* UMR 2714 CNRS-bioMérieux IFR128 BioSciences Lyon-Gerland, Ecole Normale Supérieure de Lyon, Lyon, France; and Laboratoire de Biométrie et Biologie Evolutive, UMR CUR 5558, Université Claude Bernard Lyon 1, Lyon, France
Correspondence: E-mail: francois.mallet{at}ens-lyon.fr.
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Abstract |
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Key Words: HERV-W paralogous gene orthologous gene envelope endogenous retrovirus hominoid
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Introduction |
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Insertion of retroviral elements can be a source of gene novelties, by providing either new protein-coding regions or regulatory elements. Human chromosomes contain thousands of retrovirus-like sequences, representing about 8% of the genome (International Human Genome Sequencing Consortium 2001). It is generally admitted that these Human Endogenous RetroViruses (HERVs) derive originally from rare events of germ-line infections by exogenous retroviruses leading to proviral DNA integration. The infectious retrovirus founding the contemporary HERV-W family (Blond et al. 1999) entered the human ancestor genome after the divergence between Catarrhini and Platyrrhini (Kim, Takenaka, and Crow 1999; Voisset et al. 2000), i.e., less than 40 MYA (Goodman et al. 1998). The spread of the HERV-W family into the genome essentially results from events of intracellular retrotransposition of transcriptionally active copies, a phenomenon mediated either by their own reverse transcriptase (RT) machinery or by RT from LINE elements (Costas 2002; Pavlicek et al. 2002). Generally, due to the absence of a selective pressure, HERV-W elements have accumulated inactivating substitutions (frame-shifts, nonsense mutations), leading to complex multicopy families whose transmission is exclusively Mendelian. Thus, the contemporary HERV-W family consists of collections of heterogeneous elements, ranging from full-length defective proviruses (gag, pol, and env genes flanked at both extremities by two long terminal repeats [LTRs]) to isolated LTRs derived from recombination events.
HERV-W elements were found to be transcribed at various levels in several pathological and physiological contexts, e.g., certain tissues of multiple sclerosis (Perron et al. 1997) and schizophrenia-affected (Karlsson et al. 2001) individuals, testis (Mi et al. 2000), and placenta (Blond et al. 1999; Mi et al. 2000). The HERV-W transcripts detected in the placenta mainly result from the expression of a unique locus, termed ERVWE1 (OMIM accession number 604659). These transcripts are matured through a specific splicing strategy (Blond et al. 1999) and lead to the expression of an envelope glycoprotein in vivo (Voisset et al. 2000). Interestingly, this locus was shown to be the only copy of the whole family having retained a complete env open reading frame (ORF) (Voisset et al. 2000). The gag and pol elements of this ERVWE1 provirus contain stop codons and frameshift mutations. The fact that, contrarily to gag and pol, the env ORF has not become a pseudogene led us to propose that this env gene had been selectively preserved (Blond et al. 1999). The Env ERVWE1 envelope exhibits fusogenic properties (Blond et al. 2000; Mi et al. 2000) and was shown to be involved in trophoblast differentiation (Mi et al. 2000; Frendo et al. 2003). We have identified orthologs of the human ERVWE1 locus in chimpanzee, gorilla, orangutan, and gibbon, and we have shown that they contain a functional env ORF (Mallet et al. 2004). These different findings strongly suggest that the ERVWE1 env gene has been recruited to play a role in placental physiology (Mallet et al. 2004).
To better understand how this locus was domesticated during primates evolution, we have searched for the hallmarks of selective pressures acting on the ERVWE1 env gene. Patterns of insertions or deletions (indels) and base susbtitutions have been evaluated using (H)ERV-W intra- and interspecies data. The comparison of the proviral human ERVWE1 locus with HERV-W paralogous copies has allowed us to identify an ERVWE1-specific signature consisting of a four amino acid deletion in the intracytoplasmic tail of the glycoprotein. This deletion event has been found to be crucial for the envelope fusogenic activity. ERVWE1 comparison with human paralogs and chimpanzee, gorilla, orangutan, and gibbon orthologs demonstrates the existence of a selective pressure to maintain the env reading frame open and of a relatively high rate of evolution of the encoded protein.
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Materials and Methods |
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Consensus sequences of the HERV-W env gene and the RT pseudogene were computed with Seaview (Galtier, Gouy, and Gautier 1996) with a majority threshold at 50%. The env genes were delimited from start to stop codon : 1,617 base pairs (bp) for the human ERVWE1 locus (accession number AC000064 from nucleotide 35879 to 37495). The approximate boundaries of the RT pseudogenes were defined by similarity with functional RTs from other retroviral families. This RT region spanned 1,500 bp in the human ERVWE1 locus (accession number AC000064 from nucleotide 32703 to 34202).
Orhologous ERVWE1 loci accession numbers : Pan troglodytes (AY101586, AY101587), Gorilla gorilla (AY101588, AY101589), Pongo pygmaeus (AY101590, AY101591), and Hylobates pileatus (AY101592, AY101593).
Phylogenetic and Comparative Analysis of (H)ERV-W Elements
GenBank queries and sequences retrieval were made under ACNUC using Query (Perriere and Thioulouse 1996). Sequence alignments were computed with ClustalW (Thompson, Higgins, and Gibson 1994). Phylo_win (Galtier, Gouy, and Gautier 1996) was used to build phylogenetic trees. Distances between sequences were computed under the Kimura two-parameters model (Kimura 1980) and under the Li model for synonymous (KS) and nonsynonymous (KA) substitutions (Li 1993). We performed a maximum-likelihood test of selection, using the codon substitution models proposed by Yang et al. (2000), as implemented in the PAML package (http://abacus.gene.ucl.ac.uk/software/paml.html). Likelihood values and parameters were estimated under models M0, M1, M2, M3, M7, and M8 (Yang et al. 2000).
Cell-Cell Fusion Assay
Envelope glycoprotein expression plasmids were transfected by calcium phosphate precipitation into TELCeB6 expressing ß-galactosidase cells as previously described (Blond et al. 2000). Three hours after transfection, cells were harvested by a 10 min incubation at 37°C with 0.02% versene in phosphate-buffered saline (PBS) (Invitrogen life technologies, Cergy, France) and seeded in 2.2-cm diam wells at a density of 6 x 104 /well. After adhesion of the transfected cells, HeLa indicator cells (3 x 105 /well) were overlaid. The determination of the fusion activity of the transfected envelope glycoproteins was performed after 20 h of coculture. The total number of syncytia in one well and the number of nuclei in each syncytia were determined.
FACS Analysis
TE671 cells were washed in PBS and harvested by a 10 min incubation at 37°C with 0.02% versene in PBS. 106 cells were stained for 1 h at 4°C with an anti-Env specific monoclonal antiboby (6A2B2) at 1/100 dilution in PBA (PBS with 2% fetal calf serum and 0.1% sodium azide). Cells were washed once with PBA and incubated with fluorescein isothiocyanate-conjugated antibody (Dako, Trappes, France). Cells were washed twice with PBA and counterstained with propidium iodide (20 µg/ml). Fluorescence of living cells was analyzed with a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, San Jose, Calif.).
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Results |
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Interestingly, the comparison of ERVWE1 env with the putative ancestral sequence reveals a deletion corresponding to a loss of four codons. No other HERV-W env sequence contains this 12-bp deletion, indicating it is clearly specific to the ERVWE1 locus. This deletion probably results from a recombination event between both elements of a tandemly repeated sequence or from DNA polymerase slippage, maintaining the reading frame open (fig. 1A). To evaluate the significance of this 12-bp sequence, the four codons LQMV deduced from human HERV-W paralogous env sequences were inserted in the phCMV-ERVWE1-env expression vector previously used to demonstrate the Env fusion capacity (Blond et al. 2000). Properties of wild-type envelope glycoprotein and LQMV-derived mutant were compared in an heterologous cell-cell fusion assay. The env expression vectors were transfected in TELCeB6 producer cells phenotypically characterized by a blue nucleus as expressing ß-galactosidase. A limited amount of producer cells was overlaid with an excess of indicator HeLa cells, characterized by uncolored nuclei. If the glycoprotein exhibited fusogenic properties, the coculture of both indicator and producer cells would lead to the formation of syncytia containing generally one blue nucleus and ten white nuclei. A high fusogenic activity is observed for the wild-type envelope, characterized by hundreds of syncytia per well and an average of 40 nuclei per syncytia containing one or two blue nuclei, as previously observed (Blond et al. 2000; Mallet et al. 2004). Conversely, no syncytia is produced with the ERVWE1-LQMV glycoprotein in this coculture assay, indicating the loss of the fusogenic phenotype for this mutant envelope (fig. 1B).
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Pattern of Substitution and Indels Along the ERVWE1 Locus in Hominoids
The regional variations in substitution rate along the complete provirus were analyzed in the ERVWE1 orthologs from the five hominoid species. Kimura distances along the ERVWE1 locus were calculated globally in the whole lineage by summing all branch lengths in the phylogenetic tree. The substitution rate profile (measured with a 326-bp sliding window) reveals two main features, a peak of divergence for both proviral regulatory regions (5' and 3' LTRs) and a low divergence along the env gene (fig. 2A). Overall, in the five species, the rate of substitution over the env ORF (1,617 bp) is about 0.050 substitutions per site, compared to 0.079 substitutions per site for the entire ERVWE1 provirus (8,815 bp, env excluded) and 0.089 substitutions per site for the RT pseudogene (1,500 bp). The differences of substitution rates between the env gene and both the corresponding provirus and RT regions are significant (2, 1 df, P < 0.001), consistent with a negative selective pressure acting on the env ORF.
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There is a possible ascertainment bias here since the human ERVWE1 env gene was identified because it contains an intact ORF. We therefore recomputed the number of nonsense substitutions and indels expected in ERVWE1 env in the whole phylogenetic tree, excluding all branches leading to the human lineage. The expected occurrences (four stop codons, five indels) were still found to be significantly higher (P = 0.053 and P = 0.011, respectively) than the observed zero values. Hence, the analyses of ERVWE1 orthologous sequences in hominoids provide evidence of the action of a selective pressure to preserve the env ORF as a functional cellular gene.
Synonymous and Nonsynonymous Substitutions in env ERVWE1
To determine the nature of the selective pressure on ERVWE1 env, the ratio of nonsynonymous (KA) to synonymous (KS) substitutions rate was analyzed. The KA/KS ratio obtained for env ERVWE1 over the whole phylogenetic tree is 0.8 (fig. 3A). This value is close to the theoretical value expected in case of neutral evolution and therefore does not reveal any obvious selective pressure on the protein sequence. However, the KA/KS ratio analysis over the whole gene may be obscured when both positive and negative selective pressures affect different parts of a gene. We therefore measured the KA/KS ratio of the whole tree over a 180-bp sliding window (fig. 3B). Two main regions emerge in the env ORF, corresponding to approximately the first 500 bp and to the last 1,100 bp. The 3' part of the gene appears to be subject to a negative selective pressure (KA/KS about 0.6). To test the significance of this low KA/KS ratio in the 3' part of the gene, we have performed a maximum-likelihood test of selection, using the codon substitution models proposed by Yang et al. (2000). The KA/KS ratio estimated under the model M0 (that assumes a constant KA/KS ratio along the sequence) is 0.56. The model M0 (log-likelihood l = 1841.41) is significantly more likely (P < 0.05) than the null hypothesis of neutral evolution (i.e., KA/KS = 1; l = 1843.46), which confirms that the 3' part of the gene is under purifying selective pressure. More complex models (assuming heterogeneity of KA/KS along the sequence or among branches of the phylogenetic tree) do not significantly improve the likelihood of the data. In the 5' region, we observe a relatively high KA value, a low KS value, and the resulting KA/KS ratio is above one, suggesting a positive selection. However, likelihood-ratio test comparisons indicate that this KA/KS ratio is not significantly different from the null hypothesis for neutral evolution and hence no obvious selective pressure, neither positive nor negative, could be inferred.
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Discussion |
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The systematic comparison of env paralogous sequences reveals an env ERVWE1-specific 12-bp deletion that results in a four amino acid shortened cytoplasmic tail of the Env transmembrane subunit. We have shown that this deletion is crucial for the envelope fusogenic activity. This suggests that this deletion might have played a major role in the domestication of this retroviral gene. Conversely, the four additional amino acids persisting in all other env paralogous pseudogenes may represent a vestige of the intracytoplasmic tail sequence of the infectious ancestor. Thus, the local modification of the ERVWE1 envelope cytoplasmic tail may result from constraints due to the acquired physiological role (reduction of the pathogenic potential) and/or from an adaptation to the host cellular environment (optimization of functions).
The ability to pseudotype retrovirus vectors with a variety of envelope proteins, e.g., modified retroviral envelopes or vesicular stomatitis virus G glycoprotein, has been used for years to significantly broaden the tropism of replication-defective retrovirus vectors (Kafri 2004). Similarly, the in vivo incorporation of ERVWE1 Env into pathogenic retroviruses would be dramatically detrimental to the host by broadening the tropism of the virus. Thus, the ERVWE1 Env cytoplasmic tail may reduce such a possibility as illustrated by the inability of this envelope glycoprotein to be incorporated on Murine Leukemia Virus particles (Blond et al. 2000; An, Xie, and Chen 2001). Conversely, it was observed that ERVWE1 Env was able to pseudotype Human Immunodeficiency Virus (HIV)-based retroviral vectors in vitro (An, Xie, and Chen 2001; Lavillette et al. 2002). A modification of the ERVWE1 Env cytoplasmic tail, consisting of a 53 carboxy-terminal amino acid deletion, induced a 20-fold increase in viral titers of HIV pseudotypes (Lavillette et al. 2002). This suggested that the pseudotyping process, which involved the native ERVWE1 envelope, was inefficient. Interestingly, the carboxy-terminal end of the mutant shortened tail takes place just one residue upstream from the specific ERVWE1 env signature, i.e., the site of the 12-bp deletion. Nevertheless, whether these differences in pseudotyping efficiency were sequence-dependent or length-dependent deserves further investigation.
The capacity of infectious retroviruses to fuse virus-cell membranes is dependent on an envelope maturation step consisting of cleavage of the intracytoplasmic tail by the viral protease (Rein et al. 1994). Conservation of this regulatory feature of the Env-mediated fusion for millions of years would have required (1) conservation of an HERV-W active protease and (2) temporal and spatial coordinated expression of pro and env genes. This does not reflect the HERV-W family portrait, as (1) the ERVWE1 locus expressed in placenta contains a pro pseudogene and (2) the human genome did not contained any pro HERV-W genes in a favorable translational context (Voisset et al. 2000; Pavlicek et al. 2002). The absence of fusogenic property of the LQMV modified envelope, proposed to mimic the infectious ancestor, could be correlated with the absence of HERV-W protease activity. Hence, one may speculate that the ERVWE1 Env cytoplasmic domain evolved so as to bypass the cleavage requirement (constitutive fusogenic envelope) or to adapt cellular protease(s). In addition, contrary to the proposed involvement of a cytoplasmic helical structure in the mechanism of cell-cell fusion (Taylor and Sanders 2003), the fusogenic cellular envelope exhibited an altered helix, which was restored in the nonfusogenic LQMV mutants. Further investigations will be required to decipher the respective contribution of the cytoplasmic tail maturation and structure in the fusion process mediated by the cellular envelope.
To conclude, we have identified hallmarks of the action of natural selection that demonstrate the selective preservation of the env gene of the retrotransposed ERVWE1 proviral locus. Identification of env ERVWE1-specific traits suggests mechanisms underlying the process of evolution from a retroviral envelope toward a cellular gene.
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Acknowledgements |
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Footnotes |
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