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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Abstract |
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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P30, X41), all related to RNAs also expressed in parasitized Spodoptera littoralis larvae (A.-N. Volkoff and others, unpublished results).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 RTPCR, 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 RTPCR analysis, only the K29-related gene was transcribed in long-term infected Sf9 cells.
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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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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 AflIIIBglII 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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K19 and K29 cDNA sequences were identical except for the presence in K29 of the second intron sequence. RTPCR 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.
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References |
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Received 19 October 2000;
accepted 29 November 2000.
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