An infectious progenitor for the murine IAP retrotransposon: emergence of an intracellular genetic parasite from an ancient retrovirus

Abstract

Mammalian genomes contain a high load of mobile elements among which long terminal repeat (LTR)- retrotransposons may represent up to 10% of the genomic DNA. The murine intracisternal A-type particle (IAP) sequences, the prototype of these mammalian "genetic parasites," have an intracellular replicative life cycle and are responsible for a very large fraction of insertional mutagenesis in mice. Yet, phylogenetic analyses strongly suggest that they derive from an ancestral retrovirus that has reached the germline of a remote rodent ancestor and has been "endogenized." A genome-wide screening of the mouse genome now has led us to identify the likely progenitor of the intracellular IAP retrotransposons. This identified "living fossil"-that we found to be present only as a single fully active copy-discloses all the characteristics of a bona fide retrovirus, with evidence for particle formation at the cell membrane, and release of virions with a mature morphology that are infectious. We show, by generating appropriate chimeras, that IAPs derive from this element via passive loss of its env gene, and gain of an endoplasmic reticulum targeting signal, resulting in its "intracellularization" and in the gain of transpositional activity. The identification within the mouse genome of the still active retroviral progenitor of the IAP endogenous mobile elements and the experimental dissection of the molecular events responsible for the shift in its life cycle provide a conclusive illustration of the process that has led, during evolution, to the generation of very successful intracellular retrotransposons from ancient retroviruses.

Figure 1.

Structure of IAPs and related elements. (A, top) Genomic organization of an IAP LTR-retrotransposon, with the LTRs flanking the three ORFs homologous to the retroviral gag, pro, and pol genes. (Bottom) Electron microscopy of 293T cells transfected with an expression vector for the retrotransposition competent IAP(92L23) copy, disclosing numerous typical IAP VLPs accumulated in the cisternae of the ER. (Pm) Plasma membrane; (ERm) endoplasmic reticulum membrane. (B) Phylogeny of LTR-retroelements, based on their reverse transcriptase (RT) domain. The tree was determined by the neighbor-joining method using the seven blocks of conserved residues found in the RT domain of all retroelements and was rooted using non-LTR retrotransposons. The retroviruses listed include alpha- (ALV), beta- (MMTV, JSRV, MPMV), gamma- (MLV, GALV), delta- (HTLV-1), epsilon- (WDSV) retroviruses; lentiviruses (HIV-1); and foamyviruses (PFV-1). All sequences are readily accessible from GenBank and previous reports (e.g., Malik and Eickbush 2001). (C) Search for IAP-related retroelements containing a coding-competent env gene. BLAST search in the C57BL/6 mouse genome identifies the already known IAPE-A, -B, and -C elements, together with the yet-unreported IAPE-D subfamily. Sequences of the representative IAPE-D element and the homologous conserved domains in other IAP and IAPE elements are indicated in light blue; non homologous domains between IAP and IAPE elements are indicated in purple; non homologous domains between IAPE-D and the other IAPE subfamily members are indicated in dark blue. The limits of the conserved domains between IAPs and IAPEs were determined by DotPlot analysis (see IAP/IAPE-D dot plot analysis on the right; window size = 15, threshold = 45). For each subfamily, the number of copies in the C57BL/6 genome is indicated together with the number of full-length coding copies.

Figure 2.

Characterization of the IAPE Env glycoprotein. (A) Schematic structure and primary sequence of the IAPE-D1 Env protein. The SU and TM subunits are delineated, with the canonical furin cleavage site (R/K-X-R/K-R) between the two subunits indicated in red. The hydrophobic signal peptide, fusion peptide, and the transmembrane domain are shaded in gray. The G₃₈₄ residue, mutated to E in the IAPE-D2 Env, is underlined. (B) Successive confocal images of a living cell stained for IAPE Env, demonstrating its localization at the cell surface. Human HeLa cells were grown on glass coverslips, transfected with the IAPE-D1 Env expression vector, and stained 48 h post-transfection, without permeabilization, with a rabbit anti-IAPE Env antiserum and an Alexa-Fluor 488-conjugated anti-rabbit IgG secondary antibody. (C) Infectivity assay of SIV particles pseudotyped with the IAPE-D Env proteins. Supernatants of human 293T cells cotransfected with an expression vector for the SIV core proteins, a lacZ gene-marked defective retroviral vector, and an expression vector for IAPE-D1, -D2, or -D2 E384G Env proteins (IAPE-D2*), or a control plasmid (no env) were used to infect human, murine, or hamster target cells. Viral titers, corresponding to the number of LacZ⁺ cell foci per milliliter of supernatant, are indicated as follows: (−) <10; (+) 10–100; (++) 100–1000; (+++) >1000. (D) Western blot analysis of SIV virions contained in the supernatant of transfected cells, pseudotyped (or not) with the IAPE-D1 Env protein. The corresponding samples from the infectivity assay in C were analyzed using a rabbit anti-IAPE Env antiserum. Supernatants were treated or not with peptide-N-glycosidase F (PNGaseF) as indicated.

Figure 3.

Structure of the IAPE-D1 provirus and characterization of its gene products. (A) Genomic organization of the IAPE-D1 provirus, cloned under the control of the CMV promoter (in black) and structure of the corresponding viral transcripts. (SD) Splice donor site; (SA) splice acceptor site. The splice sites of the IAPE subgenomic transcripts (2 and 3) were determined by sequencing the RT-PCR products obtained using total RNAs and the two sets of primers indicated. (B) Northern blot analysis of total RNA extracted from 293T cells transfected with pCMV-β (ctrl) or the IAPE-D1 expression vector (IAPE), using a probe (shown in A) complementary to a 3′ domain of the IAPE genome. (C) Western blot analysis of IAPE-D1 proteins. Whole-cell extracts and virion-containing supernatants of human 293T cells transfected with IAPE-D1 (WT), a protease-deficient mutant (pro⁻), or a control plasmid (ctrl) were analyzed by Western blotting, using rabbit anti-IAPE Gag or anti-IAPE Env antisera, as indicated. (D, 1–4) Electron microscopy of cells transfected with IAPE-D1 and of the released viral particles. (1) Representative low-magnification image of transfected 293T cells, with particles budding at the plasma membrane. The nucleus (Nu) and plasma membrane (Pm) are indicated. (2) High-magnification view of budding and extracellular particles. Prominent spikes, corresponding to the Env protein, are indicated (arrows); (inset) view of an extracellular mature particle, with a condensed central core. (3–4) Immunogold labeling of IAPE particles in transfected 293T cells using the anti-IAPE Gag (3) or anti-IAPE Env (4) rabbit antiserum and a secondary antibody linked to gold beads, observed by electron microscopy. Gold beads are preferentially associated with viral particles (439 ± 165 gold beads/μm² associated with VLPs vs. 5.6 ± 3.0 and 2.8 ± 2.6 gold beads/μm² associated with cytoplasm and VLP-free extracellular medium for the anti-IAPE Gag antiserum and 302 ± 119 gold beads/μm² associated with VLPs vs. 18.1 ± 9.1 and 4.2 ± 2.4 gold beads/μm² associated with cytoplasm and VLP-free extracellular medium for the anti-IAPE Env antiserum).

Figure 4.

Infectivity and functional characterization of IAPE-D1 and mutant derivatives. (A) Rationale of the infectivity assay and viral titer of IAPE-D1. 293T cells were cotransfected with an expression vector for the wild type (see Fig. 3A) or mutant IAPE-D1 derivatives (or pCMV-β as a control) and a neo-marked defective IAPE reporter. Supernatants from the transfected cells were collected 48 h post-transfection and used to infect HeLa target cells. After a 3-d growth period, infection events were detected upon G418 selection of the cells, and viral titers were quantified by counting the number of G418^R clones per milliliter of supernatant (mean values ± SD, n = 3). (B) Structure of de novo integrated IAPE proviruses. The complete characterization of integrated IAPE elements and their insertion sites was performed using individual HeLa clones obtained after infection and G418 selection. A provirus insertion and the corresponding empty, pre-insertion site are schematized at the top, with the sequences of three characterized IAPE de novo insertions shown below. Target-site duplications of 6 bp (TSD, light gray) are found in all cases, associated with reconstituted 5′-LTRs. (C) IAPE genes required for infection. Infectivity assays were performed with either the wild-type IAPE-D1 copy or the same element rendered defective for gag, pro (via in-frame deletions, from nucleotides 1909–2313 and from nucleotides 2876–3139, respectively), pol (via introduction of a stop codon at nucleotide 3802), or env (via an out of frame deletion from nucleotide 7194–7453). The number of G418^R clones obtained per milliliter of supernatant for each construct is indicated. (D) Rationale of the assay and evidence for CTE activity in IAPE-D. The reporter vector contains a cat gene flanked by splice donor and acceptor sites (SD and SA) placed under the control of a simian virus 40 promoter (prom) and polyadenylation signal (pA), and sequences to be tested for CTE activity are inserted as indicated. The presence of a CTE should promote the export of unspliced RNAs from the nuclei of cells transfected with the reporter plasmid, leading to a detectable chloramphenicol acetyltransferase (CAT) activity, whereas the absence of a CTE-like activity should lead to the export of spliced RNA and no CAT activity. 293T cells were transiently transfected with the reporter vector containing the indicated IAPE fragments, placed in the forward or reverse orientation (numbers indicate the nucleotide positions of the 5′- and 3′-ends of each fragment in the IAPE sequence). Forty-eight hours post-transfection, cells were lysed, and CAT activity was determined as described in Methods. The mean ratio of CAT activity between the reporter vector containing an IAPE sequence (or a control MPMV CTE) versus the empty vector (no CTE) was calculated from two to four independent experiments (A.U., arbitrary units; error bars indicate standard deviation).

Figure 5.

Recovery of an extracellular life cycle for an IAP chimera. (A) Structure of the wild-type and chimeric IAPs and morphology of associated VLPs. The IAP-MA^IAPE element was constructed as illustrated by replacing the IAP Gag N terminus with that of IAPE. (1–3) Electron microscopy of 293T cells transfected with either wild-type or chimeric IAPs. (1) Representative low-magnification image of wild-type IAP particles accumulated in the cisternae of the ER. No particle can be observed at the cell membrane or in the extracellular space. (2) Low-magnification image of cells transfected with the chimeric IAP, disclosing particles budding at the cell membrane. No particle can be observed in the ER. (3) High-magnification images of chimeric IAP-associated particles. Images represent (from left to right) budding, free immature, and free mature particles. (B) Immunofluorescence confocal analysis of human HeLa cells transfected with wild-type or chimeric IAPs, fixed, permeabilized, and stained with the anti-Gag antibody (in green). Nuclei are stained with TO-PRO-3 iodide (in blue). (C) Western Blot analysis of whole-cell lysates or cell supernatants from 293T cells transfected with either the wild-type (WT) or the chimeric IAP (MA^IAPE). (Bottom right) Detection of RT activity in the corresponding cell supernatant, using the PERT assay. (D) Functional characterization of wild-type and chimeric IAPs. For the intracellular retrotransposition assay, HeLa cells were cotransfected with the wild-type or chimeric IAP, and a neo^TNF-marked defective IAP reporter in which a (backward) neomycin resistance gene—with the neo ORF interrupted by a (forward) intron—becomes functional only after splicing out of the intron, upon achievement of a complete replicative cycle. Retrotransposition events are detected upon G418 selection of the transfected cells, and frequencies are expressed as numbers of G418^R clones per transfected cells in selection (mean values ± SD, n = 4); see Dewannieux et al. (2004) and Supplemental Figure S2 for a detailed description of the assay. For the infectivity assay, 293T cells were cotransfected with the same IAP plasmids as above, but with in addition the IAPE-D1 Env expression vector. Supernatants from the transfected cells were collected and used to infect naive HeLa cells, as in Figure 4A, and the viral titers were determined upon G418 selection of the target cells (mean values ± SD, n = 3).

Figure 6.

Structural and functional IAPE to IAP transition. (A) Structure of the wild-type and chimeric IAPEs and morphology of associated VLPs. The IAPE-MA^IAP element was constructed as illustrated by replacing the IAPE Gag N terminus with that of IAP. Representative low-magnification images of 293T cells transfected with the wild-type IAPE disclose particles budding at the cell membrane (left). No particle can be observed at the level of the ER. Low-magnification images of cells transfected with the chimeric IAPE (right) disclose particles accumulated in the cisternae of the ER, with a high-magnification image of a particle budding into a cisternae (bottom right inset). No particle can be observed at the level of the cell membrane. (B) Immunofluorescence confocal analysis of human HeLa cells transfected with wild-type or chimeric IAPEs, fixed, permeabilized, and stained with the anti-Gag antibody (green). Nuclei are stained with TO-PRO-3 iodide (blue). (C) Western blot analysis of whole-cell lysates or cell supernatants from 293T cells transfected with either the wild-type (WT) or the chimeric IAPE (MA^IAP). (Bottom right) Detection of RT activity in the corresponding cell supernatant, using the PERT assay. (D) Functional characterization of wild-type and chimeric IAPE. Infectivity was assayed as in Figure 4 either with the wild-type or the chimeric IAPE (mean values ± SD, n = 3). For the retrotransposition assay, HeLa cells were transfected with the wild-type or chimeric IAPE element in which the neo^TNF indicator gene was inserted into the env gene, thus rendered defective (see structures in Supplemental Fig. S2). Retrotransposition frequencies were determined upon G418 selection of the transfected cells, as in Figure 5D (mean values ± SD, n = 3).

Figure 7.

A model for the “intracellularization” of infectious retroviruses. A bona fide infectious retrovirus—for example, the IAP/IAPE progenitor—is “endogenized” upon infection of the germline of a remote ancestor and Mendelian transmission to the following generations, thus resulting in a so-called Endogenous RetroVirus (ERV) (de Parseval and Heidmann 2005; Bannert and Kurth 2006). This ERV may retain the characteristics of retroviruses, that is, produce infectious extracellular particles with a functional envelope protein, which are prone to horizontal transmission and can also amplify in their proper host genome by reinfection of the germline (the IAPEs). “Intracellularization” is expected to correspond to a further adaptation, in which the produced virus-like particles are no more able to exit or to re-enter the cell. For IAPs this has been achieved via the conversion of the plasma membrane targeting function of the Gag MA domain with the acquisition of a signal for Gag-targeting to the ER membrane, and the loss of the env gene. The resulting strictly intracellular life cycle resembles that of primitive Ty-like LTR-retrotransposons (although both classes of elements should be evolutionarily distinct) and is associated—at least in the case of the IAP element—with high-efficiency retrotransposition. For both types of elements, that is, the infectious IAPEs and the strictly intracellular IAPs, defective elements resulting from genetic drift (e.g., point mutation, deletion, recombination, reverse transcription error, DNA-editing) can be complemented in trans by active copies, resulting in the present-day multiple-copy IAP and IAPE subfamilies, with a single “surviving” functional IAPE element, but ∼300 full-length autonomous IAP copies still present due to their high “multicopying” activity.