1 Unité des Virus Emergents, Faculté de Médecine de Marseille, IFR48-IRD UR034, 27 boulevard Jean Moulin, 13005 Marseille, France
2 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
Correspondence
Xavier de Lamballerie
Xavier.de-Lamballerie{at}medecine.univ-mrs.fr
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ABSTRACT |
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The sequences reported in this article have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers AF411835, AY223844, AY223845, AY223846, AY223847 and AY223848.
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INTRODUCTION |
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We report here a novel dataset that definitively establishes the existence of genetic transfer from RNA viruses to eukaryotic cells. Our observations involve flavivirus-related RNA viruses. Flaviviruses are enveloped viruses, the genome of which is a ssRNA molecule of positive polarity encoding a polyprotein secondarily cleaved to form structural (capsid- and envelope-associated) and non-structural (NS1NS5) proteins. The evolutionary lineage includes more than 30 arthropod-borne viruses pathogenic for humans (e.g. Yellow Fever, Dengue, West Nile, Tick-borne encephalitis virus) but also non-vectored viruses and viruses only isolated in mosquitoes, such as Cell Fusing Agent virus (CFAV) (Cammisa-Parks et al., 1992) and Kamiti River virus (KRV) (Sang et al., 2003
; Crabtree et al., 2003
). The presence of DNA sequences related to CFAV and KRV in the genome of Aedes mosquitoes is reported and evolutionary implications are discussed.
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METHODS |
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Preparation of clonal C6/36 cell lines.
Serial dilutions in culture medium of a suspension of C6/36 cells were prepared. One hundred microlitre aliquots of the dilution containing an average of 1 cell per ml were distributed in 96-well microplates and incubated at 28 °C for 7 days. Wells containing a single colony were selected, cells were transferred to 11 cm2 wells and grown until cellular confluence.
Extraction of nucleic acids.
Cultured cells were centrifuged at 2500 g and the resultant pellet was washed twice with Hanks' balanced salts solution. Individual mosquitoes were crushed using a mixer mill MM300 (Qiagen) in 200 µl Hanks' solution. DNA and RNA were extracted using the proteinase K/phenol/chloroform method (Sambrook et al., 1989) and the RNA Now kit (Biogentex), respectively.
PCR protocols
Flavivirus-like NS3 sequence in the genome of C6/36 cells.
PCR was performed using DNA template that had been extracted as described above, under standard conditions and without a reverse transcription step using the X1 (5'-YIRTIGGIYTITAYGGIWWYGG-3')/X2 (5'-RTTIGCICCCATYTCISHDATRTCIG-3') primer set and a recombinant Taq polymerase (Invitrogen). The X1/X2 primer set was designed to hybridize with NS3 nucleotide patterns conserved in most of the flavivirus sequences available to date.
Flavivirus-like NS5 sequence in the genome of Aedes aegypti.
PCR was performed using DNA template that had been extracted as described above, under standard conditions and without a reverse transcription step using the PF1S (5'-TGYRTBTAYAACATGATGGG-3')/PF2R (5'-GTGTCCCADCCDGCDGTRTC-3') primer set and a recombinant Taq polymerase. The PF1S/PF2R primer set was designed to hybridize with NS5 nucleotide patterns conserved in most of the flavivirus sequences available to date.
Specific detection of cell silent agent (CSA) inserts in the genome of individual Aedes albopictus mosquitoes and in C6/36 subclones.
Conducted under similar conditions with primer sets CSA_NS3_S1/R1 (NS3 region: CSA_NS3_S1, 5'-GATCATCGTGCGCAGCTTTATGG-3'; CSA_NS3_R1, 5'-CCTTGGTTTCAGAAACAATGACC-3') and CSA_seq#2_S1/R1 (NS5 C-terminal region: CSA_seq#2_S1, 5'-AATTAGCAAGGAAGACTTGC-3'; CSA_seq#2_R1, 5'-GTGAGGTTCTTTCCTCAAGA-3').
Detection of CSA2 inserts in the genome of individual Aedes aegypti mosquitoes.
Conducted under similar conditions using the primer set PF1S/PF2R.
PCR of the cytochrome oxidase 1 (CO1) gene of Aedes mosquitoes.
DNA was extracted from cell lines or individual mosquitoes as described above and amplified under standard conditions using primers COI-1S (5'-TACACAAGAAAGWGGAAAAAAGGAA-3') and COI-1R (5'-GTAATTCTGAATAASTATGTTCTGC-3').
Digestion by nucleases.
DNA extracted from C6/36 cells was digested at 37 °C for 2 h in the appropriate buffer using either 50 U bovine pancreas DNase I, 1040 U of restriction enzymes (PvuII or AluI) or 10 µg bovine pancreas RNase A ml1 (all enzymes from Roche Molecular Biochemicals).
Genome walking.
This was performed using the Universal GenomeWalker kit and the Advantage Genomic Polymerase mix (Clontech Laboratories).
Sequencing.
Sequencing of both DNA strands of amplicons was performed using PCR primers (and additional primers deduced from the sequence for long fragments), dRhodamine DNA sequencing kit and an ABI Prism 377 sequence analyser (Perkin Elmer).
RT-PCR identification of mRNAs.
RNA was extracted from C6/36 cells using the RNA Now kit (Biogentex) and incubated at 37 °C for 2 h with 50 U bovine pancreas DNase I. mRNAs were captured, washed and eluted using the PolyAtract system 1000 kit (poly T magnetic beads; Promega). mRNAs were reverse transcribed at 42 °C for 90 min in the presence of 200 U MuMLV Superscript II RNase H reverse transcriptase (Invitrogen) and random hexaprimers (Roche Molecular Biochemicals). PCR was performed using overlapping sets of primers spanning the ORF of CSA (primers available upon request to the corresponding author). Direct PCR using the same primers was used as a control to exclude the presence of contaminating DNA.
Southern blot.
DNA was extracted from C6/36 and Aedes w-albus cells as described above, digested by either XbaI, NotI or SmaI restriction enzymes and run on a 0·8 % agarose gel (overnight, 40 V in TAE buffer). The gel was subsequently treated with 0·24 M HCl (10 min) and a denaturation buffer (0·5 M NaOH, 1·5 M NaCl, 2x 15 min). Transfer on to a nylon membrane (Hybond-N; Amersham Biosciences) was performed overnight using the capillary transfer method and the denaturation buffer. After neutralization (0·5 M Tris/HCl, pH 7·2, 1·5 M NaCl, 5 min), DNA was fixed (80 °C for 1 h, followed by UV cross-linking). A 32P-labelled probe was prepared by the nick-translation method using a 599 nt PCR product located at the NS1NS2 junction of CSA and hybridized to the immobilized DNA [overnight, at 67 °C in CHURCH buffer (7 % SDS, 0·5 M NaHPO4, pH 7·2)]. Washings and exposure to X-ray film were performed as described elsewhere (Sambrook et al., 1989).
Sequence analysis.
The CFAV genomic sequence was obtained from GenBank (accession no. NC_001564) and the KRV sequence was provided by M. B. Crabtree (Centers for Disease Control and Prevention, Fort Collins, CO, USA). This sequence is now available in GenBank under AY149904. Other sequences of flaviviruses used for phylogenetic reconstruction are the same as reported previously (de Lamballerie et al., 2002).
Sequences reported here were deposited in GenBank under accession numbers, AF411835 (sequence #1), AY223844 (#2), AY223845 (#3), AY223846 (#4), AY223847 (#5) and AY223848 (#6).
Alignments and phylogenetic reconstruction.
Alignments of nucleotide or amino acid sequences were generated with the help of the CLUSTAL W (v1.74) program (Thompson et al., 1994) and pairwise genetic distances were estimated with the program MEGA v2.0 (Kumar et al., 2001
).
A phylogenetic tree was reconstructed using the alignment of the CSA ORF amino acid sequence with the corresponding sequences of representative member viruses of the flavivirus lineage, with the pairwise distance algorithm and the neighbour-joining method implemented in MEGA. The robustness of the resulting branching patterns was tested by bootstrap analysis with 1000 replications.
BLAST.
The relatedness of newly characterized sequences to sequences deposited in databases was assessed by the Basic Local Alignment Search Tool (Altschul et al., 1990) implemented via the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/blast/) against the complete GenBank database. The BLASTN (nucleotide querynucleotide database comparison), BLASTP (protein queryprotein database comparison) and BLASTX (nucleotide queryprotein database comparison) algorithms were used. Relatedness to KRV sequence was tested using a local database of flavivirus polyproteins and the BLAST program implemented in the DNATools (v5.2.014) software program.
Sequence repeats.
These were sought with DNATools (v5.2.014).
Hydropathy plots.
Profiles of the protein encoded by CSA ORF and the homologous region of KRV polyprotein were produced in Microsoft Excel using the amino acid hydropathy values determined by Kyte & Doolittle (1982), and a sliding window of 11 aa. Aligned sequences were exported with all alignment-generated gaps maintained (with a hydropathy value of zero). This permitted the comparison of hydropathy plots of sequences of unequal lengths.
Evaluation of divergence dates for mosquitoes and viruses.
The putative age of origin of the Diptera [225 to 280 millions of years ago (mya)] has been previously evaluated from fossils, biogeographical history or molecular clock work (Simmons & Weller, 2001
; Gaunt & Miles, 2002
). This corresponds to a
60 % nucleotide divergence for the CO1 gene sequences of the most divergent diptera (see Fig. 4
). If we put forward the hypothesis that evolution of the CO1 gene within this order conforms approximately to a molecular clock, the observed
9 % divergence between the CO1 sequences of Aedes albopictus and Aedes w-albus (this study, accession no. AY223849) indicates a date of divergence
3442 mya.
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RESULTS |
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Extension of sequence by genome walking and comparison with flaviviral sequences
The sequence initially characterized was extended using genome walking. Results are presented in Fig. 1. A 10 623 nt sequence was obtained (Aedes albopictus sequence #1) that includes an ORF of 1557 aa homologous to a region of CFAV and KRV genomes (59 and 67 % identity, respectively), starting in the N-terminal part of the NS1 gene and ending shortly after the NS4ANS4B junction.
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Identification of other sequences related to flaviviruses
Genome walking using primers located in motif (a 117 nt sequence located in the 3'-region of sequence #1) led to the characterization of a new sequence of interest (sequence #3), which is not contiguous to sequence #1. Sequence #3 includes a 1037 nt sequence related to KRV NS4B/NS5 (70 % aa identity, BLASTP E value=5e153). In order to identify additional flavivirus-related sequences, primers were designed from the NS5 gene of CFAV/KRV and tested on C6/36 DNA extracts. This led to the identification of an 852 nt sequence (sequence #2) similar to the C-terminal region of the KRV NS5 (70 % nt identity using coding positions 1+2), which is not contiguous to sequence #3. Genome walking extension of sequence #2 showed that the NS5-like insert is contiguous to a 334 nt region homologous to the KRV envelope (57 % aa identity, BLASTP E value=4e31).
Evidence for integration into the cell genome
Overall, approximately two-thirds of a flavivirus-like genome were characterized, comprising a patchwork of sequences of various sizes. In order to test fully the hypothesis of integration into the cellular genome, we investigated the nature of the flanking sequences. We compared the latter with those short partial sequences that are available for Aedes albopictus, plus other insect sequences from GenBank. We found strong evidence that CSA sequences are present in the Aedes albopictus genome, at three different positions. In sequence #1 (first integration site), characterization of the 3'-non-viral flanking sequence led to the identification of the first 213 aa of an ORF with significant similarity (BLASTP E value=7e42) to the agCP8252 protein of Anopheles gambiae. Both deduced proteins contain a plant homeo domain (PHD) finger motif that folds into an interleaved type of zinc finger chelating two zinc ions. They are significantly related (BLASTP E value<4e06) to the capsid proteins of LTR-retrotransposons of the Pao/Ninja lineage. In sequence #2 (second integration site), the 3'-non-viral flanking sequence contains a 266 nt sequence that is 98 % identical to the Aedes albopictus microsatellite AaUM1.3 (BLASTN E value=1e137). Moreover, sequences #1 and #3 (third integration site) contain the conserved motif . This was also identified in cellular sequences #4 and #5, which contain ORFs with cognate specific coding sequences in insect genomes. In sequence #4, a 602 aa ORF is closely related (BLASTP E value<1e154) to the agCP7521 protein of Anopheles gambiae. It includes a zinc-binding motif that is the central catalytic domain of an integrase related to the retrotransposon Copia of Drosophila melanogaster9 (BLASTP E value=1e93). In sequence #5, a 241 aa ORF is related to the agCP7781 protein of Anopheles gambiae (BLASTP e value=5e76) and the aad53951 sulfate transporter of drosophila melanogaster (BLASTP E value=1e35).
In addition to investigations based on sequence determination and analysis, direct evidence for integration of the CSA sequence into the Aedes albopictus genome was provided by Southern blotting. A probe located at the NS1NS2 junction of CSA specifically hybridized to 25, 19 and 13 kb restriction fragments of DNA extracted from Aedes albopictus and digested with NotI, XbaI and SmaI, respectively. Hybridization with DNA extracted from Aedes w-albus cells was not observed (Fig. 4).
Analysis of CSA inserts in subclones of C6/36 cells
Having demonstrated the presence of CSA in the genome of C6/36 cells, we investigated whether all individual cells within the cell line contained this integration. We produced 50 new clonal lines from C6/36 cells that were tested using specific primers designed from the NS3 and NS5 regions of CSA. All clones tested positive for both the NS3 and NS5 insertion sites.
Analysis of CSA inserts in Aedes albopictus mosquitoes
Wild Aedes albopictus mosquitoes from Thailand, Texas (USA), Cameroon and Madagascar and laboratory-bred mosquitoes from Japan, Thailand, Madagascar, Italy and France were tested for the presence of CSA sequences using primers specific for the NS3 and NS5 regions. Amplification and sequencing of the CO1 gene was used as a control for the quality of DNA and identification of the mosquito (Simmons & Weller, 2001). Among 130 mosquitoes assigned as valid samples, 97·7 % tested positive for at least one integration site, including those from all geographical origins tested, all stages of development (eggs, larvae, nymphs and adults) and both males and females.
Analysis of other Aedes species
The presence of CSA sequences in other Aedes species was examined in the closest relatives of Aedes albopictus (Singh & Bhat, 1971) [species Aedes w-albus (Shouche & Patole, 2000
) and Aedes aegypti] with CSA-specific NS3 and NS5 primers and NS3 and NS5 degenerate primers (X1/X2 and PF1S/PF2R). In Aedes w-albus cell culture, all results were negative. However, when laboratory-bred Aedes aegypti mosquitoes were tested with flavivirus NS5 consensus primers, a CSA2 sequence distinct from CSA but related to CFAV and KRV was identified in the ten individuals tested. It was also found in all five wild specimens from Senegal that were tested and in the A20 Aedes aegypti cell line. The sequence was extended by genome walking and consisted of a 1479 nt long flavivirus-like region (Aedes aegypti sequence #1), encoding a 492 aa ORF (61 % aa identity to KRV, BLASTP E value=0·0) related to the NS5 of flaviviruses and flanked by non-viral sequences (Fig. 1
). The 3'-flanking region is related to the agCP11637 protein of Anopheles gambiae (BLASTP e value=2e9) and transposon i of drosophila melanogaster (BLASTX E value=5e5), indicating integration into the genome of Aedes aegypti.
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DISCUSSION |
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The main insert includes an ORF that corresponds to approximately one-half of the flavivirus polyprotein. Its integration within the cellular genome was confirmed by Southern blotting. The encoded 1557 aa polyprotein is homologous to the NS1, NS2A, NS2B, NS3 and NS4A of flaviviruses. This region includes the well-characterized enzymic domains of the viral helicase and serine protease. These motifs are conserved and the expression of the polyprotein is likely to lead to functional enzymes. This is a different situation to that observed in sequence #2, in which the sequence homologous to the flavivirus polymerase is truncated and includes numerous stop codons, formally excluding the possibility that a functional enzyme is generated. The subsistence of the long NS1NS4B ORF along the evolution of Aedes albopictus and the conservation of enzymic domains suggests that the corresponding proteins are expressed. This hypothesis is reinforced by the detection of the corresponding mRNAs in C6/36 cells. Although the presence of the corresponding proteins has not yet been fully investigated and is necessary for definitive confirmation, the presence of mRNAs constitutes strong evidence that the captured genes are likely to be used by host cells.
An important point was to determine what proportion of cells contained the viral inserts. A large number of subclones of the cell line were produced and all tested positive for the presence of inserts. Because the C6/36 cell line is clonal (Igarashi, 1978), this implied the presence of insertions in the original parent cell (and therefore in the mosquito larvae used for establishing the cell line). This led us to test laboratory-bred and wild Aedes albopictus mosquitoes, for which positive results were obtained with mosquitoes from all geographical origins tested, all stages of development and both males and females. Only 2·3 % tested negative for all integration sites; however, these results may represent technical artefacts because they were obtained from dried insects rather than fresh material. Nevertheless, the presence of integrated sequences in the majority of individuals is of great interest because it demonstrates that the integration of CSA sequences is not an artefact due to the manipulation of the C6/36 cell line and exists in wild mosquitoes. It is likely to be associated with vertical transmission of the sequence through generations of mosquitoes rather than current infection of Aedes albopictus mosquitoes by the viral form of CSA.
The discovery of the CSA2 sequences in the genome of Aedes aegypti mosquitoes provides the first example of flavivirus-like sequence detected in the dsDNA of mosquitoes. The NS5-like sequence characterized is different from that of Aedes albopictus and does not include the functional motifs of a polymerase. As observed in the case of Aedes albopictus, the analysis of non-viral flanking sequences provides strong evidence for integration in the cellular genome and the flavivirus-like sequence is present in laboratory-bred and wild mosquitoes. The implications of the presence of different flavivirus-related sequences in different mosquitoes are important from an evolutionary point of view and are discussed further below.
The presence of flaviviral-like sequences in mosquito genomes could be explained by one of two hypotheses: either (i) these sequences were integrated into the genomes of Aedes spp. mosquitoes following infection by the corresponding RNA viruses or (ii) flaviviruses originate from the genome of Aedes spp. The latter hypothesis is contradicted by several observations. Firstly, the organization of these genes as a unique ORF in the genomes of flaviviruses is not observed in insect genomes, where they constitute independent inserts. In addition, with the remarkable exception of Aedes albopictus sequence #1, these genes are truncated (Aedes albopictus sequences #2 and #3 and Aedes aegypti sequence #1) or contain multiple stop codons (Aedes albopictus sequence #2). Also, there is a complete absence of overlap between sequences of the different CSA inserts identified, which strongly suggests a single original integration, followed by several reshuffling events. Secondly, our current knowledge of flaviviruses and mosquito evolution suggests that the split between Aedes albopictus, Aedes aegypti and Aedes w-albus (3442 mya; Fig. 5
) is significantly more ancient than the most recent common ancestor of CSA, CFAV and KRV (probably around
3500 ya and certainly less than 350 000 ya). Therefore, a cellular origin of flaviviruses would require the conservation of an intact ORF within Aedes spp. genomes throughout several million years, with a sudden disappearance from the genomes of both Aedes albopictus and Aedes aegypti mosquitoes during the recent millennia. This is a very improbable evolutionary scenario. Thirdly, the polymerases of numerous viruses that specifically infect mammalian cells (pestiviruses, hepaciviruses and Tamana bat virus) are phylogenetically related to those of flaviviruses (de Lamballerie et al., 2002
) and there is little chance that these viruses originated from the genome of mosquitoes in recent times; for example, it has been suggested that the common ancestor of hepaciviruses GB virus A and C existed 35 mya in primates (Charrel et al., 1999
).
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In summary, we have reported for the first time the presence of a multigenic sequence from a non-retroviral RNA virus in a eukaryotic cell, and have demonstrated that it takes the form of DNA integrated into the cellular genome itself. Our observations in mosquitoes shed new light upon previous studies, which have found DNA forms of RNA viruses in cells from species as wide-ranging as mammals and nematodes, suggesting that this phenomenon is in fact more common than previously thought. Previous findings described viruses with a ssRNA genome of negative polarity (Filoviridae) or with a segmented ssRNA genome of negative or ambisense polarity (Arenaviridae, Bunyaviridae), while flavivirus genomes are ssRNAs of positive polarity. RNA viruses are characterized by their extreme genetic diversity and their propensity to evolve quickly, and thus may represent a source of evolution for eukaryotic cells, which are genetically much more stable. In the case of CSA, the integration into the majority of mosquitoes tested, the perfect conservation of enzymic motifs and the detection of mRNAs suggests that an advantage may be conferred upon Aedes albopictus from the expression of the viral proteins. This finding could have major implications in terms of evolutionary theory since it represents an entirely different method apart from the accepted processes for the generation of genetic diversity in eukaryotic cells. In addition, it will be of major importance to determine whether RNA viruses infecting humans are able to transmit genes to infected somatic cells and what consequences this could have. The mechanisms of reverse transcription and integration remain to be determined precisely. They will be discussed in another article in preparation, together with the report of experimental data demonstrating that generation of cDNAs can be observed following the infection of mosquito cells by present-time flavivirus-related viruses such as CFAV.
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ACKNOWLEDGEMENTS |
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Received 25 November 2003;
accepted 23 February 2004.