APOBEC-Mediated Editing of Viral RNA

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Science  30 Jul 2004:
Vol. 305, Issue 5684, pp. 645
DOI: 10.1126/science.1100658


Retroviral DNA can be subjected to cytosine-to-uracil editing through the action of members of the APOBEC family of cytidine deaminases. Here we demonstrate that APOBEC-mediated cytidine deamination of human immunodeficiency virus (HIV) virion RNA can also occur. We speculate that the natural substrates of the APOBEC enzymes may extend to RNA viruses that do not replicate through DNA intermediates. Thus, cytosine-to-uracil editing may contribute to the sequence diversification of many viruses.

Viral sequence variation frequently plays an essential role in viral replication and pathogenesis. For instance, sequence changes can underlie alterations in viral phenotype, escape from host immune responses, and the acquisition of drug resistance. Usually, such changes are due to the fixation of copying errors made during genome replication. Sequence variation may also arise through the post-synthesis editing of RNA or DNA: Measles, hepatitis delta, and polyoma viral RNAs, for example, are presumed substrates for adenine deamination (1). Editing of human immunodeficiency virus (HIV) DNA by the human cytidine deaminase APOBEC3G (hA3G) has also received much recent scrutiny. HIV particles carrying this enzyme yield minus-strand reverse transcripts in which multiple cytosine (C) residues are deaminated to uracil (U). The resulting hypermutation [guanine (G) to adenine (A) on plus strands] and cDNA instability combine to inhibit viral infectivity. Normally, the viral protein Vif protects HIV from hA3G by inducing its degradation and exclusion from virions (2).

Beyond hA3G, mammalian genomes harbor a variety of additional RNA/DNA cytidine deaminases (3). The founder member of this APOBEC family is APOBEC1 (human A1, or hA1), the catalytic subunit of a complex that deaminates C6666 in apolipoprotein B mRNA in human gastrointestinal tissue to yield a premature stop codon. To determine whether other APOBEC proteins can mutate HIV and/or regulate infectivity, wild-type or vif-deficient (△vif) viruses were produced in the presence of various family members, and viral infectivities were measured (Fig. 1A) (4, 5). As expected, hA3G profoundly inhibited the △vif virus but had a mild effect on its wild-type counterpart. Surprisingly, whereas hA1 had negligible effects, rat A1 (rA1) was a potent suppressor of both viruses. Although the basis for the dichotomy between rA1 and hA1 (which share ∼70% amino acid sequence identity) is not understood, we speculate that either hA1 is intrinsically incapable of influencing HIV infection or that 293T cells lack cellular cofactors critical for this activity (6).

Fig. 1.

(A) Infectivity of HIV produced in the presence of hA3G, hA1, or rA1; error bars show standard deviation. WT, wild type. (B) Plus-strand cDNA mutation spectra for the indicated virus/APOBEC pairs; n = the total number of sequenced base pairs. (C) rA1 deaminates viral RNA. Clones (n = 21) were analyzedfor each virus, with all changes marked onto a single horizontal line representing the entire amplicon; C-to-T changes are indicated by asterisks.

Because hA3G induces excessive C-to-U deamination of minus-strand HIV cDNA, we asked whether this extended to rA1. Viral cDNAs were recovered from infected cells, and a nef/3′–long-terminal-repeat (LTR) fragment was then amplified by polymerase chain reaction (PCR), cloned, and individual sequences determined (Fig. 1B). The mutations induced by hA3G were ∼95% G-to-A transitions (5). However, this was only the predominant mutation (∼70%) induced by rA1, with many C-to-T mutations also being evident (∼22%). These particular mutations could have arisen through deamination of unpaired plus-stranded cDNA or virion RNA.

To address the latter possibility, cell-free virions were purified and a nef/3′-LTR region of the genomic RNA was converted to cDNA in vitro, amplified by PCR, cloned, and sequenced (Fig. 1C). There was a clear accumulation of C-to-T (U in the template RNA) changes in the RNAs of HIV produced in the presence of rA1 (17 events in 23,982 bases, a frequency close to that seen with DNA sequencing). Thus, HIV RNA as well as DNA can be a substrate for APOBEC-mediated deamination, making it plausible that C-to-U editing of either could contribute to the sequence diversity that marks natural HIV infection (7). In both cases, this will require expression of APOBEC proteins and their presumed cofactors in the natural targets of HIV infection, namely T lymphocytes and macrophages.

These observations allow speculation (i) that APOBEC-mediated C-to-U editing may contribute to the sequence variation of viruses that replicate entirely through RNA, of which there are many; and (ii) that additional cellular RNA substrates might exist for the APOBEC enzymes. Both possibilities will require further rigorous experimental investigation. It has also been demonstrated that hA3G inhibits the association of hepatitis B virus RNA with viral cores—in this case, in the absence of viral RNA/DNA deamination (8). Thus, APOBECs have the potential to affect their substrates through at least three mechanisms: DNA editing, RNA editing, and a nonediting pathway. The possibilities for APOBEC involvement in the regulation and dysregulation of cell function (6, 7), as well as in innate resistance to invading nucleic acids, may therefore be considerable.

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