Background
Humans and nonhuman primates (NHPs) possess seven APOBEC3 (A3) enzymes, which restrict various viruses, notably HIV in humans and SIV in NHPs. These antiviral proteins inhibit viral infection by inducing G>A mutations in viral genomes and/or through mutation-independent mechanisms. HIV and SIV counteract A3 enzymes via their virion infectivity factor (Vif), which promotes A3 proteasomal degradation. Despite this, G>A mutations remain prevalent in viral genomes. A3G mainly induces GG>AG mutations, which frequently generate stop codons, while other A3 enzymes induce GA>AA changes, are less mutagenic, and do not produce new stop codons.
Methods
We analyzed all HIV and SIV sequences available at the Los Alamos National Laboratory (LANL) databases and publicly available RNA-seq data from Sequence Read Archive (SRA). Sashimi plots were used to visualize mRNA splicing. Deaminase activity and dinucleotide substrate preferences were assessed via oligo-based deaminase assays. Oxford Nanopore Technologies (ONT) MinION Long-read sequencing incorporating adaptive sampling (AdS) was used to obtain full-length A3 isoforms.
Results
Our analyses revealed species-specific G>A hypermutation and mRNA splicing patterns of A3 enzymes. Specifically, we found mRNA splicing defects in Old-World monkey A3Gs resulting in a significantly reduced level of GG>AG mutations in SIV compared to HIV. Furthermore, a 48-hour ONT MinION sequencing run on rhesus macaque PBMCs using AdS depletion achieved 8x-302x coverage of A3 enzymes despite their low expression. This analysis identified novel variants of A3A, A3C, A3G, and A3H, and revealed additional splicing defects in these genes and many other genes across human and NHP models.
Conclusions
A3 and many other genes undergo species-specific splicing, highlighting the need for understanding the functional relevance of these molecular differences across animal models and humans to ensure that research findings in animal models are translatable. We are currently conducting functional assays to assess the role of A3 splice variants in species-specific anti-viral immunity. Our long-term goal is to extend this study to other immune genes.
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