Analysis of Emerging Variants in Structured Regions of the SARS-CoV-2 Genome

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two sets of SARS-CoV-2 genomic sequences to identify emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, 20 variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5ʹ untranslated region (UTR), including a group of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-ss molecular switch in the 3ʹUTR. Finally, 5 variants destabilize structured elements within the 3ʹUTR hypervariable region, including the S2M (stem loop 2 m) selfish genetic element, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. Our analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences.

The highly conserved stem-loop II motif is important for the lifecycle of astroviruses but dispensable for SARS-CoV-2

“…2A ). Because VA1-s2m 18, 24 was viable despite alteration of the stop codon, this finding suggests that the extension of ORF2 by 16 amino acids does not by itself compromise the viability of VA1. Finally, we determined if we could recover a mutant in which two G-C base pair sites were inverted compared to WT.…”

“…At 96 hpi, viral RNA and titers were reduced for VA1-s2m 12, 18, 24, 33 (RNA: 3.6-fold, P= 0.0014; virus titer: 4.4-fold, P< 0.001), VA1-s2m 12, 33 (RNA: 1.5-fold, P= 0.049; virus titer: 4.9-fold P< 0.001) and VA1-s2m 9 (RNA: 3.3-fold, P< 0.001; virus titer: 6.8-fold, P< 0.001). VA1-s2m 18, 24 had no significant differences in RNA or viral titer (both P> 0.06). These results suggest that some of the recombinant viruses have a growth defect, with VA1-s2m 9 having the greatest reduction at 96 hpi.…”

“…S2). Both reversion mutants yielded viable virus in 7 out of 9 replicates for VA1-s2m 12,33 and 9 out of 9 replicates for VA1-s2m 18,24 (Table 1, Fig. 2A).…”

“…Disruption of the VA1 s2m was achieved through deletion (s2m ∆25-41 ), and single (VA1-s2m 24 and VA1-s2m 33 ) or double substitutions (VA1-s2m 12,18 and VA1-s2m 24,33 ) that are predicted to significantly alter the secondary structure of the s2m. The loss of the single or double G-C binding pairs was complemented with compensatory substitutions that are predicted to restore the s2m secondary structure: VA1-s2m 18,24 , VA1-s2m 12,33 , and VA1-s2m 12,18,24,33 . VA1-s2m 32 to VA1-s2m-WT, VA1-s2m 12,18,24,33 was lower at 48 and 96 hpi (48 hpi P= 0.045, 96 hpi P= 0.0014).…”

“…Interestingly, the reference SARS-CoV-2 genome and all subsequent variants of concern encode a unique genetic variant with a uracil at position 32 in the s2m that is distinct from other s2m sequences and is predicted to perturb the secondary structure [19][20][21][22][23] . Additional genetic variants or deletions of the s2m have been periodically detected from clinical isolates [23][24][25][26] .…”