APOBEC3 (Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3) proteins are a family of cytosine deaminases known for their role in restricting the replication of diverse viral families, and best characterised for their activity against retroviruses. APOBEC3 genes are exclusive to placental mammals, while APOBEC5 is present in marsupial mammals. However, its antiviral activity is unknown. Here we investigate whether marsupial APOBEC5 proteins are an example of convergent evolution with functional antiviral equivalence to placental APOBEC3.
Sequence and phylogenetic analysis of mammalian APOBEC-family proteins reveal that APOBEC5 proteins possess the key conserved sequence motifs required for cytosine deaminase activity and form a discrete clade of proteins that are present only within marsupial genomes. To assess the function of APOBEC5, we analysed the activity of marsupial APOBEC5 proteins from the South American Grey short-tailed opossum (Monodelphis domestica) and the Australian Tasmanian devil (Sarcophilus harrisii). Rifampicin mutagenesis analysis revealed that APOBEC5 is significantly mutagenic when expressed in E. coli, with genomic mutation hotspots at positions previously observed for other APOBEC-family proteins. When APOBEC5 was co-expressed in mammalian 293T cells with an infectious molecular clone of the retrovirus, murine leukemia virus (MLV), a trend in the reduction of viral particle release was observed compared to cells expressing MLV alone. To find signatures of APOBEC3-like activity against ancient retroviral infections we performed a hypermutation analysis of endogenous gammaretroviruses and betaretroviruses within the opossum genome. This revealed APOBEC3-like activity in the analysed gammaretroviruses but not the betaretroviruses, with strand-biased deamination with a predominantly GY dinucleotide context.
These data suggest that marsupials have convergently evolved APOBEC3-like antiviral activity against some but not all retroviruses, while possessing mutagenic APOBEC5 but not APOBEC3 genes. Our findings provide new insights into the evolution of antiviral innate immunity in marsupials.