Hidden evolutionary constraints dictate the retention of coronavirus accessory genes

Abstract

Coronaviruses exhibit many mechanisms of genetic innovation, including the acquisition of accessory genes that originate by capture of cellular genes or through duplication of existing viral genes. Accessory genes influence viral host range and cellular tropism, but little is known about how selection acts on these variable regions of virus genomes. We used experimental evolution of mouse hepatitis virus (MHV) encoding a cellular AKAP7 phosphodiesterase and an inactive native phosphodiesterase, NS2, to model the evolutionary fate of accessory genes. After courses of serial infection, the gene encoding inactive NS2, ORF2, unexpectedly remained intact, suggesting it is under cryptic constraint uncoupled from the function of NS2. By contrast, AKAP7 was retained under strong selection but rapidly lost under relaxed selection. Experimental evolution also led to altered viral replication in a cell-type-specific manner and changed the relative proportions of subgenomic viral RNA in plaque-purified viral isolates, revealing additional mechanisms of adaptation. Guided by the retention of MHV ORF2 and similar patterns in related betacoronaviruses, we analyzed ORF8 of SARS-CoV-2, which is proposed to have arisen via gene duplication and contains premature stop codons in several globally successful lineages. As with MHV ORF2, the coding-defective SARS-CoV-2 ORF8 gene remained largely intact in these lineages, mirroring patterns observed during MHV experimental evolution, challenging assumptions on the dynamics of gene loss in virus genomes, and extending these findings to viruses currently adapting to humans.

Keywords: SARS-CoV-2; coronaviruses; evolutionary genetics; experimental evolution; viral gene expression; virus evolution.

Figure 1.. Experimental evolution of mouse hepatitis virus reveals hidden selective constraints.

A) Schematic of the MHV genome with ORF2 and its PDE protein product NS2, highlighted. B) Schematic of the MHV^AKAP7 genome, showing the inactive NS2 protein product and insertion of the AKAP7 gene in place of ORF4. C) Replication of wild-type MHV, MHV^NS2mut , MHV^AKAP7, and MHV^AKAP7mut in macrophages and L2 fibroblasts 24 hours post-infection at an MOI of 0.01. Statistical testing was conducted by 2-way ANOVA. One experiment representative of three is shown. D) Schematic of the experimental evolution protocol using conditions of strong and relaxed selection. E-F) PCR analysis of AKAP7 at passage 1 to 10 in macrophages and L2 fibroblasts, respectively. The black arrow indicates full-length intact AKAP7, while red arrows indicate AKAP7 amplicons with deletions. PCR analysis of AKAP7 in the remaining two macrophage and L2-passaged virus populations is available in Figure S1A-D. G) Replication in macrophages 24 hpi (MOI=0.01) of p0 MHV^AKAP7 and p10 MHV^AKAP7 populations from L2 fibroblasts and macrophages. Merged data from three independent experiments is shown and was subjected to statistical testing by 2-way ANOVA. H) Replication of plaque purified isolates in macrophages. Values are the average titer of all plaques. Statistical testing was done by unpaired t-test. I) Replication of plaque purified isolates in L2 fibroblasts. Values are the average titer of all plaques. Statistical testing was done by unpaired t-test. J-K) PCR analysis of ORF2 in macrophages and L2 fibroblasts, respectively. Images are representative of all three virus populations. Additional data related to this Figure can be found in Figure S1.

Figure 2.. Nanopore direct cDNA sequencing shows retention of ORF2 and near-complete loss of AKAP7 following serial passage.

A-C) Relative coverage depth plots of ORF2 in purified plaque isolates from passage 0 MHV^AKAP7, and passage 10 macrophage and L2 fibroblast purified plaques. D-E) Relative coverage depth plots of AKAP7 in purified plaque isolates from passage 0 MHV^AKAP7, and passage 10 macrophage and L2 fibroblast purified plaque isolates. PCR analysis of AKAP7 in plaque-purified virus isolates, as well as coverage plots from additional L2-derived isolates that were sequenced are available in Figure S2. See also Table S1.

Figure 3.. Experimental evolution of MHV^AKAP7 alters relative expression of viral subgenomic RNA.

A-C/E-G) Indicated sgRNAs plotted as a percentage of total subgenomic RNA for p0, L2 p10, and macrophage p10 passaged viruses. Individual L2 plaques are consistently colored across plots. D) Percentage of total reads with leader-body junctions that included the AKAP7-specific junction sequence. Run-statistics for all direct cDNA ONT sequencing are available in Table S1. Read-counts underlying the subgenomic RNA analysis are available in Table S2.

Figure 4.. ORF8 is retained in SARS-CoV-2 lineages despite mutations that disrupt protein coding.

A) Schematic of SARS-CoV-2 genome. B) Schematic (with ORF8 expanded, not to scale) of ORF8, with mutations resulting in loss of subgenomic mRNA synthesis (BA.5) or an early stop codon (B.1.1.7 and XBB.1.x) indicated. Figure S3 contains the histograms showing the epidemic curve of each lineage. C) Deletion length distribution - this displays the size of all deletions in ORF8 in each lineage from 0 to >100 nucleotides. Table S3 contains the raw data underlying this plot. D-F) Proportion of genomes from lineages with a deletion at each position in SARS-CoV-2 ORF8. Figure S4 contains these plots for the XBB lineages we analyzed.

Figure 5.. SARS-CoV-2 ORF8 is recurrently retained following acquisition of an early stop codon.

A-F) Line plots of percent nucleotide content for the indicated SARS-CoV-2 lineages over time throughout the course of their circulation in humans. Dark lines are the mean percentage for all sequences collected on that date, and faded lines are the 95% confidence interval. Dashed vertical line is the date by which 50% of sequences assigned to the designated lineage were collected, and the solid line is the 90% cutoff. Figure S3 contains the histograms showing the epidemic curve of each lineage analyzed. Figure S4 contains plots of deletions at each position in ORF8 for the XBB lineages we analyzed.