The Anti-HIV-1 Cytidine Deaminase APOBEC3G Is a Cellular Site-Specific RNA Editing Enzyme

Background:The APOBEC3 (A3) family of cytidine deaminases in primates is comprised of seven homologous enzymes that are structurally related to the RNA editing enzyme APOBEC1. APOBEC3G (A3G) is a restriction factor for HIV-1 and endogenous retroviruses, and is highly expressed in T lymphocytes. Encapsidation of A3G into HIV-1 particles is essential for its antiviral activity which leads to hypermutation of its cDNA in target cells, and requires RNA binding by A3G to form a ribonucleoprotein complex with viral proteins. A3G can also reduce HIV-1 production in producer cells independently of its DNA deaminating activity. A3G has homologous N-and C-terminal catalytic domains (NTD and CTD) but only the CTD is active for deamination of ssDNAs. The zinc-coordinating catalytic residues as well as non-catalytic residues in A3G-NTD are known to bind RNA and this interaction is required for A3G's binding to the HIV-1 nucleocapsid for recruitment into nascent virions as well as for A3G dimerization. A3G binds to DNA and RNA substrates with similar affinity. Thus far, studies have demonstrated DNA deamination by A3G whereas deamination has not been observed in HIV-1 RNA or synthetic RNA oligonucleotides, thereby, ruling out the RNA editing function of A3G. We recently described that the structurally related enzyme A3A induces widespread site-specific C-to-U RNA editing of cellular transcripts in pro-inflammatory macrophages and in monocytes exposed to hypoxia and/or interferons. We hypothesized that A3G may also have RNA editing function, which may play a role in HIV-1 restriction.

Methods:To determine if A3G is capable of RNA editing, we transiently overexpressed the protein in 293T cells, a model routinely used by various labs to study A3G function and its mode of HIV-1 restriction, and then performed transcriptome-wide RNA sequencing (RNA-Seq), Sanger sequencing and site-directed mutagenesis.

Results: RNA sequencing analysis showed site-specific RNA editing in hundreds of genes' transcripts, including approximately 200 genes that acquire protein recoding. The transcripts edited by A3G are largely distinct from those edited by A3A. We find that several host genes including NMT1, CHMP4B, MAPK1, ACIN1, MED1, NFAT5, RBM14 which areinvolved in HIV-1 infection acquire pathogenic recoding RNA mutations by A3G-mediated RNA editing. By performing Sanger sequencing of PCR-amplified cDNA, we validated site-specific, non-synonymous C-to-U RNA editing for 21 of 21 (100%) tested sites in 20 genes that we had selected for experimental confirmation. As expected no genomic mutations were seen in the DNA sequences corresponding to the RNA-edited sites in 11 tested genes. The discovery of A3G's RNA editing function prompted us to study the role of the N-terminal domain in RNA editing. We made mutations in the zinc-coordinating and non-catalytic residues in both N-terminal and C-terminal domains of A3G. We demonstrate that mutating zinc-coordinating residues in either N- and C-terminal domains of A3G in 293T cells greatly reduce or abolish editing in its target transcripts.

Conclusions: We demonstrate a novel RNA editing function for the A3G cytidine deaminase. Our study shows that the RNAs of genes involved in HIV-1 replication, assembly, transcription and infectivity are targets of A3G-mediated RNA editing. This result raises the possibility that the editing of host transcripts may be a novel mechanism by which HIV-1 infection is inhibited by A3G. Our findings suggest a previously unrecognized role for the N-terminal domain of A3G in RNA editing. A3G is the second of the seven members of the APOBEC3 family of cytidine deaminases and the first two-domain cytidine deaminase for which a previously unrecognized RNA editing activity has been discovered. It suggests that other APOBEC3 proteins may also possess hitherto unknown RNA editing activity that may underpin some of their biological roles. Our findings have the potential to significantly expand on the role of C-to-U RNA editing in epitranscriptomic regulation in T lymphocytes, define specific gene targets of A3G-mediated RNA editing and open new avenues of inquiry on the functions of APOBEC3 genes in HIV-1 restriction.