Restriction by APOBEC3 proteins of endogenous retroviruses with an extracellular life cycle: ex vivo effects and in vivo"traces" on the murine IAPE and human HERV-K elements
- Cécile Esnault†1,
- Stéphane Priet†1, 2,
- David Ribet†1, 3,
- Odile Heidmann†1 and
- Thierry Heidmann1Email author
© Esnault et al; licensee BioMed Central Ltd. 2008
Received: 24 June 2008
Accepted: 14 August 2008
Published: 14 August 2008
APOBEC3 cytosine deaminases have been demonstrated to restrict infectivity of a series of retroviruses, with different efficiencies depending on the retrovirus. In addition, APOBEC3 proteins can severely restrict the intracellular transposition of a series of retroelements with a strictly intracellular life cycle, including the murine IAP and MusD LTR-retrotransposons.
Here we show that the IAPE element, which is the infectious progenitor of the strictly intracellular IAP elements, and the infectious human endogenous retrovirus HERV-K are restricted by both murine and human APOBEC3 proteins in an ex vivo assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. In silico analysis of the naturally occurring genomic copies of the corresponding endogenous elements performed on the mouse and human genomes discloses "traces" of APOBEC3-editing, with the specific signature of the murine APOBEC3 and human APOBEC3G enzymes, respectively, and to a variable extent depending on the family member.
These results indicate that the IAPE and HERV-K elements, which can only replicate via an extracellular infection cycle, have been restricted at the time of their entry, amplification and integration into their target host genomes by definite APOBEC3 proteins, most probably acting in evolution to limit the mutagenic effect of these endogenized extracellular parasites.
The APOBEC family of cytosine deaminases includes numerous members that can deaminate cytosine to uracil within DNA and/or RNA molecules. Among these enzymes, the APOBEC3 sub-family has been discovered when human APOBEC3G (hA3G) was reported to restrict HIV replication (; reviewed in ). Human hA3G has been shown to trigger extensive deamination of cytosine in the negative viral DNA strand during reverse transcription and to lead to deleterious G-to-A mutations considered as the hallmark of APOBEC3-editing activity. Subsequently, several other human APOBEC3 proteins – including APOBEC3A (hA3A) , APOBEC3B (hA3B) [4, 5], APOBEC3C (hA3C) , APOBEC3DE (hA3DE) , APOBEC3F (hA3F) [7–9] and APOBEC3H (hA3H)  – have been shown to exhibit antiviral effects against a variety of viruses, including numerous retroviruses – i.e. HIV, SIV, MLV, HTLV and foamy viruses –, hepatitis B virus and adeno-associated virus (AAV) (for review ). In contrast to humans, the mouse genome encodes only one APOBEC3 (mA3) protein, which, like human APOBEC3 proteins, displays antiviral effects . Aside from the antiviral function of APOBEC3 proteins against exogenous viruses, some inhibitory effects have been reported on intracellular targets (for review ) and several studies support the notion that the primary function of APOBEC3 proteins could be to prevent the propagation of mobile elements. Indeed, mammalian genomes have accumulated numerous transposable elements which account for > 45% of the genomic DNA [13, 14]. These elements can be grouped into two main classes: the strictly intracellular non-LTR (Long Terminal Repeat) retrotransposons, namely long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), which account for ~30% of each mammalian genome, and the LTR-containing retroelements (including the endogenous retroviruses, ERVs), accounting for ~10% of the genomes and closely related to retroviruses. The life cycle of ERVs includes the formation of virus-like particles (VLPs) that, in several instances – but not systematically – can remain strictly intracellular as observed for the well-characterized murine intracisternal A-particle (IAP) and MusD elements (the so-called "intracellularized" ERVs, [15–18]), or that can bud at the cell membrane for an extracellular cycle as observed for the recently identified murine intracisternal A-particle-related envelope-encoding (IAPE; ) and the human endogenous retrovirus HERV-K(HML2) elements [19, 20]. Although most of these elements are no longer active due to the accumulation of inactivating mutations, some of them are still functional and have been cloned, thus allowing direct ex vivo assay of the effect of APOBEC proteins on their mobility. Accordingly, several APOBEC3 proteins, including hA3A, hA3B, hA3C and hA3F have been demonstrated to restrict the retrotransposition of the human LINE-1 (L1) elements [3, 21, 22], as well as the L1-dependent transposition  of the human Alu SINE elements . Moreover, although no effect on the retrotransposition of L1 elements was observed in the presence of hA3G [21, 25–27], reports have shown that hA3G can prevent the retrotransposition of Alu elements [27, 28] by sequestering Alu RNAs in cytoplasmic high-molecular-mass (HMM) ribonucleoprotein complexes . Similarly, the cloning of active copies for the intracellular murine IAP and MusD elements [15, 17] made possible to demonstrate susceptibility of these retroelements to murine APOBEC3 and to most of the human APOBEC3 proteins [24, 26, 29]. In addition, in silico analyses of the naturally present genomic copies of these elements in the murine genome have revealed "traces" of APOBEC3 editing on these elements (; see also ), thus supporting the physiological relevance of the observed ex vivo assays, and the genomic impact of APOBEC3 protein activity.
Here we take advantage of the recent identification of the infectious progenitor of the intracellularized IAP retrotransposon, namely IAPE, to analyze the possible restriction of a bona fide murine ERV, in a state close to that at the time of its initial endogenization step when the element still behaved as an infectious retrovirus, having not yet reached its highly adapted "intracellularized" state . In parallel, we performed a similar analysis on the human progenitor of the HERV-K(HML2) family members that we had "reconstituted", resulting in the Phoenix element which proved to be a bona fide endogenous retrovirus, the element being able to enter cells by infection and integrate with all the characteristic features of the genomic copies presently found in the human genome . These two functional human and murine "extracellular" ERVs were used to assess the effects of APOBEC3 proteins on mammalian endogenous retroviruses in appropriate ex vivo assays, and refined in silico analyses of the naturally present copies of these elements in their target host genomes finally unambiguously demonstrated "traces" of APOBEC3 editing, with identifiable signatures. Altogether, the data show that APOBEC3 proteins play a role not only on the intracellular retrotransposons found in humans and mice, but also on their retroviral "progenitors" endowed with an extracellular life style, thus de facto filling the gap between the described effects of APOBEC3 proteins on bona fide exogenous retroviruses on the one hand and intracellular retroelements on the other.
Results and discussion
Restriction of murine and human infectious ERVs by APOBEC3 proteins
Traces of APOBEC3 past activity on resident IAPE and HERV-K elements in the murine and human genome
The restriction effects of APOBEC proteins on endogenous retroelements have essentially concerned retrotransposons with a strictly intracellular life cycle, namely the LINE/SINE non-LTR retrotransposons, and LTR-retrotransposons including the yeast Ty1 element [36, 37], and the IAP and MusD murine elements [3, 24, 26, 33]. In these cases severe restriction has been observed, both in ex vivo assays and by in silico analysis of the traces that APOBEC proteins have left through DNA edition in the course of reverse transcription of the retroelements . Here we show that similar effects take place at the level of endogenous retroviruses with an extracellular life cycle, with an unambiguous restriction of the murine IAPE by a murine APOBEC3 protein, and of the human HERV-K element by a human APOBEC3 protein. Taking into account that an infectious IAPE retrovirus with an extracellular life cycle has been the progenitor of the IAP element, the restriction observed for IAP by mA3 appears simply to be the consequence of the restriction that initially controlled the progenitor infectious IAPE invading the rodent ancestor, with the effect being maintained in the evolution of the endogenized IAP retroelements. Although it concerns a heterologous – and therefore not necessarily very relevant-situation, it is noteworthy that the human hA3A protein can control the murine intracellular IAP retroelement, a property not observed for the IAPE infectious progenitor. This is most probably relevant to the localization of the hA3A protein – in the nucleus – and to its rather atypical mode of action – not involving editing of the reverse transcribed DNA – which identifies this restriction factor as more specifically devoted to intracellular retrotransposons, consistent with the absence of reported effects of this factor on – most – infectious retroviruses (reviewed in ). Finally, in silico analysis of the genomic copies of the elements demonstrates that APOBEC3 editing has taken place in evolution for these amplified elements, with clear-cut evidence for a severe heterogeneity in the extent of the editing process.
The human (HERV-K) and murine (IAPE-D) neo-marked ERV copies (pBS CMV-Kcons Stop Env neoAS and pCMV RU5 IAPE neoAS, respectively), the VSV-G and IAPE-D env expression vectors, and the neo-marked autonomous murine IAP retrotransposon (pGL3-IAP92L23 neoTNF) have been previously described [17–19]. The APOBEC3 expression plasmids were obtained from M. Malim (hA3A), the NIH AIDS Research and Reference Reagent program (hA3B, hA3C and hA3F), Open Biosystems (hA3DE), A. Hance (hA3G), and N. Landau (mA3). A plasmid expressing a defective hA3G gene (with a premature stop codon) was used as a negative control. All the APOBEC3 ORF-containing fragments were re-cloned into the pcDNA6 expression plasmid (Invitrogen).
Retrotransposition and infection assays
Retrotransposition assays with the neo-marked IAP were as described previously . For the infection assays, 293T cells seeded in 60-mm-diameter plates were transfected using the Lipofectamine Plus kit (Invitrogen) with 4.5 μg of the neo-marked env-defective murine or human ERV, 0.5 μg of the IAPE or VSV-G env expression vector, and 5 μg of the APOBEC3 expression vector to be tested. Supernatants were harvested 48 h post-transfection, filtered through 0.45-μm pore-size PVDF membranes, supplemented with Polybrene (4 μg/ml), and used to infect HeLa target cells by spinoculation (1.200 g for 2.5 h at 25°C). Infection events were detected upon G418 selection of target cells and viral titers quantified as the number of G418R clones per mL of supernatant [18, 19].
Analysis of integrated proviral DNAs
Cellular DNA from 20–25 individual G418R clones was used to PCR-amplify a 996 bp fragment encompassing the env to neo gene region (nt 6783–7779) of the IAPE-D element and a 2049 bp fragment spanning the neo to gag gene region (nt 1093–3142) of the HERV-K element (initial 3 min denaturation step at 94°C; 40 cycles: 94°C, 50 sec; 60°C, 50 sec; 68°C, 150 sec). PCR reactions were performed with sets of appropriate primers in 50 μl containing 0.5 μg of cellular DNA, 1× Buffer II and 1.5 U AccuPrime Taq DNA polymerase (Invitrogen). The PCR products were electrophoresed on agarose gels, purified with the Nucleospin Extract II kit (Macherey-Nagel) and a ~800 bp or a ~1600 bp fragment was sequenced (Applied Biosystem sequencing kit) for IAPE-D and HERV-K, respectively.
Human and Mouse genome analyses
IAPE-D, IAPE-A and HERV-K endogenous retroviruses were extracted from the mouse and human genome sequence databases (Mouse GoldenPath mm8, February 2006 assembly and Human GoldenPath hg18, March 2006 assembly; http://genome.ucsc.edu/) by using as a querying probe the sequence of the previously described functional IAPE-D1 copy , the sequence of the IAPE-A copy with intact gag-pol open reading frames (chr14-0436, ), and the sequence of the HERV-K element (Phoenix-derived; ) used in the cell-based infection assay. Twenty sequences displaying the highest homology to their cognate probe were selected for the IAPE-D and HERV-K elements. Twenty sequences with the highest homology to the IAPE-A sequence and localized on the Y chromosome were also selected to be used as a control (see Results). Alignments were performed using the ClustalW and Editsequence softwares and consensus sequences generated. Quantitative analysis of the nucleotide substitutions within the IAPE-A, IAPE-D and HERV-K elements was performed using Excel and Hypermut 2.0 (available at the http://www.hiv.lanl.gov/ website) softwares, on the full-length retroviruses. The localization of the analyzed sequences within the mouse and human genomes are given in additional file 1.
Significance levels for the data in Figures 2 and 3 were calculated using the Kruskal Wallis test (GrapPrism software package). More refined analyses for the occurrence of the G-to-A versus C-to-T mutations were performed using a Poisson regression in a log-linear model. The genmod procedure of the SAS software was used (version 9.1, SAS Institute Inc, Cary, NC). The observed distributions of the G-to-A mutations among the GA, GC, GG and GT contexts for HERV-K or the GXA, GXC, GXG and GXT contexts for IAPE were compared to the distribution of these di- or trinucleotides by the chi square test.
The authors wish to thank Anne Aupérin for help in the statistical analyses and Christian Lavialle for critical reading of the manuscript. This work was supported by the CNRS, a grant from the Ligue Nationale contre le Cancer (Equipe labellisée) and fellowships from the CNRS to SP and the Association pour la Recherche sur le Cancer (ARC) to DR.
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