Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines

Anne-Nathalie Volkoff1, Janick Rocher1, Pierre Cérutti1, Marc C. P. Ohresser1, Yves d’Aubenton-Carafa2, Gérard Devauchelle1 and Martine Duonor-Cérutti1

Laboratoire de Recherches de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-les-Alès, France1
CNRS, Centre de Génétique Moléculaire, 91198 Gif-sur-Yvette, France2

Author for correspondence: Anne-Nathalie Volkoff. Present address: Department of Entomology, S-225 Agricultural Sciences Center N., University of Kentucky, Lexington, KY 40546-0091, USA. Fax +1 859 323 1120. e-mail nvolk2{at}pop.uky.edu


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An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3'-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.


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Polydnaviruses (PDVs) are unusual insect viruses associated with parasitic Hymenoptera and characterized by polydisperse double-stranded DNA genomes. Viral genomes are integrated in the wasp genome and transmitted as integrated proviral DNA (Stoltz, 1990 ). Virus replication takes place in the reproductive tracts of female wasps. Viral particles are stored in female oviducts and then injected into the lepidopteran host during oviposition. In the lepidopteran host, no virus morphogenesis has been observed (Blissard et al., 1986 ), but PDVs are transcriptionally active. Few PDV genes have been characterized, and the function of viral gene products in the lepidopteran host remains poorly understood although they are essential for parasitoid development, and are responsible for host immune-suppression (Webb, 1998 ).

To identify Hyposoter didymator ichnovirus (HdIV) genes expressed in the lepidopteran host, Sf9 (Spodoptera frugiperda; ATCC CRL1711) cells were infected with HdIV, and RNAs were extracted for cDNA synthesis at 24 h post-infection (p.i.) (Volkoff et al., 1999 ). Seven distinct viral cDNAs were identified (S6, D8, M24, C28, K29, P30, X41), all related to RNAs also expressed in parasitized Spodoptera littoralis larvae (A.-N. Volkoff and others, unpublished results).

The seven viral cDNAs were digested with EcoRI, 32P-labelled and used as probes for mRNA expression in non-infected or HdIV-infected Sf9 cells. All cDNAs hybridized with viral RNAs expressed in HdIV-infected cells from 2 h until 10 days p.i. (data shown only for K29; Fig. 1A, lanes 2 and 3). However, at 10 days p.i. only RNAs related to K29 were detected (Fig. 1A, lanes 4 to 9). To confirm this result by RT–PCR, total RNAs were extracted for cDNA synthesis from non-infected, 48 h and 3 year infected Sf9 cells and from parasitized larvae [5 days post-parasitism (p.p.)]. cDNA templates were used for PCR amplification with sets of primers specific to each of the cDNAs. PCR was done with Taq DNA polymerase (Promega) under standard conditions. Amplification products were obtained only with the K29-specific primers from long-term infected Sf9 cells DNA (Fig. 1B, lane 4; data are provided only for M24 as results with the other cDNAs were identical). So, of the cDNAs tested by Northern blot and RT–PCR analysis, only the K29-related gene was transcribed in long-term infected Sf9 cells.



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Fig. 1. The K-related gene is transcribed in HdIV-infected lepidopteran cells. (A) Northern blot analysis of total RNAs (10 µg per lane) extracted from Sf9 cells. Hybridization with 32P-labelled EcoRI-digested K29 (lanes 1 to 8) or total HdIV DNA (lane 9). RNAs were extracted from non-infected cells (lane 1) and from cells at different times p.i. (lanes 2 to 9: h, hours; d, days; m, months; y, years). (B) RT–PCR products amplified with K29-specific primers (upper panel) and M24-specific primers (lower panel). cDNA templates (1/20 fraction) synthesized from RNAs (3 µg) from uninfected Sf9 cells (lane 1), infected Sf9 cells 48 h p.i. (lane 2), parasitized larvae 5 days pp (lane 3) and infected Sf9 cells, 3 years p.i. (lane 4). The other lanes show control PCR products amplified from 30 ng plasmid K29 or M24 (lane 5) and genomic HdIV (lane 6). Numbers in primer nomenclature indicate primer location in genomic nucleotide sequence. (C) Northern blot analysis of total RNAs (10 µg per lane) extracted from different HdIV-infected cell lines: parasitized S. littoralis haemocytes (lanes 3 and 4); in vitro infected S. littoralis haemocytes (lanes 5 and 6); T. ni cells (lane 7). Hybridization with 32P-labelled EcoRI-digested K29. For (A) and (C), 24 h exposure; underneath each lane, the number of passages the infected cells underwent before RNA extraction is indicated.

 
To determine whether K-related transcripts persist in long-term infection of other HdIV permissive insect cells, Trichoplusia ni cells (High Five, InVitrogen) and cell lines established from haemocytes of the H. didymator rearing host Spodoptera littoralis (Sl2a and Sl2b; Volkoff et al., 1999 ) were infected with HdIV particles. After several weekly sub-cultures, total RNAs were extracted and probed with K29 cDNA. Total RNAs were also extracted from two ‘naturally infected’ cell lines, named Slp1 and Slp2. The Slp1 and Slp2 cell lines were established from S. littoralis haemocytes collected from 23 parasitized larvae 8 days p.p. They were obtained as described for Sl2a and Sl2b (Volkoff et al., 1999 ) and underwent more than 100 passages before analysis. These RNAs derived from in vivo haemocyte infections were analysed to verify that detection of K-related transcripts was not unique to in vitro infections. Northern blot analysis showed that K29-related transcripts were present in all the analysed in vitro or naturally infected lepidopteran cell lines after several months of subculture (Fig. 1C, lanes 3 to 7).

To determine whether the lack of transcripts other than K29-related in the long-term infected Sf9 cells was due to the absence of viral DNAs encoding these genes or to a failure to transcribe some viral genes, we attempted to detect viral DNA related to the different cDNAs. PCR was done using the primers designed for expressed genes and total DNA from infected cell cultures, extracted according to Sambrook et al. (1989) . By PCR, only K29-related DNA was detected in long-term HdIV-infected Sf9 cells (Fig. 2A; as in Fig. 1B data are provided only for M24), thus suggesting that of the sequences tested only the K29-related segment was maintained. However, we cannot exclude the maintenance of genes for which sequences are unavailable and/or persistence of non-coding viral sequences as our analyses focused only on sequences known to be expressed. This result demonstrated that only part of the HdIV genome was retained, as reported in other systems (Gundersen-Rindal et al., 1999 ; Kim et al., 1996 ; McKelvey et al., 1996 ), with the same part of the genome, the K29 genomic DNA, detected in infected lepidopteran cell lines.



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Fig. 2. (A, B) Maintenance of the K-gene. (A) PCR products amplified with the K-specific primers (upper panel) and M24-specific primers (lower panel) from HdIV DNA and transformed Sf9 cells DNA. (B) Southern blot analysis of HdIV DNA hybridized with 32P-labelled EcoRI-digested K29. Upper-left panel: total HdIV DNA undigested (U), digested with BamHI (Ba), HindIII (H) and BglII (Bg) (2 µg, 1 day exposure). Upper-right panel: total undigested DNA from S. littoralis larvae (L, non-parasitized; Lp, parasitized 5 days) and from Sf9 cells (Sf9, non-infected; 5d and 3y, infected 5 days and 3 years) (1 µg, 6 days exposure). I, integrated; OC, open circular; SH, super-helical forms of viral DNA. Lower panel: cellular DNA from Sf9 cells, long-term infected Sf9 cells (Sf9-3y), Sl and Slp cells; undigested (U), digested with BamHI (Ba), BglII (Bg) and HindIII (H) (10 µg, 3 days exposure). The 1·3 kbp BglII restriction fragment is indicated by an arrowhead (see text). (C) K-gene splicing. Alignment of genomic (gen), K29 and K19 cDNAs sequences. The genomic AflIII restriction site is highlighted. Arrows indicate limits of a region involved in a stable secondary structure. Intron sequences are in small characters and splicing sites are in bold characters. The start of transcription (ATG) is underlined.

 
To address the status of K29 genomic DNA in long-term infected Sf9 cells, total DNAs were extracted from non-parasitized and parasitized S. littoralis larvae (5 days p.p.), non-infected and HdIV-infected Sf9 cells (5 days and 3 years p.i.) and Sl2a and Slp1 cells, and then probed with K29 cDNA. K29 genomic DNA, as shown by Southern blotting, was located in a unique circular molecule from the HdIV genome (Fig. 2B, upper-left panel, lane U). In early infection (i.e. 5 days p.i.), K29 hybridized with circular viral DNA (Fig. 2B, upper-right panel, lanes 2 and 4) while in long-term infected Sf9 cells (3 years p.i.) or in Slp cells, the hybridization signal was associated only with high molecular mass DNA (Fig. 2B, upper-right panel, lane 5; lower panel, lanes U). Total cellular DNA was digested with BglII and HindIII, both present as a single site in the K29 sequence, and with BamHI, present as a single site upstream of K29 (P. Cerutti and others, unpublished results). When digested cellular DNA was probed with K29, a broad range of bands hybridized (Fig. 2B, lower panel). These results suggested that viral DNA was maintained as integrated DNA rather than as high molecular mass episomal concatemer. Southern blots also suggest that the K29-related segment was integrated into many sites in chromosomal DNA, although we do not know how many copies of the K29 segment are retained in each cell. This question will be addressed by analysis of cloned long-term infected cells. Even though our results strongly suggest integration of the K29-containing HdIV segment, formal demonstration of integration would require characterization of the chromosomal sequences flanking putative integration sites. The detection of a BglII restriction fragment about 1·3 kbp in size both in digested HdIV and cellular DNA (Fig. 2B, arrow) suggests that putative integration sites are located on both sides of BglII sites. Future studies will also be required to know whether maintenance concerns the whole or partial molecule. Integration of PDV DNA in an insect cell genome has been demonstrated for one segment of the bracovirus associated with Glyptapanteles indiensis (Gundersen-Rindal & Dougherty, 2000 ). In lepidopteran hosts, PDV genomes have been reported to persist as viral circular molecules throughout the time of parasitization (Stoltz et al., 1988 ; Strand et al., 1992 ; Theilmann & Summers, 1986 , 1988 ). However, most studies concerned the whole viral genome and did not focus on individual viral segments. It is possible that some segments efficiently integrate early into lepidopteran chromosomal DNA, while the majority persist as super-helical molecules that are lost over time. Conversely, cell lines may differ karyotypically from their antecedents, as shown for Sf9 cells (Gerbal et al., 2000 ), so considerable DNA rearrangements might reasonably be expected resulting in a different status for viral DNA in insect cell lines compared to parasitized larvae.

On Southern blots, K29 cDNA hybridized with a single HdIV molecule (Fig. 2B). By screening a genomic library (P. Cerutti and others, unpublished results), a 735 bp AflIII–BglII fragment from a K29-related genomic clone was isolated. Sequence alignment of this genomic restriction fragment (GenBank acc. no. AF241775), K29 cDNA (1646 bp; GenBank AF191723) and K19 (1527 bp; GenBank AF237946), a K29 cross-hybridizing cDNA, revealed the presence in the K-gene of two short intron sequences, 122 and 119 bp long (Fig. 2C). K19 and K29 cDNA sequences were identical except for the presence of the second intron sequence in K29, thus indicating that K19 corresponded to the fully spliced K gene transcript.

K19 cDNA contains a short open reading frame, 321 bp long (Fig. 3A, B). Sequences preceding the first putative initiation ATG codon were in good agreement with the Kozak consensus sequence. The first intron was placed 31 bp upstream of the ATG initiation codon, while the second was located 90 bp downstream from the start codon. The positions of the introns relative to the putative ATG initiation codon were conserved between the K and M HdIV genes, and also between the HdIV genes and the Campoletis sonorensis ichnovirus gene VHv1.1 (Fig. 3C). The first 52 nucleotides following the ATG codon of the K gene shared 68% identity with the same region of the M24-related gene (Fig. 3C). The K19 cDNA 3'-untranslated region (3'-UTR) represented more than two-thirds of the RNA length and contained repeated sequences arranged in a tandem array. Dot-plot matrix analysis revealed 13 similar repeated sequences that differed in size, but with conserved nucleotide motifs (Fig. 3B). The significance of this 863 bp repeated AT-rich region (71% AT) in the mRNA 3'-UTR remains to be investigated.



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Fig. 3. Sequence analysis of the K19 cDNA. Complete K19 sequence was obtained by adding the 203 bp from the K29 5'-end to the K19 clone. (A) Schematic of K19 cDNA. Intron positions are indicated by lower vertical arrows. Upper vertical arrows indicate start and stop of translation as the putative polyadenylation signal. (B) Nucleotide and predicted peptide sequences. Intron positions are indicated by arrowheads. Repeated amino acid sequences in the open reading frame are highlighted. Repeated sequences in the 3'-untranslated region are aligned and conserved nucleotides are underlined (except mismatched nucleotides). Start and stop codon are in bold characters; putative polyadenylation signal is boxed. (C) Comparison of the intron position with other ichnovirus genes. Nucleotide identities between HdIV K- and M- gene sequences (indicated by asterisks, line HdIde) showing conservation of intron position relative to ATG codon, and also with Campoletis sonorensis ichnovirus gene VHv1.1 (GenBank acc. no. U41656). Only parts of the sequences are reported; missing regions are indicated by ‘...x nt...’. Intron sequences are in small characters, and splicing sites are in bold characters. The start of transcription (ATG) is underlined.

 
The predicted polypeptide encoded by the K19 ORF has a molecular mass of 11·8 kDa. The polypeptide precursor sequence begins with a putative 16 amino acid signal peptide and has 56% probability of being secreted (PSORT analysis; Nakai, 1991 ). Cognate polypeptides were expected to contain 13% proline residues and 11% glycine residues. The predicted protein contains a 10 amino acid glycine-rich sequence repeated twice (Fig. 3). Comparison with nucleotide and peptide sequences from several databases revealed no significant homologies, allowing no prediction of function for this protein.

K19 and K29 cDNA sequences were identical except for the presence in K29 of the second intron sequence. RT–PCR analysis using different sets of primers and RNA templates from infected cells led to amplification of both K19 and K29 cDNA-related fragments (not shown). The major amplification product corresponded in size to the fully spliced K19 cDNA, which is most likely more abundant. However, the presence of an intron-containing cDNA was puzzling since pre-RNAs are usually rapidly spliced and exported to the cytoplasm for translation. The presence of K29 could be explained by a defective mutation leading to the retention of the intron as often observed in lower eukaryotes with short introns (Sterner et al., 1996 ). However, in the K-gene the short introns have splice signals with good fit to Drosophila consensus sequences (including the branch point and pyrimidine stretch) except for the exonic parts of the sites. Another explanation might be that accessibility of trans-acting factors to the splice sites may be impaired by the formation of a stable RNA structure. However, extensive evaluation of the stable folding propensity for sequences belonging to the whole K-gene revealed only few stable putative secondary structures, none involving the second intron (data not shown). Another possibility not yet investigated is alternative splicing, which is frequently exploited by virus genomes. This would result in two different transcripts encoding different proteins.

In this study, we have identified an HdIV gene that is not only maintained in long-term infected lepidopteran cell lines, but is also transcribed. Relative to genomic organization, the K-gene does not appear to belong to a gene family such as the previously described M24-related gene (Volkoff et al., 1999 ). Whatever the status (integrated or episomal) of HdIV DNA in the infected lepidopteran cells, the question of why the K29-related gene is specifically retained and transcribed in long-term infected cells is of paramount importance. Specific maintenance of the K-gene could be due to particular sequences or motifs specific to this molecule (e.g. homologies with lepidopteran sequences that would allow homologous recombination, replication origin...). Or it could be linked to a particular function, which is still unknown, of the encoded protein. The biological significance of K-related transcripts in long-term infected lepidopteran cells is questionable since parasitoid development is accomplished within 10 days, followed immediately by the death of the parasitized lepidopteran host. Future studies are required to determine whether or not viral DNA has a different status in infected cell lines compared to parasitized lepidopteran larvae. Similarly, it will be important to understand if there is any biological significance to retention of some viral segments and not others.


   Acknowledgments
 
We thank R. Bros and B. Limier for providing S. littoralis larvae, S. Berger for the precious ready-to-use buffers, and A. Gregoire, R. Vincent and M. Ogliastro for the constructive discussions. Special thanks to Don Stoltz and Bruce Webb for their time and their helpful comments on this work.


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Received 19 October 2000; accepted 29 November 2000.



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