Department of Applied and Molecular Ecology, Waite Campus, The University of Adelaide, Glen Osmond, South Australia 5064, Australia
Correspondence
Sassan Asgari
sassan.asgari{at}adelaide.edu.au
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY150370 for CrCRT and AY150371 for CrHs70.
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INTRODUCTION |
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The general view is that these particles have originated from a pathogenic ancestral virus(es) that has lost some viral properties and become part of the wasp genotype during evolution. Recent discussions on the endosymbiotic relationship of virus and wasps (Whitfield & Asgari, 2002) suggest that PDVs may constitute vehicles for the transfer of genetic information from the wasp to the host. In this scenario, genes encoded by the circular DNA include proteins involved in host manipulation, whereas genes encoding structural particle proteins are not circularized and are mainly expressed during particle production. To identify the origin of PDVs, it is therefore essential to isolate and characterize proteins that are a structural part of PDVs. The genes expressed from PDV DNA in the host that have been identified so far do not show any similarity to other known viruses.
Since circular DNAs from PDVs appear to lack genes encoding structural virus proteins, we used antibodies against purified PDVs from the wasp Cotesia rubecula to perform immune screens of peptides from cDNA fragments from wasp ovaries. These screens revealed particle proteins from the wasp C. rubecula that had similarities to calreticulin and heat-shock protein 70 (Hsp70) (CrCRT and CrHs70, respectively). The association of chaperones with virus production and assembly has been explored in various systems (Sullivan & Pipas, 2001). Calreticulin, found mainly in the endoplasmic reticulum (ER), is a widely expressed calcium-binding protein that acts as a molecular chaperone (Michalak et al., 1992
). However, calreticulin has also been localized in other cellular compartments, such as in the cytoplasm and nucleus, the cell surface and as secreted forms. There is growing evidence suggesting that calreticulin may mediate a broader array of cellular functions.
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METHODS |
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Polydnaviruses were purified from female wasps as described previously (Beckage et al., 1994). Briefly, isolated ovaries were macerated in PBS using micro-scissors followed by centrifugation at 750 g for 5 min to exclude large cellular debris. The homogenate was then passed through a 0·45 µm filter (Ministart; Sartorius) using a 3 ml syringe. Viral particles were pelleted by centrifuging the filtrate at 12 000 g for 20 min and resuspended in PBS.
Western blotting and elution of antibodies.
Protein samples were separated by 10 or 12 % SDS-PAGE (Laemmli, 1970) and transferred on to nitrocellulose membranes as described (Sambrook et al., 1989
). Anti-CrPDV (1 : 5000) (Asgari & Schmidt, 1994
), anti-Hsp70 (see below) and anti-calreticulin (1 : 3000; B. L. Lee, Pusan National University, South Korea) antibodies were used as probes followed by detection with alkaline phosphatase-conjugated anti-rabbit IgG antibodies (1 : 5000).
To obtain specific anti-Hsp70 antibodies, purified phages containing the gene encoding CrHs70 were plated and induced for protein production by overlaying with an IPTG-soaked membrane. The expressed proteins transferred on to the membrane were probed with anti-CrPDV antibodies (1 : 5000) and then detected by a secondary antibody. The anti-Hsp70 antibodies were eluted from the membrane as described (Smith & Fisher, 1984). Eluted antibodies were used (without dilution) in a Western blot containing purified CrPDVs to confirm the presence of Hsp70 in the particles.
Isolation of RNA and slot blotting.
Total RNA was extracted from ovaries and wasp carcasses devoid of ovaries as described (Chomczynski & Sacchi, 1987). RNA (3 µg) samples were blotted on to a nylon membrane under denaturing conditions according to the manufacturer's instructions (BioRad). The blot was hybridized with a 32P-labelled PCR product encoding CrCRT for 20 h at 65 °C. A P. rapae 18S ribosomal DNA fragment probe (R. Glatz, unpublished) was used as a control.
Screening a cDNA library using anti-CrPDV antibodies.
A C. rubecula ovary/venom gland expression cDNA library was constructed using a SMART cDNA library construction kit (Clontech) according to the manufacturer's instructions. Plaques formed on plates (2·5x104 in total) were induced for protein expression by overlaying plates with nitrocellulose membranes soaked in 10 mM IPTG and dried. Induction was carried out at 42 °C for 4 h. Membranes were removed and washed in TBST for 5 min. Membranes were then blocked for 1 h in milk blocking solution to which anti-CrPDV antibodies had been added (1 : 5000). Positive plaques were detected by a secondary antibody and isolated for rescreening. Confirmed positives were recircularized as plasmids according to the manufacturer's instructions (Clontech). Inserts were sequenced in both directions.
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RESULTS AND DISCUSSION |
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Heat-shock protein 70
The cDNA obtained for CrHs70 was 2086 bp in length encoding 656 amino acids (Fig. 2B). A putative cleavage site was found between amino acids 16 and 17 using PSORT II (Fig. 2B
). Sequence homology searches revealed a high similarity to the Hsp70 and Hsc70 proteins from various organisms. The highest similarities were to Rivulus marmoratus (Teleostei, 83·4 %) and Artemia franciscana (Crustacea, 84·3 %) (Fig. 5
). The predicted size for CrHs70 is 71·4 kDa. Western blot analysis using specific anti-Hsp70 antibodies recognized a protein of approximately 71 kDa in size in purified C. rubecula particles (Fig. 4B
).
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Apart from mediating correct assembly of proteins, these proteins may also be involved in several other cellular functions, such as protein transport across membranes and vesicle transport along microtubules and virion assembly. The expression of molecular chaperones is usually up-regulated during virus infection and replication, probably not as a result of stress due to infection, but rather as a requirement for some viral functions. Certain viruses encode their own specific chaperones, whereas others rely on the host cellular chaperones. More studies are required to elucidate the functional role of these molecules in polydnavirus biology. However, since PDVs cannot be replicated in vitro (even their in vivo replication is atypical of a virus), a possible role of these proteins in virus production is not possible using traditional assays. Moreover, it remains to be seen how conserved these structural proteins are within the virus families to pinpoint the closest relatives and ancestral origins of these unique particles.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 25 September 2002;
accepted 6 December 2002.
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