Murine APOBEC1 Is a Powerful Mutator of Retroviral and Cellular RNA In Vitro and In Vivo

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Abstract

Mammalian APOBEC molecules comprise a large family of cytidine deaminases with specificity for RNA and single-stranded DNA (ssDNA). APOBEC1s are invariably highly specific and edit a single residue in a cellular mRNA, while the cellular targets for APOBEC3s are not clearly established, although they may curtail the transposition of some retrotransposons. Two of the seven member human APOBEC3 enzymes strongly restrict human immunodeficiency virus type 1 in vitro and in vivo. We show here that ssDNA hyperediting of an infectious exogenous gammaretrovirus, the Friend–murine leukemia virus, by murine APOBEC1 and APOBEC3 deaminases occurs in vitro. Murine APOBEC1 was able to hyperdeaminate cytidine residues in murine leukemia virus genomic RNA as well. Analysis of the edited sites shows that the deamination in vivo was due to mouse APOBEC1 rather than APOBEC3. Furthermore, murine APOBEC1 is able to hyperedit its primary substrate in vivo, the apolipoprotein B mRNA, and a variety of heterologous RNAs. In short, murine APOBEC1 is a hypermutator of both RNA and ssDNA in vivo, which could exert occasional side effects upon overexpression.

Introduction

Human APOBEC3 molecules deaminate cytidine residues in single-stranded DNA (ssDNA).1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 Particularly vulnerable to the action of the cytoplasmic APOBEC3F and APOBEC3G cytidine deaminases (hA3F and hA3G) is the cDNA of the human immunodeficiency virus type 1 (HIV-1).3, 4, 5, 6, 7, 9, 11, 12 Other members of the seven-gene human APOBEC3 locus on chromosome 22 have little or no effect.13, 14, 15 To overcome this sensitivity, HIV-1 encodes a gene product, the Vif protein, that effectively neutralizes hA3F and hA3G.3, 4, 5, 6, 7, 9, 11, 12 In the absence of Vif, cytidine residues in neo-synthesized minus-strand DNA are edited, resulting in multiple C  U mutations. These show up as so-called G  A hypermutants when compared with the viral plus strand as reference. The vif gene is a hallmark of all members of the large lentiviral subgroup of retroviruses but one. By contrast, no other retrovirus group, including the hepadnaviruses, encodes an additional gene product that has any homology whatsoever with vif. There is no consensus as to whether the Bet protein of foamy virus counters APOBEC3 activity.16, 17 Given the lack of sequence homology, it is not a Vif ortholog.

The human APOBEC3 proteins belong to the larger APOBEC1, APOBEC2, and AID family of mammalian enzymes, of which APOBEC1 is the prototype, that encode a distinct zinc finger motif with clear homology to cytidine, adenosine, and even some bacterial guanosine deaminases.18, 19, 20, 21, 22

Both humans and mice encode single APOBEC1 (hA1, mA1) and APOBEC2 (hA2, mA2) genes.23, 24, 25 While no substrate or function has been identified for any A2, hA1 specifically deaminates residue C6666 in human apolipoprotein B (apoB) mRNA when part of a multiprotein complex.26, 27, 28 Although human APOBEC1 expression is generally confined to the small intestine, for the mouse, rat, rabbit, and dog APOBEC1, there is a much larger tissue distribution, including hepatocytes and spleen,29, 30, 31, 32, 33 following from the fact that there are two promoters for distinct transcripts for the mouse, whereas there is a single promoter in humans.23, 31, 34 Despite this, the human and mouse proteins are strictly colinear, there being 71% amino acid identity.

A broader substrate specificity for APOBEC1 deaminases is suggested by hA1-associated DNA editing in an Escherichia coli mutator assay.35, 36 Using another heterologous system, it was recently shown that rat A1, but not human A1, curtailed HIV-1 replication in vitro, the restriction being accompanied by cytidine editing of minus-strand DNA at a frequency of ∼ 1 × 10 2 and an even lower editing of cytidines in virion RNA (∼ 4 × 10 3).37

The impact of mouse APOBEC3 (mA3) on the replication of murine retroviruses remains unclear. Friend–murine leukemia virus (Fr–MLV), which encodes the strict minimum of three retroviral genes, gag, pol, and env, is apparently resistant to mA3 even when overexpressed by the powerful cytomegalovirus immediate early promoter.38 Some reports suggest that mA3 may be packaged, but there is no consensus,39 while it was recently suggested that the MLV protease could cleave mA3, although this issue remains open too.40 Increased spread of mouse mammary tumor virus in mA3−/− mice has been observed even though no deamination of viral cDNA was found.41

We sought to examine the impact of all three mouse APOBECs on the replication of MLV in culture and in vivo. Fr–MLV was chosen as it causes frank disease within as short as 2 months of infection in 100% of newborn mice.42 It is shown here that although mA1 and mA3 have little impact on MLV replication, a small fraction of viral genomes does indeed undergo massive editing of nascent DNA by both enzymes. Interestingly, mA1 can extensively edit viral RNA, while hA1 cannot, in vitro, indicating that the two enzymes are not strictly orthologous. Edited MLV genomes were identified in vivo and probably arose as a result of APOBEC1 as opposed to APOBEC3. In a general context, murine APOBEC1 is able to hyperedit its primary substrate in vivo, the apoB mRNA, and a variety of heterologous RNAs. In conclusion, in this study, we have demonstrated that murine APOBEC1 is a hypermutator of both RNA and ssDNA in vivo.

Section snippets

mA1 and mA3 but not mA2 can edit MLV cDNA

The impact of the three mouse APOBEC molecules on MLV infectivity was assessed in a simple growth assay. Human 293T cells were transfected with an infectious molecular clone of MLV in the presence or absence of the three mouse APOBEC genes, mA1, mA2, and mA3. Seventy-two hours post-transfection, viruses with and those without packaged APOBEC molecules were harvested and used to infect NIH 3T3 cells. No difference in the kinetics of replication out to 12 days was observed whatsoever as revealed

Discussion

In the absence of the HIV-1 vif gene product, hA3G and hA3F molecules are packaged during budding, resulting in deamination of cDNA in the next round of infection.3, 4, 5, 6, 7, 9, 11, 12 By comparison, gammaretroviruses, such as MLV, lack a vif ortholog or analog. With the use of exquisitely sensitive 3D-PCR, the present findings show that a small proportion of MLV genomes may undergo massive ssDNA editing (i.e., G  A hypermutants) by homologous and heterologous APOBEC deaminases in vitro. Most

Plasmids

Murine APOBEC1, APOBEC2, and APOBEC3 cDNAs were a gift from N. Landau, while human APOBEC1, hA2, hA3G, hACF, and mACF were obtained from N. Navaratnam; hA3A was obtained from B. Cullen. The different human APOBEC3 cDNAs were obtained from an IMAGE clone cDNA library and were subcloned in the pcDNA3.1D/V5-His-TOPO expression vector (Invitrogen, France). The MLV strain 57 infectious molecular clone has been reported,42 while pNL4.3 and pSV2tat plasmids encode for HIV-1 and tat, respectively, and

Note added in proof

Since this manuscript was submitted, two articles have shown that in Fr–MLV-resistant mice, the Rfv3 restriction factor might be murine APOBEC3.62, 63 However, as Fr–MLV is restricted by multiple genes, it is not clear whether murine APOBEC3 is a major restriction factor in feral mice. By contrast, the present study shows that mA1 and mA3 are minor players in Fr–MLV-sensitive mice.

Acknowledgements

This work was supported in part by grants from the Pasteur Institute, Agence Nationale de Recherche sur le SIDA, Fondation pour la Recherche Médicale, Sidaction, Association pour la Recherche contre le Cancer, and Fondation de France/Comité Leucémie. We thank Monica Sala, Chantal Jacquet, and Marc Plays for their help with mouse experiments.

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