School of Biological Sciences, University of Surrey, Guildford GU2 5XH, UK1
Author for correspondence: Michael J. Carter.Fax +44 1483 300 374. e-mail m.carter{at}surrey.ac.uk
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We wished to examine potential nuclear involvement with particular reference to non-structural proteins. We have expressed regions of ORF 1a and 1b in baculovirus and raised polyclonal antisera. These have been used to determine the intracellular location of these proteins within astrovirus-infected cells.
That section of the genome spanning the nuclear addressing motif (residues 643940; hereafter referred to as the nuclear addressing region, NAR), was amplified from ORF 1a of strain A2/88 Newcastle and inserted into the baculovirus shuttle vector pAcHLT-B (PharMingen), fusing it in-frame to a tag of histidine residues. This region comprised about 30% of ORF 1a and was chosen to express all sequences downstream of the protease (assuming this to have a size similar to the proteases of other positive-strand RNA viruses). The whole of ORF 1b was also amplified and treated similarly. Fidelity of both constructs was confirmed by sequence analysis before they were transferred to baculovirus by recombination as described (Willcocks & Carter, 1993 ). Recombinant viruses were harvested at 4 days and plaque purified. High-titre stocks of each were prepared and checked for induction of novel proteins on polyacrylamide gels.
Each of the recombinant baculoviruses induced proteins not seen in mock-infected Sf21 cells, or in those infected with either wild-type AcNPV or recombinant AcNPV expressing an unrelated protein.
Recombinant virus expressing the NAR (NARbaculovirus) induced a 45 kDa protein from 2 to 5 days post-infection (p.i.). This was larger than expected from the region cloned (equivalent to 39 kDa) and may reflect post-translational modifications. Many proteins destined for the nucleus are heavily glycosylated (Hart et al., 1988 ).
Sf21 cells infected with recombinant NARbaculovirus were separated into cytoplasmic and nuclear fractions at 4 days p.i. by the method of Penman (1966) . Cells were swollen on ice in low-osmotic-strength buffer (LOS; 10 mM TrisHCl, pH 7·5, 10 mM NaCl, 1·5 mM MgCl2), and gently homogenized to rupture the plasma membranes. This was followed by centrifugation at 1500 g for 5 min to pellet the nuclear material. Nuclei were then washed in LOS buffer plus 1% Nonidet P40 and 0·2% sodium deoxycholate to remove perinuclear membranes (Penman, 1966
) and recovered by centrifugation as before. Both supernatants were pooled to form the cytoplasmic fraction, and proteins from both fractions were analysed by PAGE. Kenacid blue total staining showed that the expressed NAR protein behaved in a similar manner to the cell histones and segregated almost entirely with the nuclear fraction (Fig. 1
). Thus, the NAR may be functional in insect cells.
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Recombinant proteins were purified from infected cells. These were washed at 4 °C in 10 mM TrisHCl, 10 mM NaH2PO4, pH 7·6, 150 mM NaCl, 1% Triton X-100, resuspended in denaturing lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM TrisHCl, pH 7·6) and lysed at room temperature for 30 min. Attempts to purify the proteins using non-denaturing conditions failed to dissolve the recombinant proteins. The cell lysate was clarified at 10000 g for 10 min and diluted to 6 M with respect to urea. The recombinant protein was then recovered via its affinity tag using nickelNTA resin chromatography; beads were washed and the bound protein was eluted with 500 mM imidazole. Finally, urea was removed by dialysis (24 h) against PBS (four changes) at 4 °C. This resulted in the flocculent precipitation of the purified protein which was then redissolved in PBS containing 5% glycerol and 0·1% Triton X-100. Purity and concentration were assessed on polyacrylamide gels. ORF 1bbaculovirus gave lower yields than NARbaculovirus. Antisera to each protein was raised in New Zealand White rabbits: animals received (intramuscularly) 450 µg of purified protein in Freund's complete adjuvant; 225 µg was administered similarly 2 weeks later and 225 µg was given (intravascularly) 2 weeks after that. Serum was collected at 0 and 42 days from each rabbit and tested for reactivity in Western blot and immune fluorescence using mock-infected, wild-type- and recombinant baculovirus-infected Sf21 cells. Both sera showed strong reaction with the recombinant proteins to which they had been raised and both also had minor reactivity to some baculovirus proteins. The antisera were initially used to examine the proteolytic processing of these regions of ORF 1a and 1b. CaCo-2 cells were grown and infected with HAst-1 as previously described (Willcocks et al., 1990 ). Cells were harvested at 18 to 96 h p.i. and proteins analysed by Western blotting using a 1:100 dilution of the rabbit anti-recombinant protein antiserum. Detection was performed using HRP-conjugated goat anti-rabbit immunoglobulin G. The results obtained from both antisera are shown in Fig. 2
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In contrast, analysis using sera raised to the ORF 1bbaculovirus protein suggested that little cleavage takes place in products from this region (Fig. 2 b). The same high molecular mass band was observed in this analysis as in that using the NARbaculovirus serum, supporting the identification of this band as a readthrough product of ORFs 1a and 1b. A second band of 59 kDa was also observed which remained relatively stable throughout the infection. This corresponds closely to the size expected for the complete ORF 1b product (59 kDa) and suggests that cleavage of the readthrough precursor might occur at or close to the ORF 1a/1b boundary.
Astrovirus-infected CaCo-2 cells were also studied by immunofluorescence to locate proteins derived from ORFs 1a and 1b. Mock- and HAst-1-infected CaCo-2 cells were harvested by scraping from the culture dish at 1896 h p.i. Cells were then transferred to coverslips and fixed in ice-cold acetone, air-dried and then rehydrated in PBS before they were incubated for 1 h at 37 °C in 1:200 dilution of primary antibody. For this analysis we have used anti-NARbaculovirus antiserum, anti-ORF 1bbaculovirus antiserum or anti-astrovirus structural protein polyclonal antiserum (I. Grant, Dako, UK). Rabbit pre-immune serum was used as a control. Cells were washed and incubated in a 1:160 dilution of FIT-conjugated goat anti-rabbit immunoglobulin G. Finally, the cells were washed extensively and incubated for 5 min with antifading reagent (1,4-diazabicyclo[2.2.2]octane; Sigma) and mounted in glycerolPBS for examination under a fluorescence microscope.
The results are shown in Fig. 3. CaCo-2 cells form confluent monolayers joined by tight junctions. The individual cells have prominent nuclei and relatively little cytoplasm. Harvested monolayers stained with Giemsa to identify the nuclei are shown in Fig. 3(a)
. Fluorescence was indistinct before 24 h p.i. and increased from 2472 h. The anti-astrovirus structural protein antiserum stained the cell cytoplasm with a clear concentration ringing the nucleus (Fig. 3b)
. This has also been reported in bovine astrovirus infection (Aroonprasert et al., 1989
). Occasional cells also revealed a nuclear inclusion as expected from previous reports. However, such cells were few in number and in the majority the nuclei were left unstained (Fig. 3b)
In contrast, cells stained with NARbaculovirus antiserum showed prominent bright accumulations of stain within the majority of cell nuclei (Fig. 3c)
. These areas were comparatively large, accounting for as much as 50% of the nuclear area in some cases. Some diffuse cytoplasmic staining was also observed. Finally, cells stained with serum raised to the recombinant ORF 1b protein showed a diffuse and predominantly cytoplasmic staining with little suggestion of accumulation around the nucleus (Fig. 3d)
. These antisera clearly had some cross-reactivity with CaCo-2 cell proteins, and some products were recognized in the Western blots of uninfected cell proteins presented in Fig. 2
. Since immunofluorescent reaction with mock-infected cells was very weak, it could only be demonstrated using extended exposure times. In these cases the distribution of fluorescence was the same regardless of the serum used and results obtained using serum raised to the recombinant NAR protein are presented in Fig. 3(e)
: in contrast to the virus-infected cells, fluorescence was weak and spread diffusely through the cell. Finally, rabbit pre-immune serum failed to induce significant fluorescence with either infected or mock-infected cells.
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References |
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Received 26 April 1999;
accepted 3 August 1999.