Conserved Structural Motifs of Two Distant IAV Subtypes in Genomic Segment 5 RNA

Abstract

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

In silico analysis of local RNA secondary structure in influenza virus A, B and C finds evidence of widespread ordered stability but little evidence of significant covariation

“…IAV had an overall average z-score (z avg ) of −0.51 ± 1.14 and −0.53 ± 1.14 for the positive and negative strand, respectively, IBV had −0.56 ± 1.11 and −0.60 ± 1.07, and ICV had −0.63 ± 1.12 and −0.63 ± 1.08. The negative trend observed in these data indicate some potential for influenza being inherently structured and are in-line with previous predictions performed on influenza, which found similar skews in predicted z-score 31 .…”

“…Subsequent work challenged this finding 43 , however the significance of covariation in these RNAs remains a point of contention. Similarly, previous studies posited the existence of conserved structural elements which were (at least for IAV) subjected to subsequent structural probing 20 , 21 , 23 , 24 , 44 – 46 and functional analyses 13 , 14 , 17 – 19 , 22 , 25 , 31 . No motifs with statistical evidence of covariation were found in IAV, and the few hits we did observe were in ICV; indeed, the only motif with wide conservation (across clades) was found in ICV.…”

Structural and Functional RNA Motifs of SARS-CoV-2 and Influenza A Virus as a Target of Viral Inhibitors

“…The correlation between the function and the structure of the influenza virus RNA has been intensively studied for many years. The RNA secondary structures have been studied in vitro for segment 5: vRNA5 (strain A/Vietnam/1203/2004 (H5N1)), strain A/California/04/2009 (H1N1)), (+) RNA5 (strain A/California/04/2009 (H1N1)); segment 7: vRNA7 (strain A/Vietnam/1203/2004 (H5N1)); and segment 8: vRNA8 (strain A/Vietnam/1203/2004 (H5N1), strain A/California/04/2009 (H1N1)) [ 41 , 42 , 43 , 44 , 45 ] ( Figure 6 ). Through the use of methods such as chemical mapping, isoenergetic microarrays, RNase H cleavage, assessment of base pairing probabilities, and conservation of canonical base pairs in strains, the complex nature of vRNA secondary structures has been revealed [ 130 ].…”

“…Some structural RNA motifs were experimentally determined in vitro [ 34 , 36 , 37 ], and a compensatory mutagenesis study of the RNA structural motifs of the influenza A virus was published [ 38 , 39 , 40 ]. Recently, in vitro secondary structure models based on experimental data were proposed for full-length vRNA5, vRNA7, and vRNA8 of A/Vietnam/1203/2004 (H5N1) and vRNA5 and vRNA8 of A/California/04/2009 (H1N1) [ 41 , 42 , 43 , 44 , 45 ], as well as segment 5 (+) RNA of A/Vietnam/1203/2004 [ 46 ]. A secondary structure for vRNA8 of A/California/04/2009 (H1N1) formed in the presence of cellular and viral components was proposed [ 47 ].…”

Universal and strain specific structure features of segment 8 genomic RNA of influenza A virus—application of 4-thiouridine photocrosslinking

“…Many of influenza’s viral processes have been tied to RNA structures, and key motifs are highly conserved across strains ( 7 , 8 , 9 , 10 , 11 ). Previously, the secondary structure of three entire viral genomic segments (8, 7, and 5) of A/Vietnam/1203/2004 (H5N1) was determined ( 12 , 13 , 14 , 15 ). Connections between viral RNA structure variances and their pandemic potential have been shown in several cases ( 16 , 17 , 18 ).…”