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.
authors.affiliation: RT Biochemistry Section, Basic Research Laboratory, National Cancer Institute-Frederick, National Institutes of Health, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Joanna
authors.initials: J
authors.lastname: Sztuba-Solinska
authors.affiliation: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Larissa
authors.initials: L
authors.lastname: Diaz
authors.affiliation: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Mia R
authors.initials: MR
authors.lastname: Kumar
authors.affiliation: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Kolb
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Wiley
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Lucas
authors.initials: L
authors.lastname: Jozwick
authors.affiliation: Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Disease, National Institutes of Health, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Jens H
authors.identifier: http://orcid.org/0000-0002-7800-6045
authors.initials: JH
authors.lastname: Kuhn
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Gustavo
authors.identifier: http://orcid.org/0000-0001-5062-1938
authors.initials: G
authors.lastname: Palacios
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Sheli R
authors.identifier: http://orcid.org/0000-0001-8976-8056
authors.initials: SR
authors.lastname: Radoshitzky
authors.affiliation: RT Biochemistry Section, Basic Research Laboratory, National Cancer Institute-Frederick, National Institutes of Health, Fort Detrick, Frederick, MD 21702, USA legrices@mail.nih.gov.
authors.forename: Stuart F
authors.initials: SF
authors.lastname: J Le Grice
authors.affiliation: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Fort Detrick, Frederick, MD 21702, USA johnsonreed@mail.nih.gov.
authors.forename: Reed F
authors.initials: RF
authors.lastname: Johnson
mesh_terms: D029043:Ebolavirus / Q000235:genetics*; D016679:Genome, Viral; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019142:Hemorrhagic Fever, Ebola / Q000378:metabolism / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D006360:Heat-Shock Proteins; D034741:RNA, Small Interfering; D012367:RNA, Viral