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Relationships between polydnavirus genomes and viral gene expression
Journal of Insect Physiology 44 (1998) 785–793
Relationships between polydnavirus genomes and viral gene expression Bruce A. Webb b
a,*
, Liwang Cui
a,b
a Department of Entomology, University of Kentucky, Lexington, Kentucky 40546, USA Current address: Entomology Department, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA
Received 1 October 1997; accepted 23 October 1997
Abstract Polydnavirus genomes and viral gene functions are atypical for viruses. Polydnaviruses are the only group of viruses with segmented DNA genomes and have an unusual obligate mutualistic association with parasitic Hymenoptera, in which the virus is required for survival of the wasp host and vice versa. The virus replicates asymptomatically in the wasp host but severely disrupts lepidopteran host physiology in the absence of viral DNA replication. It is not surprising then that viral gene expression is divergent in its two insect hosts and that differences in viral gene expression are linked to these divergent functions. Some viral genes are expressed only in the wasp host while other viral genes are expressed only in the lepidopteran host and are presumed to be involved in the disruption of host physiological systems. Our laboratory has described the expression and regulation of a family of viral genes implicated in suppressing the lepidopteran immune system, the cys-motif genes. In conjunction with these studies we have described the physical organization of additional viral gene segments. We have cloned, mapped and begun the sequence analysis of selected viral DNA segments. We have noted that some viral DNA segments are nested and that nested viral DNA segments encode the abundantly expressed, secreted cys-motif genes. Conversely, other viral segments are not nested, encode less abundantly expressed genes and may be targeted intra-cellularly. These results suggest that nesting of segments in polydnavirus genomes may be linked to the levels of gene expression. By extension, the unique, segmented organization of polydnavirus genomes may be associated, in part, with the requirement for divergent levels of viral gene expression in lepidopteran hosts in the absence of viral DNA replication. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Campoletis sonorensis; Heliothis virescens; Polydnavirus; Segmented genome; Cysteine-rich gene; Gene expression
1. Introduction Polydnaviruses (PDVs) are obligate symbionts of some parasitic Hymenoptera and replicate asymptomatically from integrated proviral DNA only in specialized ‘calyx’ cells of the female reproductive tract (Stoltz, 1993; Stoltz et al., 1995). Polydnaviruses have been described from some of the ichneumonids and braconids and comprise the two genera in this family, Ichnovirus and Bracovirus, respectively. The PDV life-cycle is characterized by asymptomatic virus replication from proviral DNA in a parasitic wasp followed by pathogenic virus infection in the wasp’s larval lepidopteran host. Thus, PDV replication and function is inextricably
* Corresponding author. Tel: (606) 257 7415. Fax: (606) 323 1120. E-mail: [email protected]. 0022–1910 /98 /$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 1 1 - 0
linked to the life-cycle of the parasitic wasp (Stoltz, 1993). Virus is introduced from the female reproductive tract into parasitized insects during oviposition. A species-specific subset of viral genes is expressed in parasitized insects in the absence of viral DNA replication. In both PDV genera, viral gene expression in the lepidopteran host disrupts some host physiological systems and is required for successful development of the endoparasitic wasp (Edson et al., 1981; Lavine and Beckage, 1995; Strand and Pech, 1995a). Thus, the PDV–wasp parasite– lepidopteran host system provides an unusual example of an obligate mutualistic association between a virus and a parasitic wasp that functions to the extreme detriment of the parasite’s lepidopteran host. As a result of this mutually obligate association, PDVs are found in every individual of an infected species and do not replicate outside of their associated wasp host (Stoltz et al., 1986; Stoltz, 1990, 1993). To emphasize this point,
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PDVs replicate only in some of the wasp cells carrying proviral DNA and persist, evolutionarily, on the basis of their incorporation in the wasp genome. PDVs are the only viruses with segmented DNA genomes (Stoltz et al., 1995). Moreover, segmentation of PDV genomes is often extreme. Segmented RNA viruses may have up to 12 segments with an aggregate genome size of about 20 kb, while the Campoletis sonorensis polydnavirus (CsPDV) has at least 28 DNA segments with genome size estimates in excess of 250 kb (Krell et al., 1982; Fleming and Krell, 1993). Although a segmented genome is a hallmark of PDVs, we have little understanding of the evolutionary pressures that have driven, or alternatively, allowed segmentation of the viral genome. One hypothesis ventured is that genome segmentation (and the presence of repeated DNA and viral gene families) enhances the capability of the virus to generate genetic variability through recombination (Dib-Hajj et al., 1993; Summers and Dib-Hajj, 1995). While the importance of mechanisms for generating genetic diversity is unquestionable, it does not account for recently described PDV segments with extensive sequence identity (Xu and Stoltz, 1993). Specifically, mapping of cross-hybridizing Hyposoter fugitivus PDV segments suggests that some smaller DNA segments are excised from larger DNA segments, an organizational pattern known as ‘segment nesting’ (Cui and Webb, 1997a; Xu and Stoltz, 1993). Segment nesting increases the number of PDV segments without significantly increasing the virus‘ sequence complexity. However, nesting does increase the representation of nested sequences within the genome. Apparently, at least some PDV genomes are comprised of nested DNA segments, although the extent of segment nesting and its functional significance remained enigmatic. Stoltz and Whitfield (1992) proposed that the unique genomic organization of PDVs is a consequence of their unique viral life cycles in which PDVs play an integral and essential role in insect host–parasite systems. In this paper, we partially describe the contributions of nested PDV segments to genome complexity, and begin to evaluate the relationships between segment nesting and PDV gene expression. The CsPDV is the type species of the Ichnovirus genus (Stoltz et al., 1995) and has been described in the most detail. CsPDV segments exist as circular, supercoiled DNA molecules (superhelical DNAs) in virions, and as linear proviral molecules when integrated in the wasp genome. Segments are named alphabetically by the size of the circular molecules from the smallest segment A (~6 kb) to the largest X (~16 kb) (Krell et al., 1982; Blissard et al., 1986a). Viral segments are present in nonequimolar amounts with some present in much greater molar amounts than others. Early studies described significant hybridization between segments (Blissard et al., 1986b), which raised the possibility that the viral gen-
ome is composed of families of segments with smaller segments derived from larger DNA segments through partial replication or intra-molecular recombination (i.e. segment nesting). Since not all CsPDV segments crosshybridize to any one probe, a ‘master’ template segment does not give rise to all segments but it seemed possible that the entire viral genome could be encoded by a few template segments (Blissard et al., 1986b). Subsequent studies that described multiple, cross-hybridizing gene families (Blissard et al., 1987, 1989; Dib-Hajj et al., 1993; Cui and Webb, 1996) and a ubiquitous 540-bp repetitive sequence (Theilmann and Summers, 1987, 1988) in the CsPDV genome suggested that crosshybridization between CsPDV gene segments could be caused by related genes on different segments rather than segment nesting. Molecular analyses of segment B demonstrated that it was integrated in the wasp genome and was not nested (Fleming and Summers, 1986, 1991). That four other segments hybridized to different wasp genomic loci in Southern blots also favored the hypothesis that the CsPDV genome was largely a unique sequence with each segment integrated at a different location (Fleming and Krell, 1993). However, sequence data that would document the nature and extent of the sequence homology between cross-hybridizing CsPDV segments have not been reported. We have revisited the issue of segment nesting in CsPDV and report here that many cross-hybridizing segments appear to be nested. We have begun to test the hypothesis that an increase in gene copy number results from segment nesting and is correlated with high levels of gene expression and the mode of action of individual genes.
2. Materials and methods 2.1. Biological materials The parasitic wasp C. sonorensis and its host Heliothis virescens were reared as described by Krell et al. (1982). For virus collection, ovaries were dissected from female wasps and the viral particles were purified by centrifugation on sucrose gradients (Krell et al., 1982). For RNA extraction, hemolymph was collected and fat body was dissected from parasitized 4th-instar H. virescens larvae. To minimize hemocyte contamination, the fat body samples were washed five times in 1 ml of cold phosphate-buffered saline (PBS). To evaluate the effects of varying CsPDV in larvae, 1 l of the purified viral particles equal to 0.01, 0.05, 0.1 and 0.4 female equivalents (FE) were injected into premolt 4th-instar H. virescens larvae using a pulled glass capillary. Hemolymph was collected at 36 h postinjection (pi) and diluted in cold PBS (1:10) for protein analysis.
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2.2. DNA and RNA isolations, Southern and Northern blotting Viral DNA was isolated from purified virions by the method of Krell et al. (1982). Male wasp genomic DNA was isolated by a procedure modified from Ausubel et al. (1994). RNA was isolated from whole H. virescens larvae, hemocytes and fat body by the method of guanidinium isothiocyanate–phenol extraction using the TriReagent kit (Molecular Research Center, Chomczynski and Sacchi, 1987). Random primer labeling of DNA with 32P-dATP was done using the Prime-a-Gene system (Promega). Southern and Northern hybridizations were performed under conditions of high stringency (Cui and Webb, 1996). For repeat hybridizations of the same blot, the nylon membranes were boiled in 0.1 × SSC and 0.1% SDS for 10 min to remove the probe before reusing the blot in hybridizations. For viral genomic Southern blots, 3 g of viral DNA was electrophoresed in a 0.7% agarose gel and the blot was probed sequentially with labeled total viral DNA, VHv1.4 cDNA, VHv1.1 cDNA, segment V and segment B. To calculate the relative abundance of these hybridizing bands, the viral DNA blot probed with labeled VHv1.4 and the Northern blots probed with labeled segment V were scanned in a phosphorimager (Molecular Dynamics). 2.3. Cloning of viral segment E Viral DNA was electrophoresed in a 0.7% agarose gel and the E band was excised from the superhelical region. After purification from agarose gel with Geneclean (BIO 101), the DNA was digested by BamHI and cloned into the pZero-1 vector (Invitrogen). The insert was verified by hybridization to the viral segment E in a Southern blot. The cloned E band was further used to screen two viral DNA libraries in pZero-1 to identify related clones (Cui, unpublished). 2.4. Immunoblotting To study expression of VHv1.4 and VHv1.1 proteins in CsPDV injected H. virescens larvae, 1 l of hemolymph was separated using 10% SDS–PAGE. Protein transfer and immunoblotting with antisera against VHv1.1 and VHv1.4 proteins were performed simultaneously using previously described methods (Li and Webb, 1994). 2.5. Egg encapsulation assay To study the encapsulation ability of hemocytes, eggs were dissected from female C. sonorensis oviducts and washed extensively with PBS to remove calyx fluid components. After storing at 4°C overnight, 4–5 eggs
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were injected into each of 20–30 4th-instar H. virescens larvae, which were infected with different doses of CsPDV from viral injections performed 36 h earlier. The larvae were dissected 24 h after egg injection and evaluated for encapsulation of the eggs.
3. Results Polydnavirus genomes are comprised of non-equimolar DNA segments ranging in size from 6–21 kb (Krell et al., 1982; Blissard et al., 1986a). So far, seven DNA segments have been cloned and mapped (Table 1), with some other segments partially cloned (Blissard et al., 1986b; Cui and Webb, 1997a). At this time, two types of segments are evident. Some of the larger viral segments (e.g. W and V) appear to contain nested segments with some of the smaller segments (R, M, K, C2 and C) produced by excision from a parent segment. Nested segments hybridize to a single integration site in wasp genomic segments. The nested segments are produced from this integration site by intra-molecular recombination events that probably occur after the segment begins replicating (Cui and Webb, 1997a). Interestingly, all of the characterized genes that are encoded on nested segments are abundantly expressed, secreted and are members of the cys-motif family (WHv1.0, WHv1.6, VHv1.4 and VHv1.1). By contrast other segments (e.g. B and E) are not nested, and the only gene that has been described from this type of segment (BHv0.9) is less abundant (at the mRNA level). It appears not to be Table 1 Summary of cloned CsPDV segments Segment (size in kb)
mRNAs encoded
gene family
segment family
W (15.8) M, C2 (1) V (15.2) L2, K, C (2, 4) O1 (11.2) U, O1 (3) M1 (10.8) H (8.4) E (7.8) B (6.6)
1.6, 1.0
cys
nested; W, R,
1.4, 1.1
cys
nested; V, T,
3.2, 2.6, 2.1, 1.8
rep*
nested; R, Q,
2.1 3.2 ? 0.9
? rep* ? rep#
unique unique unique unique
(1, 3) (3) (4) (3)
(1) Blissard et al., 1989; Cui and Webb, 1997a; (2) Cui and Webb, 1996; (3) Theilmann and Summers, 1988; (4) In this paper. *These segments contain rep sequences based on hybridization studies and hybridize to mRNAs of the indicated sizes in northern blots but the cDNAs have not been isolated or sequenced. #The rep gene family is defined as polydnavirus genes that contain a 540 bp repeated sequence. This is a proposed gene family as only one gene that contains a rep sequence has been sequenced (Theilmann and Summers, 1988). However, at least four other mRNAs hybrodize to the 540 bp repeated sequence and are thought to be members of this proposed gene family.
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Fig. 1. Electrophoretic profiles of CsPDV DNA and physical maps of segment W, V, E and B. Ethidium bromide stained 0.7% agarose gel of viral genomic DNA (1 g) to show segments W, V, T, Q, M, L2, K, E, C and B. Nomenclature of viral DNA segments is according to Krell et al. (1982) and Blissard et al. (1986a). Viral segment W and V are representatives of nested viral segments, while segments E and B are representatives of unique segments. Genes expressed in parasitized H. virescens WHv1.6, WHv1.0 (Blissard et al., 1987), VHv1.4 and VHv1.1 (Cui and Webb, 1996), BHv0.9 (Theilmann and Summers, 1988) are marked on their viral segments. RC, relaxed circular form; SH, superhelical form.
secreted and is a member of the proposed rep gene family (Table 1). The restriction maps of representative members of nested and unique DNA segments are shown for comparative purposes (Fig. 1). Previous work has indicated that segment V also hybridized to other viral segments (Webb and Summers, 1990). To identify the related segments, CsPDV genomic DNA was hybridized under high-stringency to labeled VHv1.1, VHv1.4, and segment V probes (Fig. 2). The results showed that the segment V probe hybridized to five viral segments, namely, V, T, L2, K and C. Interestingly, the VHv1.4 probe hybridized to the same five segments, suggesting that VHv1.4 gene sequences or sequences closely related to the VHv1.4 probe are located on these segments. However, only segment V hybridized to the VHv1.1 probe, which indicates that the VHv1.1 gene is located only on segment V (Fig. 2). This suggests that the VHv1.4 gene is present on all of the cross-hybridizing segments while the VHv1.1 gene is found only on segment V. A similar pattern in which one gene is lost in nested segments also exist with segment W and its nested segments (Blissard et al., 1986b; Cui and Webb, 1997a). By contrast, and in agreement with previous studies (Fleming and Summers, 1986), segment B probes hybridized to viral segment B only. Segment E is also unrelated to other viral segments and hybridized only to E under high stringency (Fig. 2). We have used two criteria for the identification of nested DNA segments. First, nested DNA segments hybridize under high-stringency conditions. CsPDV has two proposed gene families that complicate analyses per-
Fig. 2. Viral genomic Southern blots. Undigested viral DNA was electophoresed and blotted to a nylon membrane. The blot was probed sequentially with total viral DNA (lane 1), VHv1.4 (lane 2), VHv1.1 (lane 3), segment V (lane 4), segment B (lane 5) and segment E (lane 6). Both VHv1.4 and segment V hybridized to viral segments V, T, L2, K and C, whereas VHv1.1 hybridized only to segment V. Segment E and B were included for comparison. To reduce the complexity of the blot, only the superhelical form of viral DNA is shown.
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formed under reduced stringency conditions. By focusing only on segments that cross-hybridize under high stringency conditions these complications are reduced. Second, nested DNA segments must hybridize to a single genomic locus. Because even high-stringency hybridizations could permit limited cross-hybridization to closely related genes (e.g. > 90% identity), the second criterion is essential to identify nested DNA segments. If cross-hybridization between segments was caused by the presence of closely related genes on different segments, Southern hybridization to wasp genomic DNA would produce a hybridization pattern indicative of integration at multiple loci in the wasp genome. By selecting restriction enzymes that have 1–3 sites in a viral DNA segment it is possible to predict the Southern hybridization patterns that would result from the integration of that viral DNA. For example, segment V has a single XbaI site. Therefore, digestion of wasp genomic DNA with XbaI should generate two hybridizing bands on a genomic Southern blot from the single site within the segment and two restriction sites that lie in the flanking wasp DNA. If the bands that cross-hybridize with segment V, T, L2, K and C were integrated at different locations rather than nested, the Southern hybridization pattern would be complex, and would have at least as many bands in a wasp genomic Southern blot as in the viral genomic Southern blot. XbaI digested wasp DNA hybridized to only two bands when probed with segment V (Fig. 3). Although there are three PstI sites in segment V, and four hybridizing bands would be expected in a wasp genomic Southern, only two bands hybridized to the segment V probe (Fig. 3). Two of the segment V PstI sites are less than 500-bp apart and apparently generate a small band that was not detected in this blot. An alternative explanation is that one of the PstI sites is in or near an integration site in wasp genomic DNA. In either event, the Southern results indicate that segment V and cross-hybridizing viral segments are found at a single locus in the wasp genome. Viral segment E is not nested, and hybridizes only to a single segment in viral genomic Southern blots and is also integrated at a single locus in the wasp genome (not shown). Using these criteria for identifying nested and unique DNA segments, we have tentatively classified the seven segments that have been cloned in their entirety as either unique or nested (Table 1). Ultimately, segment nesting will not be indisputably proven until at least the junctions from both integrated and nested recombination junctions are cloned and sequenced. Our sequence analysis of segment W and related segments indicate that segments R, M and possibly C2 are nested within segment W (Cui and Webb, 1997a). To further evaluate the potential link between segment nesting and gene expression we have analyzed the correlation between gene copy number and level of expression from segment V and the putatively nested
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Fig. 3. Wasp genomic Southern blots probed with labeled segment V. An aliquot of 10 g of male wasp DNA was digested with PstI and XbaI, electrophoresed, blotted and probed with labeled V. The hybridization profile indicates that viral segments that hybridized to segment V (Fig. 2, lane 4) are probably nested at a single chromosomal locus in the wasp genome.
segments T, L2, K and C. If the intact VHv1.4 gene is located and expressed from all of the hybridizing segments and the promoters for the VHv1.1 and VHv1.4 gene are comparable, then the VHv1.4 gene should be expressed at a higher level. Moreover, the level of VHv1.4 mRNA should be directly correlated with the number of additional copies of the VHv1.4 gene found on the nested DNA segments. Assuming that all of the cross-hybridizing segments contain functional copies of the VHv1.4 gene (as suggested by amplification of the VHv1.4 gene from all cross-hybridizing segments), it is possible to directly determine the relative abundance of the VHv1.4 and VHv1.1 genes in the viral genome. To measure the relative abundance of the VHv1.4 and VHv1.1 genes, a viral genomic Southern blot was probed with a VHv1.4 probe (Fig. 2) and each hybridizing band was quantified by direct counting with a phosphorimager. Direct counting of each hybridizing segment determined that ~60% of the signal was caused by hybridization with segment V and the other ~40% of the hybridization signal was attributable to hybridization
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with the segments T, L2, K and C (Table 2). As the VHv1.1 gene is present in a single copy only on segment V, it should be under-represented in the viral genome relative to the VHv1.4 gene in a ratio that reflects the relative abundance of the nested segments encoding only the VHv1.4 gene. Therefore, probing a viral genomic blot with a VHv1.4 probe and quantifying each hybridizing signal allows the relative abundance of each segment to be directly determined (Table 2). The relative copies of the VHv1.1 and VHv1.4 genes were then inferred based on the knowledge that VHv1.1 is found only on segment V, while VHv1.4 is present on all the nested segments. Based on quantitative hybridization of VHv1.1 and VHv1.4 probes to segment V and nested segments, we have determined that the CsPDV genome encodes 1.67 copies of the VHv1.4 gene for every copy of the VHv1.1 gene (Table 2). To determine if the VHv1.4 gene was expressed at a higher level than the VHv1.1 gene in parasitized hosts, RNA was isolated from parasitized insects, and Northern blots were probed with a segment V probe (not shown). The relative abundance of the VHv1.1 and VHv1.4 mRNAs in infected larvae was simultaneously quantified on a phosphorimager and corrected for the differences in the sizes of the two mRNAs (Table 2). The VHv1.4 gene was expressed at a higher level than the VHv1.1 gene at a ratio of 1.82 copies of the VHv1.4 mRNA for every VHv1.1 mRNA. The relative abundance of the VHv1.4 and VHv1.1 genes (1.67:1) is reflected in the relative abundance of their encoded mRNAs (1.82:1), which suggests that the promoters of the VHv1.1 and VHv1.4 genes are of approximately equal strength. The high level of homology between the VHv1.1 and VHv1.4 promoter sequences also suggests that these genes are likely coordinately regulated. These data provide some support for our assumption that the levels of expression of these genes are directly correlated to their copy numbers in the viral genome. CsPDV has been reported to infect a variety of tissues
in H. virescens larvae (Stoltz and Vinson, 1979). Virusencoded cys-motif proteins, VHv1.1 and VHv1.4, have been detected in large amounts in the plasma of parasitized insects (Li and Webb, 1994; Cui et al., 1997). These cys-rich proteins also bind to hemocytes and are subsequently internalized into these cells, but do not bind to fat body or nervous tissue. However, the site(s) of synthesis of the cys-motif proteins are not known. Northern blots of RNA samples isolated from hemocytes and fat body showed that both the VHv1.4 and VHv1.1 genes are expressed in these tissues (Fig. 4). Both mRNAs are detected in much higher amounts in hemocytes, which indicates that the predominant expression of CsPDV mRNAs is from this tissue. The data suggest that the VHv1.1 and VHv1.4 proteins detected in the hemocytes are expressed from multiple tissues that are secreted into the hemolymph and then internalized, largely by granulocytes (Cui et al., 1997). The data indicate that nesting of viral DNA segments may be associated with the differential expression of genes encoded on nested segments. Polydnavirus genome segments are also found in non-equimolar ratios, with the most abundantly expressed genes (VHv1.1, VHv1.4, WHv1.0, WHv1.6) found on the most abundant
Table 2 Phosphorimager quantitation of Southern and northern blots Relative abundance (%) of segments hybridizing to VHv1.4
Ratio of gene copy numbers (1.4:1.1)* Ratio of mRNA levels (1.4:1.1)†
V (60.0) T (8.4) L2 (7.1) K (17.4) C (7.1) 1.67 : 1 1.82 : 1
*The ratio of gene copy numbers of VHv1.4:VHv1.1 was calculated based on the relative abundance of viral segment V among the Vhybridizing bands. †The ratio of mRNA levels of VHv1.4:VHv1.1 was calculated on the relative amount of the VHv1.4 and VHv1.1 bands hybridized to segment V probe and corrected for the difference in their sizes.
Fig. 4. Northern blot showing the expression of two cysteine-rich genes in different tissues of parasitized H. virescens. Total RNA was isolated from whole H. virescens larvae at 48 h post-parasitization (pp) (P), from hemocytes (H), and from fat body (F) of H. virescens 36 h pp. An aliquot of 15 g of total RNA was loaded in each lane, electrophoresed, blotted and probed with VHv1.4 and VHv1.1 cDNA. After hybridization blots were exposed for 12 h at − 80°C with an intensifying screen. Quantitative inferences about the relative abundance of the VHv1.1 and VHv1.4 mRNAs based on this exposure are complicated by the non-linearity of autoradiography, as film has a relatively narrow range in which the signal is quantitative. The apparent differences in mobility between hemocyte and fat body mRNAs is an artifact of this blot rather than a true difference in the mRNAs (Cui et al., 1997).
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the function of viral genes is linked to the number of copies of each viral gene.
4. Discussion
Fig. 5. Western blot showing the expression of cysteine-rich gene products (VHv1.4 and VHv1.1) in H. virescens larvae injected with 0.01, 0.05, 0.1 and 0.4 FE of purified PDV at 36 h earlier. Hemolymph from control (C) and parasitized (P) H. virescens larvae (3 days pp) are also shown. An aliquot of 1 l of hemolymph was electrophoresed in 10% SDS–PAGE, transferred to nitrocellulose membrane and blotted with antisera against VHv1.1 (Li and Webb, 1994) and VHv1.4 proteins (Cui et al., 1997).
DNA segments. These observations support the idea that viral functions require high level expression of at least some viral genes and that the viral genome has been modified to meet this requirement. However, the relationships between virus infectious dose, expression of specific viral proteins and viral functional activities are not well understood. To study these relationships we manipulated the infectious virus dose and monitored levels of VHv1.4 protein in the hemolymph, the effects of virus infection on hemocyte spreading and the inhibition of encapsulation. Decreasing the infectious dose caused a corresponding decrease in the hemolymph titer of the VHv1.4 and VHv1.1 proteins and presumably of other viral proteins (Fig. 5). Decreasing the amount of virus injected, also decreased the inhibition of the host encapsulation response, which suggests that reductions in viral expression are inversely correlated with inhibition of the encapsulation response (Table 3). As the titer of virus decreased, the effects on hemocyte spreading became less pronounced (data not shown) and the infected hosts were increasingly able to mount an effective encapsulation response. These results are consistent with the idea that the titer of specific viral proteins is critical to virus function, and that in the non-replicative polydnaviruses, Table 3 Encapsulation of injected wasp eggs in fourth instar H. virescens larvae infected with different doses of purified CsPDV Treatment Control (PBS) 0.01 FE 0.05 FE 0.1 FE 0.4 FE
Immunoresponsive insects* (%) 18/21 (85.7%) 17/23 (73.9%) 7/20 (35.0%) 0/24 (0%) 0/18 (0%)
*Immunoresponsive insects are defined as insects that contained at least one encapsulated egg.
Polydnavirus genomes are unique among the DNA viruses. The life-cycle and functions of polydnaviruses in their obligate mutualistic associations with some parasitic Hymenoptera are equally distinctive. The accumulated data from the Campoletis system have begun to elucidate the relationships between the viral genome and its unusual life-cycle. The data suggest that abundantly expressed, secreted genes are associated with hypermolar, nested DNA segments while genes that are targeted intra-cellularly and expressed at lower levels are not present on nested DNA segments. Based on these data we have developed the hypothesis that the level of expression of a given CsPDV gene is directly correlated with the number of genes introduced into parasitized insects. As a result of this evolutionary pressure, polydnavirus genomes may have evolved to contain non-equimolar segment ratios and nested viral DNA segments. It is also possible that this selection pressure may have driven the initial segmentation of polydnavirus genomes. The general strategy of increasing the copy number of selected genes to increase the expression levels is well known in other systems. Protozoans, insects and vertebrates exploit variations on this theme by producing mini-satellite chromosomes, and polyploid and polytene cells (Spradling and Mahowald, 1980; Stark and Wahl, 1984). Inducible gene amplification in mammalian cultured cells is also associated with the development of antibiotic resistance (Schimke, 1984). All of these systems allow for the selective amplification of genes and are correlated with higher levels of expression of the amplified gene (Long and Dawid, 1980). Because PDV genome segments exist in non-equimolar ratios and viral genome segments are nested, some PDV genes are introduced and remain at relatively low copy numbers while other genes have consistently higher copy numbers. We have noted a correlation between gene copy number and the level of gene expression in CsPDV and developed our hypothesis based, in part, upon these observations. From studies of WHv1.0, WHv1.6, VHv1.1, VHv1.4 and BHv0.9 genes, a consistent pattern of CsPDV gene expression emerges. In parasitized lepidopteran hosts, viral genes are expressed rapidly and persistently throughout endoparasite development with little significant variation in mRNA levels from 4 h to 8 days after parasitization (Theilmann and Summers, 1987; Li and Webb, 1994; Cui and Webb, 1996). This type of expression is similar to viral ‘early’ genes that require only host transcription factors for expression. Indeed all three of the CsPDV promoters that have been tested in recombinant baculoviruses function as ‘early’ promoters (Soldevila and
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Webb, 1996). Although some variability in the apparent levels of gene expression was noted in one study (Theilmann and Summers, 1988), there is no indication that CsPDV exploits trans-activating factors that alter viral gene expression in a temporally dependent manner, as is common during the replication of many viruses (Cui and Webb, 1997b). Rather, viral genes are expressed rapidly and constitutively. Under this type of regulatory system, the level of gene expression is completely dependent upon the cis-dependent promoter activity and the number of copies of the gene (gene dosage). Viral genes expressed in parasitized insects alter the physiology of their hosts in dramatic and interesting ways. CsPDV genes from two gene families that are expressed in parasitized insects have been described. The cys-motif gene family (Dib-Hajj et al., 1993) has been characterized on the basis of a cysteine-rich motif that is found in single or double copies on the genes. The members of the cys-motif gene family (WHv1.0, WHv1.6, VHv1.1 and VHv1.4) are located on abundant DNA segments (W and V respectively) that are now thought to be parent molecules for other segments. Segment W, is the parent molecule for segments R, M and possibly C2 (Cui and Webb, 1997a). Segment V is also thought to produce nested segments L2, K and C. Members of the cys-motif gene family encode secreted proteins with similar gene structures and are the most abundantly expressed CsPDV genes in parasitized insects. The segment V-encoded proteins bind to host hemocytes and have been implicated in suppressing the host immune system (Li and Webb, 1994; Cui et al., 1997). The BHv0.9 gene is a member of the rep gene family that has been described on the basis of a 540-bp repeat sequence that is ubiquitously distributed within the viral genome and appears to be present in at least one copy on most viral DNA segments (Theilmann and Summers, 1987). By contrast, the BHv0.9 protein is not secreted and the mRNA is less abundant than the cys-motif mRNAs. Based on these data we propose that nesting of gene segments is associated with high level gene expression in parasitized insects and that nested segments are likely to encode abundantly expressed genes. The persistence of PDV gene expression in parasitized insects has been documented in several parasitoid–host systems. For both PDV genera, viral genes are expressed rapidly. However, the persistence of viral gene expression varies among the systems. Expression of the Ichnovirus, CsPDV, is rapid and at a constant level throughout endoparasite development (Theilmann and Summers, 1988; Li and Webb, 1994; Cui and Webb, 1996). By contrast, expression of bracoviruses may be transient or decreasing at later stages of parasitization (Harwood and Beckage, 1994; Asgari et al., 1996). The persistence of viral gene expression in parasitized insects is dependent on the persistence of the virus itself. In both PDV genera, viral DNA can persist without an increase
in the amount in the parasitized insects to late stages of parasitoid development (Theilmann and Summers, 1986; Strand et al., 1992). In parasitized hosts, the PDVs are able to infect many host tissues (Stoltz and Vinson, 1979). Both hemocytes and fat body have been reported to be major tissues of Bracovirus gene expression (Strand et al., 1992; Harwood and Beckage, 1994). Further, the Microplitis demolitor PDV infects all the hemocyte morphotypes of Pseudoplusia includens and causes apoptosis of the host granulocytes (Strand, 1994; Strand and Pech, 1995b). Similarly, we also found that CsPDV mRNAs are abundantly expressed in host hemocytes and to a lesser degree in fat body. Although apoptosis of hemocytes is not apparent in CsPDVinfected H. virescens, the morphology and in vitro spreading behavior of plasmatocytes and granulocytes are altered (Webb and Luckhart, 1994, 1996; Luckhart and Webb, 1996). Therefore, the inhibition of host cellular immunity associated with the pathology of host hemocytes may be a direct effect of hemocyte infection and/or targeting of hemocytes by virus-encoded proteins (Li and Webb, 1994; Cui et al., 1997). Our results have led us to reconsider the relationship between the genomic organization of PDVs and their unique biology. Our data suggest that PDV genome organization is inextricably linked to the diverse functions of PDVs in their two host insects and that segmentation in PDV genomes may have evolved to increase the copy number of essential viral genes. Viral genes must be abundantly expressed in parasitized insects in the absence of virus replication or a regulatory cascade to increase mRNA levels. Genome segmentation, segment nesting, non-equimolar segment ratios and the presence of viral structural proteins in the wasp genome are diverse manifestations of this fundamental selection pressure. Acknowledgements This work was supported by NIH AI 3314 and NSF MCB 9603504 to B.A.W. This is publication #96-08205 of the Kentucky Agricultural Experiment Station (Lexington, KY). References Asgari, S., Hellers, M., Schmidt, O., 1996. Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. Journal of General Virology 77, 2653–2662. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1994. Current Protocols in Molecular Biology. John Wiley and Sons. Blissard, G.W., Fleming, J.G.W., Vinson, S.B., Summers, M.D., 1986a. Campoletis sonorensis virus: expression in Heliothis virescens and identification of expressed sequences. Journal of Insect Physiology 32, 351–359.
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Blissard, G.W., Vinson, S.B., Summers, M.D., 1986b. Identification, mapping and in vitro translation of Campoletis sonorensis virus mRNAs from parasitized Heliothis virescens larvae. Journal of Virology 57, 318–327. Blissard, G.W., Smith, O.P., Summers, M.D., 1987. Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome. Virology 160, 120–134. Blissard, G.W., Theilmann, D.A., Summers, M.D., 1989. Segment W of the Campoletis sonorensis virus: expression, gene products, and organization. Virology 169, 78–79. Chomczynski, P., Sacchi, N., 1987. Single step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 161, 156–159. Cui, L., Soldevila, A.I., Webb, B.A., 1997. Expression and hemocytetargeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae. Archives of Insect Biochemistry and Physiology 36, 251–271. Cui, L., Webb, B.A., 1996. Isolation and characterization of a member of the cysteine-rich gene family from Campoletis sonorensis polydnavirus. Journal of General Virology 77, 797–809. Cui, L., Webb, B.A., 1997a. Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family. Journal of Virology 71, 8504-8513. Cui, L., Webb, B.A., 1997b. Promoter analysis of a cysteine-rich Campoletis sonorensis polydnavirus gene. Journal of General Virology 78, 1807–1817. Dib-Hajj, S.D., Webb, B.A., Summers, M.D., 1993. Structure and evolutionary implications of a ’cysteine-rich’ Campoletis sonorensis polydnavirus gene family. Proceedings of the National Academy of Sciences, USA 90, 3765–3769. Edson, K.M., Vinson, S.B., Stoltz, D.B., Summers, M.D., 1981. Virus in a parasitoid wasp: suppression of the cellular immune response in the parasitoid’s host. Science 211, 582–583. Fleming, J.G.W., Krell, P.J., 1993. Polydnavirus genome organization. In: Beckage, N.E., Thompson, S.N., Federici, B.A. (Eds). Parasites and Pathogens of Insects, Vol. 1 Parasites. Academic Press, NY, pp. 189–225. Fleming, J.G.W., Summers, M.D., 1986. Campoletis sonorensis endoparasitic wasps contain forms of C. sonorensis virus DNA suggestive of integrated and extrachromosomal polydnavirus DNAs. Journal of Virology 57, 552–562. Fleming, J.G.W., Summers, M.D., 1991. Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host. Proceedings of the National Academy of Sciences, USA 88, 9770–9774. Harwood, S.H., Beckage, N.E., 1994. Purification and characterization of an early-expressed polydnavirus-induced protein from the hemolymph of Manduca sexta larvae parasitized by Cotesia congregata. Insect Biochemistry and Molecular Biology 24, 685–698. Krell, P.J., Summers, M.D., Vinson, S.B., 1982. Virus with a multipartite superhelical DNA genome from the ichneumonid parasitoid Campoletis sonorensis. Journal of Virology 43, 859–870. Lavine, M.D., Beckage, N.E., 1995. Polydnaviruses: potent mediators of host insect immune dysfunction. Parasitology Today 11, 368–378. Li, X., Webb, B.A., 1994. Apparent functional role for a cysteine-rich polydnavirus protein in suppression of the insect cellular immune response. Journal of Virology 68, 7482–7489. Long, E.O., Dawid, I.B., 1980. Repeated genes in eukaryotes. Annual Review of Biochemistry 49, 727–764. Luckhart, S., Webb, B.A., 1996. Interaction of a wasp ovarian protein and polydnavirus in host immune suppression. Developmental and Comparative Immunology 20, 1–21. Schimke, R.T., 1984. Gene amplification in cultured animal cells. Cell 37, 705–713. Soldevila, A.I., Webb, B.A., 1996. Expression of polydnavirus genes under polydnavirus-promoter regulation in baculovirus recombinants. Journal of General Virology 77, 1379–1388.
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Spradling, A.C., Mahowald, A.P., 1980. Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proceedings of the National Academy of Sciences, USA 77, 1069–1074. Stark, G.R., Wahl, G.M., 1984. Gene amplification. Annual Review of Biochemistry 53, 447–491. Stoltz, D.B., 1990. Evidence for chromosomal transmission of polydnavirus DNA. Journal of General Virology 71, 1051–1056. Stoltz, D.B., 1993. The polydnavirus life cycle. In: Beckage, N.E., Thompson, S.N., Federici, B.A. (Eds). Parasites and Pathogens of Insects, Vol. 1 Parasites. Academic Press, NY, pp. 167–187. Stoltz, D.B., Beckage, N.E., Blissard, G.W., Fleming, J.G.W., Krell, P.J., Theilmann, D.A., Summers, M.D., Webb, B.A., 1995. Polydnaviridae. In: Murphy, F.A., Fauquet, C.M., Bishop, D.H.L., Ghabrial, S.A., Jarvis, A.W., Martelli, G.P., Mayo, M.A., Summers, M.D. Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses. Springer–Verlag, Wien and New York, pp. 143–147. Stoltz, D.B., Guzo, D., Cook, D., 1986. Studies on polydnavirus transmission. Virology 155, 120–131. Stoltz, D.B., Vinson, S.B., 1979. Penetration into caterpillar cells of virus-like particles injected during oviposition by parasitoid ichneumonid wasps. Canadian Journal of Micobiology 25, 207–216. Stoltz, D.B., Whitfield, J.B., 1992. Viruses and virus-like entities in the parasitic Hymenoptera. Journal of Hymenopteran Research 1, 125–139. Strand, M.R., 1994. Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens haemocytes. Journal of General Virology 75, 3007–3020. Strand, M.R., McKenzie, D.I., Grassl, V., Dover, B.A., Arken, J.M., 1992. Persistence and expression of Microplitis demolitor polydnavirus in Pseudoplusia includens. Journal of General Virology 73, 1627–1635. Strand, M.R., Pech, L.L., 1995a. Immunological basis for compatibility in parasitoid–host relationships. Annual Review of Entomology 40, 31–56. Strand, M.R., Pech, L.L., 1995b. Microplitis demolitor polydnavirus induces apoptosis of a specific hemocyte morphotype in Pseudoplusia includens. Journal of General Virology 76, 283–291. Summers, M.D., Dib-Hajj, S.D., 1995. Polydnavirus-facilitated endoparasite protection against host immune defenses. Proceedings of the National Academy of Sciences, USA 92, 29–36. Theilmann, D.A., Summers, M.D., 1986. Molecular analysis of Campoletis sonorensis virus DNA in the lepidopteran host Heliothis virescens. Journal of General Virology 67, 1961–1969. Theilmann, D.A., Summers, M.D., 1987. Physical analysis of the Campoletis sonorensis virus multipartite genome and identification of a family of tandemly repeated elements. Journal of Virology 61, 2589–2598. Theilmann, D.A., Summers, M.D., 1988. Identification and comparison of Campoletis sonorensis virus transcripts expressed from four genomic segments in the insect hosts Campoletis sonorensis and Heliothis virescens. Virology 167, 329–341. Webb, B.A., Luckhart, S., 1994. Evidence for an early immunosuppressive role for related Campoletis sonorensis venom and ovarian proteins in Heliothis virescens. Archives of Insect Biochemistry and Physiology 26, 147–163. Webb, B.A., Luckhart, S., 1996. Factors mediating short- and longterm immunosuppression in a parasitized insect. Journal of Insect Physiology 42, 33–40. Webb, B.A., Summers, M.D., 1990. Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences. Proceedings of the National Academy Sciences, USA 87, 4961–4965. Xu, D., Stoltz, D.B., 1993. Polydnavirus genome segment families in the ichneumonid parasitoid Hyposoter fugitives. Journal of Virology 67, 1340–1349.
Relationships between polydnavirus genomes and viral gene expression Bruce A. Webb b
a,*
, Liwang Cui
a,b
a Department of Entomology, University of Kentucky, Lexington, Kentucky 40546, USA Current address: Entomology Department, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA
Received 1 October 1997; accepted 23 October 1997
Abstract Polydnavirus genomes and viral gene functions are atypical for viruses. Polydnaviruses are the only group of viruses with segmented DNA genomes and have an unusual obligate mutualistic association with parasitic Hymenoptera, in which the virus is required for survival of the wasp host and vice versa. The virus replicates asymptomatically in the wasp host but severely disrupts lepidopteran host physiology in the absence of viral DNA replication. It is not surprising then that viral gene expression is divergent in its two insect hosts and that differences in viral gene expression are linked to these divergent functions. Some viral genes are expressed only in the wasp host while other viral genes are expressed only in the lepidopteran host and are presumed to be involved in the disruption of host physiological systems. Our laboratory has described the expression and regulation of a family of viral genes implicated in suppressing the lepidopteran immune system, the cys-motif genes. In conjunction with these studies we have described the physical organization of additional viral gene segments. We have cloned, mapped and begun the sequence analysis of selected viral DNA segments. We have noted that some viral DNA segments are nested and that nested viral DNA segments encode the abundantly expressed, secreted cys-motif genes. Conversely, other viral segments are not nested, encode less abundantly expressed genes and may be targeted intra-cellularly. These results suggest that nesting of segments in polydnavirus genomes may be linked to the levels of gene expression. By extension, the unique, segmented organization of polydnavirus genomes may be associated, in part, with the requirement for divergent levels of viral gene expression in lepidopteran hosts in the absence of viral DNA replication. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Campoletis sonorensis; Heliothis virescens; Polydnavirus; Segmented genome; Cysteine-rich gene; Gene expression
1. Introduction Polydnaviruses (PDVs) are obligate symbionts of some parasitic Hymenoptera and replicate asymptomatically from integrated proviral DNA only in specialized ‘calyx’ cells of the female reproductive tract (Stoltz, 1993; Stoltz et al., 1995). Polydnaviruses have been described from some of the ichneumonids and braconids and comprise the two genera in this family, Ichnovirus and Bracovirus, respectively. The PDV life-cycle is characterized by asymptomatic virus replication from proviral DNA in a parasitic wasp followed by pathogenic virus infection in the wasp’s larval lepidopteran host. Thus, PDV replication and function is inextricably
* Corresponding author. Tel: (606) 257 7415. Fax: (606) 323 1120. E-mail: [email protected]. 0022–1910 /98 /$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 1 1 - 0
linked to the life-cycle of the parasitic wasp (Stoltz, 1993). Virus is introduced from the female reproductive tract into parasitized insects during oviposition. A species-specific subset of viral genes is expressed in parasitized insects in the absence of viral DNA replication. In both PDV genera, viral gene expression in the lepidopteran host disrupts some host physiological systems and is required for successful development of the endoparasitic wasp (Edson et al., 1981; Lavine and Beckage, 1995; Strand and Pech, 1995a). Thus, the PDV–wasp parasite– lepidopteran host system provides an unusual example of an obligate mutualistic association between a virus and a parasitic wasp that functions to the extreme detriment of the parasite’s lepidopteran host. As a result of this mutually obligate association, PDVs are found in every individual of an infected species and do not replicate outside of their associated wasp host (Stoltz et al., 1986; Stoltz, 1990, 1993). To emphasize this point,
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PDVs replicate only in some of the wasp cells carrying proviral DNA and persist, evolutionarily, on the basis of their incorporation in the wasp genome. PDVs are the only viruses with segmented DNA genomes (Stoltz et al., 1995). Moreover, segmentation of PDV genomes is often extreme. Segmented RNA viruses may have up to 12 segments with an aggregate genome size of about 20 kb, while the Campoletis sonorensis polydnavirus (CsPDV) has at least 28 DNA segments with genome size estimates in excess of 250 kb (Krell et al., 1982; Fleming and Krell, 1993). Although a segmented genome is a hallmark of PDVs, we have little understanding of the evolutionary pressures that have driven, or alternatively, allowed segmentation of the viral genome. One hypothesis ventured is that genome segmentation (and the presence of repeated DNA and viral gene families) enhances the capability of the virus to generate genetic variability through recombination (Dib-Hajj et al., 1993; Summers and Dib-Hajj, 1995). While the importance of mechanisms for generating genetic diversity is unquestionable, it does not account for recently described PDV segments with extensive sequence identity (Xu and Stoltz, 1993). Specifically, mapping of cross-hybridizing Hyposoter fugitivus PDV segments suggests that some smaller DNA segments are excised from larger DNA segments, an organizational pattern known as ‘segment nesting’ (Cui and Webb, 1997a; Xu and Stoltz, 1993). Segment nesting increases the number of PDV segments without significantly increasing the virus‘ sequence complexity. However, nesting does increase the representation of nested sequences within the genome. Apparently, at least some PDV genomes are comprised of nested DNA segments, although the extent of segment nesting and its functional significance remained enigmatic. Stoltz and Whitfield (1992) proposed that the unique genomic organization of PDVs is a consequence of their unique viral life cycles in which PDVs play an integral and essential role in insect host–parasite systems. In this paper, we partially describe the contributions of nested PDV segments to genome complexity, and begin to evaluate the relationships between segment nesting and PDV gene expression. The CsPDV is the type species of the Ichnovirus genus (Stoltz et al., 1995) and has been described in the most detail. CsPDV segments exist as circular, supercoiled DNA molecules (superhelical DNAs) in virions, and as linear proviral molecules when integrated in the wasp genome. Segments are named alphabetically by the size of the circular molecules from the smallest segment A (~6 kb) to the largest X (~16 kb) (Krell et al., 1982; Blissard et al., 1986a). Viral segments are present in nonequimolar amounts with some present in much greater molar amounts than others. Early studies described significant hybridization between segments (Blissard et al., 1986b), which raised the possibility that the viral gen-
ome is composed of families of segments with smaller segments derived from larger DNA segments through partial replication or intra-molecular recombination (i.e. segment nesting). Since not all CsPDV segments crosshybridize to any one probe, a ‘master’ template segment does not give rise to all segments but it seemed possible that the entire viral genome could be encoded by a few template segments (Blissard et al., 1986b). Subsequent studies that described multiple, cross-hybridizing gene families (Blissard et al., 1987, 1989; Dib-Hajj et al., 1993; Cui and Webb, 1996) and a ubiquitous 540-bp repetitive sequence (Theilmann and Summers, 1987, 1988) in the CsPDV genome suggested that crosshybridization between CsPDV gene segments could be caused by related genes on different segments rather than segment nesting. Molecular analyses of segment B demonstrated that it was integrated in the wasp genome and was not nested (Fleming and Summers, 1986, 1991). That four other segments hybridized to different wasp genomic loci in Southern blots also favored the hypothesis that the CsPDV genome was largely a unique sequence with each segment integrated at a different location (Fleming and Krell, 1993). However, sequence data that would document the nature and extent of the sequence homology between cross-hybridizing CsPDV segments have not been reported. We have revisited the issue of segment nesting in CsPDV and report here that many cross-hybridizing segments appear to be nested. We have begun to test the hypothesis that an increase in gene copy number results from segment nesting and is correlated with high levels of gene expression and the mode of action of individual genes.
2. Materials and methods 2.1. Biological materials The parasitic wasp C. sonorensis and its host Heliothis virescens were reared as described by Krell et al. (1982). For virus collection, ovaries were dissected from female wasps and the viral particles were purified by centrifugation on sucrose gradients (Krell et al., 1982). For RNA extraction, hemolymph was collected and fat body was dissected from parasitized 4th-instar H. virescens larvae. To minimize hemocyte contamination, the fat body samples were washed five times in 1 ml of cold phosphate-buffered saline (PBS). To evaluate the effects of varying CsPDV in larvae, 1 l of the purified viral particles equal to 0.01, 0.05, 0.1 and 0.4 female equivalents (FE) were injected into premolt 4th-instar H. virescens larvae using a pulled glass capillary. Hemolymph was collected at 36 h postinjection (pi) and diluted in cold PBS (1:10) for protein analysis.
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2.2. DNA and RNA isolations, Southern and Northern blotting Viral DNA was isolated from purified virions by the method of Krell et al. (1982). Male wasp genomic DNA was isolated by a procedure modified from Ausubel et al. (1994). RNA was isolated from whole H. virescens larvae, hemocytes and fat body by the method of guanidinium isothiocyanate–phenol extraction using the TriReagent kit (Molecular Research Center, Chomczynski and Sacchi, 1987). Random primer labeling of DNA with 32P-dATP was done using the Prime-a-Gene system (Promega). Southern and Northern hybridizations were performed under conditions of high stringency (Cui and Webb, 1996). For repeat hybridizations of the same blot, the nylon membranes were boiled in 0.1 × SSC and 0.1% SDS for 10 min to remove the probe before reusing the blot in hybridizations. For viral genomic Southern blots, 3 g of viral DNA was electrophoresed in a 0.7% agarose gel and the blot was probed sequentially with labeled total viral DNA, VHv1.4 cDNA, VHv1.1 cDNA, segment V and segment B. To calculate the relative abundance of these hybridizing bands, the viral DNA blot probed with labeled VHv1.4 and the Northern blots probed with labeled segment V were scanned in a phosphorimager (Molecular Dynamics). 2.3. Cloning of viral segment E Viral DNA was electrophoresed in a 0.7% agarose gel and the E band was excised from the superhelical region. After purification from agarose gel with Geneclean (BIO 101), the DNA was digested by BamHI and cloned into the pZero-1 vector (Invitrogen). The insert was verified by hybridization to the viral segment E in a Southern blot. The cloned E band was further used to screen two viral DNA libraries in pZero-1 to identify related clones (Cui, unpublished). 2.4. Immunoblotting To study expression of VHv1.4 and VHv1.1 proteins in CsPDV injected H. virescens larvae, 1 l of hemolymph was separated using 10% SDS–PAGE. Protein transfer and immunoblotting with antisera against VHv1.1 and VHv1.4 proteins were performed simultaneously using previously described methods (Li and Webb, 1994). 2.5. Egg encapsulation assay To study the encapsulation ability of hemocytes, eggs were dissected from female C. sonorensis oviducts and washed extensively with PBS to remove calyx fluid components. After storing at 4°C overnight, 4–5 eggs
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were injected into each of 20–30 4th-instar H. virescens larvae, which were infected with different doses of CsPDV from viral injections performed 36 h earlier. The larvae were dissected 24 h after egg injection and evaluated for encapsulation of the eggs.
3. Results Polydnavirus genomes are comprised of non-equimolar DNA segments ranging in size from 6–21 kb (Krell et al., 1982; Blissard et al., 1986a). So far, seven DNA segments have been cloned and mapped (Table 1), with some other segments partially cloned (Blissard et al., 1986b; Cui and Webb, 1997a). At this time, two types of segments are evident. Some of the larger viral segments (e.g. W and V) appear to contain nested segments with some of the smaller segments (R, M, K, C2 and C) produced by excision from a parent segment. Nested segments hybridize to a single integration site in wasp genomic segments. The nested segments are produced from this integration site by intra-molecular recombination events that probably occur after the segment begins replicating (Cui and Webb, 1997a). Interestingly, all of the characterized genes that are encoded on nested segments are abundantly expressed, secreted and are members of the cys-motif family (WHv1.0, WHv1.6, VHv1.4 and VHv1.1). By contrast other segments (e.g. B and E) are not nested, and the only gene that has been described from this type of segment (BHv0.9) is less abundant (at the mRNA level). It appears not to be Table 1 Summary of cloned CsPDV segments Segment (size in kb)
mRNAs encoded
gene family
segment family
W (15.8) M, C2 (1) V (15.2) L2, K, C (2, 4) O1 (11.2) U, O1 (3) M1 (10.8) H (8.4) E (7.8) B (6.6)
1.6, 1.0
cys
nested; W, R,
1.4, 1.1
cys
nested; V, T,
3.2, 2.6, 2.1, 1.8
rep*
nested; R, Q,
2.1 3.2 ? 0.9
? rep* ? rep#
unique unique unique unique
(1, 3) (3) (4) (3)
(1) Blissard et al., 1989; Cui and Webb, 1997a; (2) Cui and Webb, 1996; (3) Theilmann and Summers, 1988; (4) In this paper. *These segments contain rep sequences based on hybridization studies and hybridize to mRNAs of the indicated sizes in northern blots but the cDNAs have not been isolated or sequenced. #The rep gene family is defined as polydnavirus genes that contain a 540 bp repeated sequence. This is a proposed gene family as only one gene that contains a rep sequence has been sequenced (Theilmann and Summers, 1988). However, at least four other mRNAs hybrodize to the 540 bp repeated sequence and are thought to be members of this proposed gene family.
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Fig. 1. Electrophoretic profiles of CsPDV DNA and physical maps of segment W, V, E and B. Ethidium bromide stained 0.7% agarose gel of viral genomic DNA (1 g) to show segments W, V, T, Q, M, L2, K, E, C and B. Nomenclature of viral DNA segments is according to Krell et al. (1982) and Blissard et al. (1986a). Viral segment W and V are representatives of nested viral segments, while segments E and B are representatives of unique segments. Genes expressed in parasitized H. virescens WHv1.6, WHv1.0 (Blissard et al., 1987), VHv1.4 and VHv1.1 (Cui and Webb, 1996), BHv0.9 (Theilmann and Summers, 1988) are marked on their viral segments. RC, relaxed circular form; SH, superhelical form.
secreted and is a member of the proposed rep gene family (Table 1). The restriction maps of representative members of nested and unique DNA segments are shown for comparative purposes (Fig. 1). Previous work has indicated that segment V also hybridized to other viral segments (Webb and Summers, 1990). To identify the related segments, CsPDV genomic DNA was hybridized under high-stringency to labeled VHv1.1, VHv1.4, and segment V probes (Fig. 2). The results showed that the segment V probe hybridized to five viral segments, namely, V, T, L2, K and C. Interestingly, the VHv1.4 probe hybridized to the same five segments, suggesting that VHv1.4 gene sequences or sequences closely related to the VHv1.4 probe are located on these segments. However, only segment V hybridized to the VHv1.1 probe, which indicates that the VHv1.1 gene is located only on segment V (Fig. 2). This suggests that the VHv1.4 gene is present on all of the cross-hybridizing segments while the VHv1.1 gene is found only on segment V. A similar pattern in which one gene is lost in nested segments also exist with segment W and its nested segments (Blissard et al., 1986b; Cui and Webb, 1997a). By contrast, and in agreement with previous studies (Fleming and Summers, 1986), segment B probes hybridized to viral segment B only. Segment E is also unrelated to other viral segments and hybridized only to E under high stringency (Fig. 2). We have used two criteria for the identification of nested DNA segments. First, nested DNA segments hybridize under high-stringency conditions. CsPDV has two proposed gene families that complicate analyses per-
Fig. 2. Viral genomic Southern blots. Undigested viral DNA was electophoresed and blotted to a nylon membrane. The blot was probed sequentially with total viral DNA (lane 1), VHv1.4 (lane 2), VHv1.1 (lane 3), segment V (lane 4), segment B (lane 5) and segment E (lane 6). Both VHv1.4 and segment V hybridized to viral segments V, T, L2, K and C, whereas VHv1.1 hybridized only to segment V. Segment E and B were included for comparison. To reduce the complexity of the blot, only the superhelical form of viral DNA is shown.
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formed under reduced stringency conditions. By focusing only on segments that cross-hybridize under high stringency conditions these complications are reduced. Second, nested DNA segments must hybridize to a single genomic locus. Because even high-stringency hybridizations could permit limited cross-hybridization to closely related genes (e.g. > 90% identity), the second criterion is essential to identify nested DNA segments. If cross-hybridization between segments was caused by the presence of closely related genes on different segments, Southern hybridization to wasp genomic DNA would produce a hybridization pattern indicative of integration at multiple loci in the wasp genome. By selecting restriction enzymes that have 1–3 sites in a viral DNA segment it is possible to predict the Southern hybridization patterns that would result from the integration of that viral DNA. For example, segment V has a single XbaI site. Therefore, digestion of wasp genomic DNA with XbaI should generate two hybridizing bands on a genomic Southern blot from the single site within the segment and two restriction sites that lie in the flanking wasp DNA. If the bands that cross-hybridize with segment V, T, L2, K and C were integrated at different locations rather than nested, the Southern hybridization pattern would be complex, and would have at least as many bands in a wasp genomic Southern blot as in the viral genomic Southern blot. XbaI digested wasp DNA hybridized to only two bands when probed with segment V (Fig. 3). Although there are three PstI sites in segment V, and four hybridizing bands would be expected in a wasp genomic Southern, only two bands hybridized to the segment V probe (Fig. 3). Two of the segment V PstI sites are less than 500-bp apart and apparently generate a small band that was not detected in this blot. An alternative explanation is that one of the PstI sites is in or near an integration site in wasp genomic DNA. In either event, the Southern results indicate that segment V and cross-hybridizing viral segments are found at a single locus in the wasp genome. Viral segment E is not nested, and hybridizes only to a single segment in viral genomic Southern blots and is also integrated at a single locus in the wasp genome (not shown). Using these criteria for identifying nested and unique DNA segments, we have tentatively classified the seven segments that have been cloned in their entirety as either unique or nested (Table 1). Ultimately, segment nesting will not be indisputably proven until at least the junctions from both integrated and nested recombination junctions are cloned and sequenced. Our sequence analysis of segment W and related segments indicate that segments R, M and possibly C2 are nested within segment W (Cui and Webb, 1997a). To further evaluate the potential link between segment nesting and gene expression we have analyzed the correlation between gene copy number and level of expression from segment V and the putatively nested
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Fig. 3. Wasp genomic Southern blots probed with labeled segment V. An aliquot of 10 g of male wasp DNA was digested with PstI and XbaI, electrophoresed, blotted and probed with labeled V. The hybridization profile indicates that viral segments that hybridized to segment V (Fig. 2, lane 4) are probably nested at a single chromosomal locus in the wasp genome.
segments T, L2, K and C. If the intact VHv1.4 gene is located and expressed from all of the hybridizing segments and the promoters for the VHv1.1 and VHv1.4 gene are comparable, then the VHv1.4 gene should be expressed at a higher level. Moreover, the level of VHv1.4 mRNA should be directly correlated with the number of additional copies of the VHv1.4 gene found on the nested DNA segments. Assuming that all of the cross-hybridizing segments contain functional copies of the VHv1.4 gene (as suggested by amplification of the VHv1.4 gene from all cross-hybridizing segments), it is possible to directly determine the relative abundance of the VHv1.4 and VHv1.1 genes in the viral genome. To measure the relative abundance of the VHv1.4 and VHv1.1 genes, a viral genomic Southern blot was probed with a VHv1.4 probe (Fig. 2) and each hybridizing band was quantified by direct counting with a phosphorimager. Direct counting of each hybridizing segment determined that ~60% of the signal was caused by hybridization with segment V and the other ~40% of the hybridization signal was attributable to hybridization
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with the segments T, L2, K and C (Table 2). As the VHv1.1 gene is present in a single copy only on segment V, it should be under-represented in the viral genome relative to the VHv1.4 gene in a ratio that reflects the relative abundance of the nested segments encoding only the VHv1.4 gene. Therefore, probing a viral genomic blot with a VHv1.4 probe and quantifying each hybridizing signal allows the relative abundance of each segment to be directly determined (Table 2). The relative copies of the VHv1.1 and VHv1.4 genes were then inferred based on the knowledge that VHv1.1 is found only on segment V, while VHv1.4 is present on all the nested segments. Based on quantitative hybridization of VHv1.1 and VHv1.4 probes to segment V and nested segments, we have determined that the CsPDV genome encodes 1.67 copies of the VHv1.4 gene for every copy of the VHv1.1 gene (Table 2). To determine if the VHv1.4 gene was expressed at a higher level than the VHv1.1 gene in parasitized hosts, RNA was isolated from parasitized insects, and Northern blots were probed with a segment V probe (not shown). The relative abundance of the VHv1.1 and VHv1.4 mRNAs in infected larvae was simultaneously quantified on a phosphorimager and corrected for the differences in the sizes of the two mRNAs (Table 2). The VHv1.4 gene was expressed at a higher level than the VHv1.1 gene at a ratio of 1.82 copies of the VHv1.4 mRNA for every VHv1.1 mRNA. The relative abundance of the VHv1.4 and VHv1.1 genes (1.67:1) is reflected in the relative abundance of their encoded mRNAs (1.82:1), which suggests that the promoters of the VHv1.1 and VHv1.4 genes are of approximately equal strength. The high level of homology between the VHv1.1 and VHv1.4 promoter sequences also suggests that these genes are likely coordinately regulated. These data provide some support for our assumption that the levels of expression of these genes are directly correlated to their copy numbers in the viral genome. CsPDV has been reported to infect a variety of tissues
in H. virescens larvae (Stoltz and Vinson, 1979). Virusencoded cys-motif proteins, VHv1.1 and VHv1.4, have been detected in large amounts in the plasma of parasitized insects (Li and Webb, 1994; Cui et al., 1997). These cys-rich proteins also bind to hemocytes and are subsequently internalized into these cells, but do not bind to fat body or nervous tissue. However, the site(s) of synthesis of the cys-motif proteins are not known. Northern blots of RNA samples isolated from hemocytes and fat body showed that both the VHv1.4 and VHv1.1 genes are expressed in these tissues (Fig. 4). Both mRNAs are detected in much higher amounts in hemocytes, which indicates that the predominant expression of CsPDV mRNAs is from this tissue. The data suggest that the VHv1.1 and VHv1.4 proteins detected in the hemocytes are expressed from multiple tissues that are secreted into the hemolymph and then internalized, largely by granulocytes (Cui et al., 1997). The data indicate that nesting of viral DNA segments may be associated with the differential expression of genes encoded on nested segments. Polydnavirus genome segments are also found in non-equimolar ratios, with the most abundantly expressed genes (VHv1.1, VHv1.4, WHv1.0, WHv1.6) found on the most abundant
Table 2 Phosphorimager quantitation of Southern and northern blots Relative abundance (%) of segments hybridizing to VHv1.4
Ratio of gene copy numbers (1.4:1.1)* Ratio of mRNA levels (1.4:1.1)†
V (60.0) T (8.4) L2 (7.1) K (17.4) C (7.1) 1.67 : 1 1.82 : 1
*The ratio of gene copy numbers of VHv1.4:VHv1.1 was calculated based on the relative abundance of viral segment V among the Vhybridizing bands. †The ratio of mRNA levels of VHv1.4:VHv1.1 was calculated on the relative amount of the VHv1.4 and VHv1.1 bands hybridized to segment V probe and corrected for the difference in their sizes.
Fig. 4. Northern blot showing the expression of two cysteine-rich genes in different tissues of parasitized H. virescens. Total RNA was isolated from whole H. virescens larvae at 48 h post-parasitization (pp) (P), from hemocytes (H), and from fat body (F) of H. virescens 36 h pp. An aliquot of 15 g of total RNA was loaded in each lane, electrophoresed, blotted and probed with VHv1.4 and VHv1.1 cDNA. After hybridization blots were exposed for 12 h at − 80°C with an intensifying screen. Quantitative inferences about the relative abundance of the VHv1.1 and VHv1.4 mRNAs based on this exposure are complicated by the non-linearity of autoradiography, as film has a relatively narrow range in which the signal is quantitative. The apparent differences in mobility between hemocyte and fat body mRNAs is an artifact of this blot rather than a true difference in the mRNAs (Cui et al., 1997).
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the function of viral genes is linked to the number of copies of each viral gene.
4. Discussion
Fig. 5. Western blot showing the expression of cysteine-rich gene products (VHv1.4 and VHv1.1) in H. virescens larvae injected with 0.01, 0.05, 0.1 and 0.4 FE of purified PDV at 36 h earlier. Hemolymph from control (C) and parasitized (P) H. virescens larvae (3 days pp) are also shown. An aliquot of 1 l of hemolymph was electrophoresed in 10% SDS–PAGE, transferred to nitrocellulose membrane and blotted with antisera against VHv1.1 (Li and Webb, 1994) and VHv1.4 proteins (Cui et al., 1997).
DNA segments. These observations support the idea that viral functions require high level expression of at least some viral genes and that the viral genome has been modified to meet this requirement. However, the relationships between virus infectious dose, expression of specific viral proteins and viral functional activities are not well understood. To study these relationships we manipulated the infectious virus dose and monitored levels of VHv1.4 protein in the hemolymph, the effects of virus infection on hemocyte spreading and the inhibition of encapsulation. Decreasing the infectious dose caused a corresponding decrease in the hemolymph titer of the VHv1.4 and VHv1.1 proteins and presumably of other viral proteins (Fig. 5). Decreasing the amount of virus injected, also decreased the inhibition of the host encapsulation response, which suggests that reductions in viral expression are inversely correlated with inhibition of the encapsulation response (Table 3). As the titer of virus decreased, the effects on hemocyte spreading became less pronounced (data not shown) and the infected hosts were increasingly able to mount an effective encapsulation response. These results are consistent with the idea that the titer of specific viral proteins is critical to virus function, and that in the non-replicative polydnaviruses, Table 3 Encapsulation of injected wasp eggs in fourth instar H. virescens larvae infected with different doses of purified CsPDV Treatment Control (PBS) 0.01 FE 0.05 FE 0.1 FE 0.4 FE
Immunoresponsive insects* (%) 18/21 (85.7%) 17/23 (73.9%) 7/20 (35.0%) 0/24 (0%) 0/18 (0%)
*Immunoresponsive insects are defined as insects that contained at least one encapsulated egg.
Polydnavirus genomes are unique among the DNA viruses. The life-cycle and functions of polydnaviruses in their obligate mutualistic associations with some parasitic Hymenoptera are equally distinctive. The accumulated data from the Campoletis system have begun to elucidate the relationships between the viral genome and its unusual life-cycle. The data suggest that abundantly expressed, secreted genes are associated with hypermolar, nested DNA segments while genes that are targeted intra-cellularly and expressed at lower levels are not present on nested DNA segments. Based on these data we have developed the hypothesis that the level of expression of a given CsPDV gene is directly correlated with the number of genes introduced into parasitized insects. As a result of this evolutionary pressure, polydnavirus genomes may have evolved to contain non-equimolar segment ratios and nested viral DNA segments. It is also possible that this selection pressure may have driven the initial segmentation of polydnavirus genomes. The general strategy of increasing the copy number of selected genes to increase the expression levels is well known in other systems. Protozoans, insects and vertebrates exploit variations on this theme by producing mini-satellite chromosomes, and polyploid and polytene cells (Spradling and Mahowald, 1980; Stark and Wahl, 1984). Inducible gene amplification in mammalian cultured cells is also associated with the development of antibiotic resistance (Schimke, 1984). All of these systems allow for the selective amplification of genes and are correlated with higher levels of expression of the amplified gene (Long and Dawid, 1980). Because PDV genome segments exist in non-equimolar ratios and viral genome segments are nested, some PDV genes are introduced and remain at relatively low copy numbers while other genes have consistently higher copy numbers. We have noted a correlation between gene copy number and the level of gene expression in CsPDV and developed our hypothesis based, in part, upon these observations. From studies of WHv1.0, WHv1.6, VHv1.1, VHv1.4 and BHv0.9 genes, a consistent pattern of CsPDV gene expression emerges. In parasitized lepidopteran hosts, viral genes are expressed rapidly and persistently throughout endoparasite development with little significant variation in mRNA levels from 4 h to 8 days after parasitization (Theilmann and Summers, 1987; Li and Webb, 1994; Cui and Webb, 1996). This type of expression is similar to viral ‘early’ genes that require only host transcription factors for expression. Indeed all three of the CsPDV promoters that have been tested in recombinant baculoviruses function as ‘early’ promoters (Soldevila and
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Webb, 1996). Although some variability in the apparent levels of gene expression was noted in one study (Theilmann and Summers, 1988), there is no indication that CsPDV exploits trans-activating factors that alter viral gene expression in a temporally dependent manner, as is common during the replication of many viruses (Cui and Webb, 1997b). Rather, viral genes are expressed rapidly and constitutively. Under this type of regulatory system, the level of gene expression is completely dependent upon the cis-dependent promoter activity and the number of copies of the gene (gene dosage). Viral genes expressed in parasitized insects alter the physiology of their hosts in dramatic and interesting ways. CsPDV genes from two gene families that are expressed in parasitized insects have been described. The cys-motif gene family (Dib-Hajj et al., 1993) has been characterized on the basis of a cysteine-rich motif that is found in single or double copies on the genes. The members of the cys-motif gene family (WHv1.0, WHv1.6, VHv1.1 and VHv1.4) are located on abundant DNA segments (W and V respectively) that are now thought to be parent molecules for other segments. Segment W, is the parent molecule for segments R, M and possibly C2 (Cui and Webb, 1997a). Segment V is also thought to produce nested segments L2, K and C. Members of the cys-motif gene family encode secreted proteins with similar gene structures and are the most abundantly expressed CsPDV genes in parasitized insects. The segment V-encoded proteins bind to host hemocytes and have been implicated in suppressing the host immune system (Li and Webb, 1994; Cui et al., 1997). The BHv0.9 gene is a member of the rep gene family that has been described on the basis of a 540-bp repeat sequence that is ubiquitously distributed within the viral genome and appears to be present in at least one copy on most viral DNA segments (Theilmann and Summers, 1987). By contrast, the BHv0.9 protein is not secreted and the mRNA is less abundant than the cys-motif mRNAs. Based on these data we propose that nesting of gene segments is associated with high level gene expression in parasitized insects and that nested segments are likely to encode abundantly expressed genes. The persistence of PDV gene expression in parasitized insects has been documented in several parasitoid–host systems. For both PDV genera, viral genes are expressed rapidly. However, the persistence of viral gene expression varies among the systems. Expression of the Ichnovirus, CsPDV, is rapid and at a constant level throughout endoparasite development (Theilmann and Summers, 1988; Li and Webb, 1994; Cui and Webb, 1996). By contrast, expression of bracoviruses may be transient or decreasing at later stages of parasitization (Harwood and Beckage, 1994; Asgari et al., 1996). The persistence of viral gene expression in parasitized insects is dependent on the persistence of the virus itself. In both PDV genera, viral DNA can persist without an increase
in the amount in the parasitized insects to late stages of parasitoid development (Theilmann and Summers, 1986; Strand et al., 1992). In parasitized hosts, the PDVs are able to infect many host tissues (Stoltz and Vinson, 1979). Both hemocytes and fat body have been reported to be major tissues of Bracovirus gene expression (Strand et al., 1992; Harwood and Beckage, 1994). Further, the Microplitis demolitor PDV infects all the hemocyte morphotypes of Pseudoplusia includens and causes apoptosis of the host granulocytes (Strand, 1994; Strand and Pech, 1995b). Similarly, we also found that CsPDV mRNAs are abundantly expressed in host hemocytes and to a lesser degree in fat body. Although apoptosis of hemocytes is not apparent in CsPDVinfected H. virescens, the morphology and in vitro spreading behavior of plasmatocytes and granulocytes are altered (Webb and Luckhart, 1994, 1996; Luckhart and Webb, 1996). Therefore, the inhibition of host cellular immunity associated with the pathology of host hemocytes may be a direct effect of hemocyte infection and/or targeting of hemocytes by virus-encoded proteins (Li and Webb, 1994; Cui et al., 1997). Our results have led us to reconsider the relationship between the genomic organization of PDVs and their unique biology. Our data suggest that PDV genome organization is inextricably linked to the diverse functions of PDVs in their two host insects and that segmentation in PDV genomes may have evolved to increase the copy number of essential viral genes. Viral genes must be abundantly expressed in parasitized insects in the absence of virus replication or a regulatory cascade to increase mRNA levels. Genome segmentation, segment nesting, non-equimolar segment ratios and the presence of viral structural proteins in the wasp genome are diverse manifestations of this fundamental selection pressure. Acknowledgements This work was supported by NIH AI 3314 and NSF MCB 9603504 to B.A.W. This is publication #96-08205 of the Kentucky Agricultural Experiment Station (Lexington, KY). References Asgari, S., Hellers, M., Schmidt, O., 1996. Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. Journal of General Virology 77, 2653–2662. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1994. Current Protocols in Molecular Biology. John Wiley and Sons. Blissard, G.W., Fleming, J.G.W., Vinson, S.B., Summers, M.D., 1986a. Campoletis sonorensis virus: expression in Heliothis virescens and identification of expressed sequences. Journal of Insect Physiology 32, 351–359.
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