Detection of genome-scale ordered RNA structure (GORS) in genomes of positive-stranded RNA viruses: Implications for virus evolution and host persistence

Abstract

Discrete RNA secondary and higher-order structures, typically local in extent, play a fundamental role in RNA virus replication. Using new bioinformatics analysis methods, we have identified genome-scale ordered RNA structure (GORS) in many genera and families of positive-strand animal and plant RNA viruses. There was remarkably variability between genera that possess this characteristic; for example, hepaciviruses in the family Flaviviridae show evidence for extensive internal base-pairing throughout their coding sequences that was absent in both the related pestivirus and flavivirus genera. Similar genus-associated variability was observed in the Picornaviridae, the Caliciviridae, and many plant virus families. The similarity in replication strategies between genera in each of these families rules out a role for GORS in a fundamentally conserved aspect of this aspect of the virus life cycle. However, in the Picornaviridae, Flaviviridae, and Caliciviridae, the existence of GORS correlated strongly with the ability of each genus to persist in their natural hosts. This raises the intriguing possibility of a role for GORS in the modulation of innate intracellular defense mechanisms (and secondarily, the acquired immune system) triggered by double-stranded RNA, analogous in function to the expression of structured RNA transcripts by large DNA viruses. Irrespective of function, the observed evolutionary conservation of GORS in many viruses imposes a considerable constraint on genome plasticity and the consequent narrowing of sequence space in which neutral drift can occur. These findings potentially reconcile the rapid evolution of RNA viruses over short periods with the documented examples of extreme conservatism evident from their intimate coevolution with their hosts.

FIGURE 1.

Distribution of Z scores for RNA viruses and controls. Distribution of Z scores of the set of 498-base fragments of control sequences (row 1), and different genera within the Picornaviridae and Flaviviridae (rows 2,3). MFEDs were calculated using NDR for sequence randomization. Each observed distribution (bars) was overlaid with a symmetrical best fit normal distribution (solid black line); the Z-score = 0 value was highlighted by vertical bar. The arrow in the enterovirus panel indicates fragments containing highly structured RNA elements of known function (see text).

FIGURE 2.

Distribution of MFEDs across the genomes of the Picornaviridae and Flaviviridae. Mean MFEDs for individual 498-base sequence fragments across the genomes of Picornaviridae (top row) and Flaviviridae (bottom row) genera that possess (Aphthovirus, Teschovirus, HGV/GBV-C, HCV) or lack (Hepatovirus, Enterovirus, Flavivirus, Pestivirus) GORS are shown. The mean value and variability between members of each genus (standard deviation) are shown as bars and lines, respectively.

FIGURE 3.

Comparison of sequence randomization methods used for calculation of MFEDs. Mean Z scores for individual 498-base sequence fragments of RNA viruses and control sequences using different sequence randomization methods. These preserve different nonrandom sequence ordering features, such as dinucleotide frequencies (NDR, NDS) or both the dinucleotide frequency and codon order (CDLR) in the open reading frame. Abbreviations for virus and control groups on the X-axis (from left) are Flaviviridae (G) HGV-like, (C) hepaci-viruses, (P) pestiviruses, (F) flaviviruses; Picornaviridae (T) teschoviruses, (A) aphthoviruses, (E) enteroviruses, (H) hepatoviruses; Controls (He) herpesviruses, (Po) poxviruses, (Ec) Escherichia coli, (Af) Archaeoglobus fulgidus, (Hu) human mRNA.

FIGURE 4.

Extended survey of MFEDs in animal and plant RNA viruses. MFEDs of sequence fragments of positive-stranded RNA viruses infecting animals and plants (top two panels), and negative-stranded RNA viruses (lower panel), divided into orders (or virion structure for plant viruses), families, and where applicable, genera. MFEDs for sequences in their native genomic configuration are shown as filled boxes; MFEDs for reverse complementary sequences are shaded (for clarity, smaller values are stacked in front).

FIGURE 5.

Relationship between host persistence with MFED and CG underrepresentation. Virus genera were ranked by MFED (left panel) or CG underrepresentation (right panel); viruses were scored as persistent (filled boxes) or nonpersistent (gray). Histograms show mean values for the genus; the error bar corresponds to one standard deviation from the mean within the virus genus/family. First letter (virus family) abbreviations are (P) Picornaviridae; (second-letter, genus abbreviations are [A] aphthovirus; [C] cardiovirus; [P] parechovirus; [H] hepatovirus; [E] enterovirus); (F) Flaviviridae ([P] pestivirus; [F] flavivirus; [C] hepacivirus; [G] HGV-like virus); (C) Caliciviridae ([S] sapovirus; [N] norovirus; [V] vesivirus; [L] lagovirus); (TA) Togaviridae, alphavirus genus; (HE) HEV-like viruses.

FIGURE 6.

Effect of simulated neutral sequence drift on MFEDs of viruses with predicted RNA structure. Coding regions of complete genome sequences of HCV genotype 1b (GenBank accession number HPCJ491), HGV/GBV-B (genotype 2; HGU94695), and FMDV (PIFMDV2) were each mutated independently 10 times to produce sequences showing a range of sequence divergence from the original (X-axis). MFEDs, using the NDR algorithm to generate 50 sequence order randomized sequences, were calculated for each and expressed as a percentage of the MFED of the starting sequence (Y-axis). The distributions of MFED values for the mutated sequences are represented as box and whisker plots (showing 95% percentile [line], standard deviation [upper and lower box], and mean values [line within box], with outliers indicated by the symbol *). For comparison, MFEDs for naturally occurring variants in each virus group expressed as a percentage of the MFED of the starting sequences were plotted using the symbol •.