The Roles of APOBEC-mediated RNA Editing in SARS-CoV-2 Mutations, Replication and Fitness

Abstract

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.

One-sentence summary: Efficient Editing of SARS-CoV-2 genomic RNA by Host APOBEC deaminases and Its Potential Impacts on the Viral Replication and Emergence of New Strains in COVID-19 Pandemic.

Fig. 1.. Experimental design of APOBEC-mediated editing of SARS-CoV-2 RNA.

(A) Diagram of the SARS-CoV-2 genomic RNA, showing the positions (box) of the seven RNA segments (–7) selected for studying the RNA editing by APOBECs. (B) Reporter vector (top) that contain each of the seven selected viral RNA segments that are transcribed into an RNA containing an AAV intron between the eGFP and the viral RNA segment. Splicing out the AAV intron yields a mature mRNA with a new spliced junction sequence (JUNC) that differs from its coding DNA, which can be used to selectively amplify either the mature mRNA or the coding DNA. (C) Three APOBEC editor vectors (top, A1–2A-A1CF, A3A, and A3G) and the Western blot showing their expression in 293T cells (bottom). A1–2A-A1CF is constructed as one open reading frame (ORF) with a self-cleavage peptide T2A inserted between A1 and A1CF, which produced individual A1 and A1CF proteins in a 1:1 ratio (26, 54). (D) Strategy of the Safe-Sequencing-System (SSS) to minimize errors from PCR amplification and sequencing. After the SARS-CoV-2 RNAs from cell extracts are reverse transcribed, the cDNAs are sequentially amplified by the UID barcode (2 cycles) and the Illumina adapter (30 cycles). This SSS approach will distinguish the C-to-U mutations caused by APOBECs from the PCR and sequencing errors (See Methods in SI).

Fig. 2.. Local sequence context at the APOBEC-edited C sites on SARS-CoV-2 RNA.

(A) Local sequences around the significantly edited target C sites (± 5 nucleotides from target C at position 0) by A1+A1CF, A3A, or A3G. The editing level of each C site was normalized to the Ctrl, and only sites with 3x or higher editing levels than the normalized value were defined as significant editing sites. (B) Analysis of local sequences around the top 30% edited C sites (or hotspot editing sites), showing predominantly AC motif for A1+A1CF, UC for A3A, and CC for A3G. (C-D) Comparison of the C-to-U editing rates (%) of different dinucleotide motifs by a particular APOBEC (panel-C) and the C-to-U editing rates (%) of a particular dinucleotide motif by the three APOBECs (panel-D). Each dot represents the C-to-U editing level obtained from the SSS results. In panel-D, statistical significance was calculated by unpaired two-tailed student’s t-test with P-values represented as: P > 0.05 = not significant; not indicated, * = P < 0.05, *** = P < 0.001, **** = P < 0.0001.

Fig. 3.. Overall features of the RNA around the most preferred APOBEC-edited sites on SARS-CoV-2.

The predicted RNA secondary structures (55) of the sequences near the top 3 highest editing C sites by A1+A1CF (A), A3A (B), and A3G (C) (See related Table S2). The editing efficiency of each site is listed at the top of each panel. In the secondary structure, the target C sites are highlighted in red, and −1 positions of the target C sites are highlighted in green for A, pink for U, and blue for C, respectively. In panel-A, the proposed canonical mooring sequences for A1+A1CF (highlighted in sky blue) contain relatively high U/A/G contents downstream of the target C.

Fig. 4.. The potential effect of APOBEC-mediated editing on SARS-CoV-2 mutations and fitness.

(A) The number of mutational events (all single nucleotide variants) on SARS-CoV-2 RNA segment 5’UTR-Orf1a (segment 1 in Fig. 1A) from the SARS-CoV-2 genome sequence data (the Nextstrain datasets from Dec. 2019 to Jan. 22nd, 2022 downloaded from the GISAID database, https://www.gisaid.org/hcov19-variants/ and https://nextstrain.org/ncov/global). The C203, C222, and C241 represent many of the C-to-U mutational events (asterisks) with the A3A-editing UC motif in the SARS-CoV-2 variants. (B) The A3A-mediated C-to-U editing rate on UC motif in the same 5’UTR-Orf1a region obtained from our cell-based editing system and the SSS analysis. The Ctrl (EV) editing levels (or background error rates) of the corresponding region are presented as negative values (%). The C203, C222, and C241 (asterisks) all showed significant editing by A3A. (C) The C-to-U mutation prevalence over time at C203, C222, and C241. The sequencing frequency is represented by C in blue and U in yellow (referred to the Nextstrain datasets: https://nextstrain.org/ncov/global). This analysis showed that SARS-CoV-2 started to acquire the C-to-U mutation at C241 in January 2020. By July 2020, 90% of the circulating viral variants carry this mutation at C241. By March 2021, almost all circulating viral variants have this mutation, suggesting the C241 to U mutation in the 5’UTR is beneficial to the viral fitness (see Fig. S9).

Fig. 5.. SARS-CoV-2 replication and virion production in cells expressing APOBECs.

(A) Overview of experiments for SARS-CoV-2 replication and viral production in the presence of APOBECs. The Caco-2 stable cell lines were constructed to express A1+A1CF, A3A, or A3G under a tetracycline-controlled promoter. The Caco-2-APOBEC stable cell lines were then infected with SARS-CoV-2 (MOI = 0.05), and the viral RNA replication and progeny production were measured at different time points. (B) Effect of each APOBEC expression on SARS-CoV-2 viral RNA replication. Measurement of relative viral RNA abundance at different time points after viral infection of the Caco-2-APOBEC stable cell lines expressing A1+A1CF, A3A, or A3G. The viral RNA abundance was measured using real-time quantitative PCR (qPCR) to detect RNA levels by using specific primers to amplify three separate viral regions, the Nsp12, S, or N coding regions (see Methods in SI). (C) Effect of each APOBEC expression on SARS-CoV-2 progeny production. Infectious viral progeny yield harvested in the medium at 48 hrs and 72 hrs post-infection was determined by plaque assay (see Methods). Statistical significance was calculated by unpaired two-tailed student’s t-test with P-values represented as: P > 0.05 = not significant, *** = P < 0.001.