A Small Stem Loop Structure Of The Ebola Virus Trailer Is Essential For Replication And Interacts With Heat Shock Protein A8
USAMRIID Frederick United States
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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 18681890 that is similar to a previously identified structure in the replicative intermediateRI 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 andHSPA8 motif 1 in virus replication.