Human APOBEC1 cytidine deaminase edits HBV DNA
© Gonzalez et al; licensee BioMed Central Ltd. 2009
Received: 8 July 2009
Accepted: 21 October 2009
Published: 21 October 2009
Retroviruses, hepadnaviruses, and some other retroelements are vulnerable to editing by single stranded DNA cytidine deaminases. Of the eleven human genes encoding such enzymes, eight have demonstrable enzymatic activity. Six of seven human APOBEC3 are able to hyperedit HBV DNA, frequently on both strands. Although human APOBEC1 (hA1) is not generally expressed in normal liver, hA1 can edit single stranded DNA in a variety of experimental assays. The possibility of ectopic expression of hA1 in vivo cannot be ruled out and interestingly, transgenic mice with A1 expressed under a liver specific promoter develop hepatocellular carcinoma. The impact of hA1 on HBV in tissue culture is varied with reports noting either reduced DNA synthesis or not, with cytidine deamination taking a low profile. We sought to examine the hA1 editing activity on replicating HBV. Using highly sensitive 3DPCR it was possible to show that hA1 edits the HBV minus DNA strand as efficiently as hA3G, considered the reference deaminase for HIV and HBV. The dinucleotide specificity of editing was unique among human cytidine deaminases providing a hallmark of use in a posteriori analyses of in vivo edited genomes. Analysis of sequences derived from the serum of two chronic carriers, indicated that hA1 explained only a small fraction of edited HBV genomes. By contrast, several human APOBEC3 deaminases were active including hA3G.
Despite being the prototypic human cytidine deaminase, human APOBEC1 (hA1) first identified in 1995 has been overshadowed by other paralogs, notably activation induced cytidine deaminase (AICDA) and the APOBEC3 gene cluster at ch22q13.1 [1–3]. Human A1 (hA1) edits a single cytidine residue in human apolipoprotein B (apoB) mRNA, a specificity that is conferred by its major interactor, ACF, its expression being confined to intestinal epithelial cells [4, 5]. By contrast, the mouse, rat, dog and horse A1s are expressed in the intestine and other organs including the liver [2, 6, 7]. This situation is probably due to an Alu insertion in a part of the hA1 gene inactivating a generalist promoter [8, 9]. RNA editing specificity can break down in rabbit A1 transgenic mice where hyperediting of the apoB mRNA was described  and subsequently noted in normal mouse intestinal tissues . For transgenic mice expressing the rabbit APOBEC1 gene under the control of a liver specific promoter, hepatic dysplasia and hepatocellular carcinomas were found . Whether this is due to RNA or DNA editing is unknown although in an E. coli DNA mutator assay, hA1 was highly mutagenic meaning that the latter cannot be ruled out . It turned out that human and mouse/rat A1 enzymes are not true orthologs in that the rodent enzymes can hyperedit both RNA and DNA, unlike hA1 that can only deaminate ssDNA . By contrast AICDA and the human APOBEC3 (hA3) show an exclusive single stranded DNA substrate specificity [14–27].
As retroviruses and hepadnaviruses replicate via a single stranded cDNA intermediate, it is not surprising that some are vulnerable to the effects of these cytidine deaminases if expressed in the target cell. While there is a huge literature on the interaction between human immunodeficiency virus (HIV) and the hA3 cluster of genes (the hA3 genes), most of them can also edit hepatitis B virus (HBV) DNA [28–33]. The role of A1 genes on retroviral replication is somewhat checkered. One report has shown that HBV replication is restricted by hA1 yet doesn't address the question of editing . By contrast, another study shows that the rat A1 deaminase hardly impacts HBV replication, even though a little cytidine deamination was found . Both hA1 and rat A1 impact HIV replication in single cycle growth assays . In the mouse A1 hardly restricts Friend murine leukemia virus replication, although using highly sensitive 3DPCR hypermutants were recovered from a small fraction of cultured cells or splenocytes from infected mice .
As the dinucleotide context of editing is frequently a hallmark of different deaminases, for example hA3G shows a strong preference for CpC, while hA3H prefers TpC, the hA1 edited genomes were so analyzed. As can be seen from Figure 1F, hA1 showed a strong preference for TpC and a strong aversion for GpC. It was neutral with respect to ApC. All other hA3 deaminases (hA3s) either avoid GpC and ApC or are neutral (hA3A, hA3C, ). Hence, along with the preference for TpC, this feature is highly distinctive of hA1 editing.
We have previously reported hA3 editing of HBV genomes from sera derived from two chronically infected patients with high viremia (> 109 DNA copies/ml, ). Single molecule analysis of these sequences showed that there was more overlap between the patient data "clouds" than with that of hA3G. Although the individual dinucleotide analyses might suggest that a few genomes are edited by hA1 in vivo (Figures 2D, E vs. 2A, B), the combined dinucleotide analysis (Figure 2C vs. 2F) showed no overlap with the hA1 edited sequence set. We have previously shown that the dinucleotide bias for individual hA3 deaminases varies with the base composition of the locus ; hence, it is not possible to extend the analysis to other edited regions of the HBV genome.
As ~20 hypermutated sequences were derived from each of the sera, the resolution of the argument is ~5%. Obviously with 10 times more patient sequences it might be able to find bona fide evidence of a little hA1 editing. By contrast, hA3G accounts for a sizeable fraction (79% and 58% respectively for patients 130.71 and 12763). Accordingly, other hA3 deaminases must be involved; yet given the similarities between the editing contexts of hA3A, hA3B and hA3C, it is not possible to be more precise. Furthermore, chronic hepatitis shows many facets; and although hA1 expression is confined to the intestinal epithelium and not the liver , its expression profile in a variety of different clinical presentations including highly inflamed cirrhotic tissue is not known.
As mentioned above, hA1 transgenic mice under the control of the liver specific apoE promoter presented with hepatic dysplasia and ultimately liver cancer; thus, the role of cytidine deamination remains open. Even though hA1 does not edit HBV at a high frequency, because hA1 can shuttle between the cytoplasm and nucleus, it is potentially a more likely pro-cancerous candidate than hA3G, which is strictly cytoplasmic.
Differential DNA denaturation PCR
APOBEC1 complementary factor
Human activation induced cytidine deaminase
hA1: Human apolipoprotein B editing complex protein 1
hA2: Human apolipoprotein B editing complex protein 2
hA3: Human apolipoprotein B editing complex protein 3
hA3A-H: Human apolipoprotein B editing complex protein 3 proteins A to H
Hepatitis B virus
Human Immunodeficiency virus type 1.
This work was supported by grants from the Institut Pasteur, ANRS and CNRS. MCG and RS were supported by a bursaries from CONACYT (Mexico) and ARC respectively.
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