB10217.",

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AU - Sztuba-Solinska, Joanna

AU - Diaz, Larissa

AU - Kumar, Mia R.

AU - Kolb, Gaëlle

AU - Wiley, Michael R.

AU - Jozwick, Lucas

AU - Kuhn, Jens H.

AU - Palacios, Gustavo

AU - Radoshitzky, Sheli R.

AU - Le Grice, Stuart F.J.

AU - Johnson, Reed F.

N1 - Funding Information: This work was supported by the NIAID Division of Intramural Research. S.L.G. and J.S.-S. were supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. A subcontractor to Battelle Memorial Institute who performed this work is: J.H.K. an employee of Tunnell Government Services, Inc. S.R.R was supported by The Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD) of the Defense Threat Reduction Agency (DTRA) (proposal # 1323839 and CCAR# CB3849). Portions of this work performed by S.R.R. and L.J. were funded by the Defense Threat Reduction Agency, CB10217.

PY - 2016

Y1 - 2016

N2 - Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.

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