Structural Characterization of the Viral and cRNA Panhandle Motifs from the Infectious Salmon Anemia Virus

Abstract

Infectious salmon anemia virus (ISAV) has emerged as a virus of great concern to the aquaculture industry since it can lead to highly contagious and lethal infections in farm-raised salmon populations. While little is known about the transcription/replication cycle of ISAV, initial evidence suggests that it follows molecular mechanisms similar to those found in other orthomyxoviruses, which include the highly pathogenic influenza A (inf A) virus. During the life cycle of orthomyxoviruses, a panhandle structure is formed by the pairing of the conserved 5 and 3 ends of each genomic RNA. This structural motif serves both as a promoter of the viral RNA (vRNA)-dependent RNA polymerase and as a regulatory element in the transcription/replication cycle. As a first step toward characterizing the structure of the ISAV panhandle, here we have determined the secondary structures of the vRNA and the cRNA panhandles on the basis of solution nuclear magnetic resonance (NMR) and thermal melting data. The vRNA panhandle is distinguished by three noncanonical U ⅐ G pairs and one U ⅐ U pair in two stem helices that are linked by a highly stacked internal loop. For the cRNA panhandle, a contiguous stem helix with a protonated C ⅐ A pair near the terminus and tandem downstream U ⅐ U pairs was found. The observed noncanonical base pairs and base stacking features of the ISAV RNA panhandle motif provide the first insight into structural features that may govern recognition by the viral RNA polymerase.

Virtual screening of gene expression regulatory sites in non-coding regions of the infectious salmon anemia virus

“…To validate our observations, the 2D models obtained in this study for segment 8 (Additional file 2: Figures S2 and Additional file 3: Figure S3) were compared with the experimental results obtained by Brinson and colleagues [5]. Our bioinformatics analysis showed that the secondary structure model obtained for segment 8 had identical regions to those described experimentally and even predicted the non-canonical U-G interactions previously detected by NMR experiments.…”

“…In these viruses, the interaction of the terminal regions of the NCRs and the subsequent formation of panhandle structures [5] are key in performing these processes.…”

“…Structurally, these conserved sequences in influenza A have been described as partially complementary and capable of interacting in cis within each segment of RNA. This interaction forms structures called panhandles [ 5 , 9 ]. In Orthomyxoviruses , transcription of the genome requires vRNA to act as a template for each genomic segment; for transcription to occur, the conformation adopted via the folding of the NCR is essential [ 9 ].…”

Influenza virus segment 5 (+)RNA - secondary structure and new targets for antiviral strategies

“…Due, however, to vRNA GU pairs being converted to CA mismatches in the cRNA, this cRNA panhandle is structurally distinct. This structural distinction between the vRNA and cRNA panhandle structures is proposed to play a key role in regulating the promoter site used for cRNA, vRNA and mRNA replication 21 . Based on experimental data and bioinformatics analysis, the secondary structure for entire (+)RNA5 is proposed here.…”

RNA Viruses: RNA Roles in Pathogenesis, Coreplication and Viral Load

“…It has been reported that the first 12 and 13 nucleotides correspond to conserved sequences in the 3’ and 5’ ends, respectively, of all segments of the influenza A vRNA [ 25 ]. Structurally, these conserved sequences in influenza A have been described as partially complementary and capable of interacting in cis within each segment of RNA, forming structures called panhandles [ 26 , 27 ] In Orthomyxoviruses, transcription of the genome requires the vRNA to act as template for each genomic segment, and for transcription to occur, the conformation adopted via the folding of the NCR is essential [ 26 ].…”