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.

Conserved microsatellites may contribute to stem-loop structures in 5′, 3′ terminals of Ebolavirus genomes

“…S2eS19). Sequence analysis showed that the nucleic acid of Ebolavirus (EBOV, SUDV, RESTV, TAFV) between their 3 0 terminal (18 nt) and 5 0 terminal (20 nt) were highly identical [42], and conserved stem-loop structures were found [42,44,45], which proved the accuracy of the prediction results in this study. Here, we displayed the relationship between such structures and microsatellites to speculate on the role of microsatellites in Ebolavirus genome.…”

“…The 5 0 , 3 0 terminal non-coding regions were thought to regulate viral transcription, replication, and assembly of new virions [23]. The 3 0 trailer of Ebolavirus were essential for replication by formed a small stem-loop structure, and participated in RNA-Protein interaction [44]. The related results suggested that conserved microsatellites in the terminal regions of Ebolavirus were well base-paired to help forming conserved stem-loop structures ( Fig.…”

Staufen1 Interacts with Multiple Components of the Ebola Virus Ribonucleoprotein and Enhances Viral RNA Synthesis

“…For example, an important cis -acting RNA structure required for EBOV transcriptional regulation is located in the 3′ extracistronic region of the viral genome and functions through interaction with the viral trans -acting factor, VP30 ( 18 – 21 ). Nevertheless, functionality of most EBOV RNA secondary structures and potential host or viral trans -acting factors that recognize these structures have not been well established ( 22 , 23 ).…”

Probing the Structures of Viral RNA Regulatory Elements with SHAPE and Related Methodologies

“…Long-range interactions have also been identified in the negative-sense RNA genome of Ebola virus (EBOV), a filovirus that causes Ebola hemorrhagic fever in humans and other mammals (Sztuba-Solinska et al, 2013 ). This section will highlight two studies in which 5′-3′ interactions in DENV and EBOV were characterized using capillary electrophoresis-based SHAPE (CE-SHAPE) and complementary biochemical techniques (Sztuba-Solinska et al, 2013 , 2016 ).…”