Processing and intracellular location of human astrovirusnon-structural proteins

Margaret M. Willcocks1, Angela S. Boxall1 and Michael J. Carter1

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


   Abstract
Top
Abstract
Main text
References
 
Human astrovirus (HAst) non-structural polyproteins are encoded in two open reading frames linked in expression by a ribosomal frameshifting event. The first of these (ORF 1a) specifies the serine protease, whilst the second (ORF 1b) encodes the virus RNA-dependent RNA polymerase. The ORF 1a product contains an unusual motif for small RNA viruses which could potentially direct proteins to the cell nucleus. We have expressed part of ORF 1a containing this motif and the whole of ORF 1b separately in recombinant baculovirus and raised specific antisera to each. We now report that expressed proteins from ORF 1a accumulate in the nucleus of both baculovirus-infected insect cells and HAst-infected CaCo-2 cells. In contrast the products of ORF 1b remain predominantly cytoplasmic.


   Main text
Top
Abstract
Main text
References
 
The astrovirus genome is single-stranded, polyadenylated positive-sense RNA (Jiang et al., 1993 ; Willcocks et al., 1994 ). The genome organization differs sufficiently from other non-enveloped positive-stranded RNA viruses of animals to warrant assignment to a separate virus family – the Astroviridae (Monroe et al., 1995 ; Carter & Willcocks, 1996 ). The RNA contains three open reading frames (ORFs 1a, 1b and 2), ORF 2 encodes the structural proteins and is located at the 3'end of the genome (Willcocks & Carter, 1993 ). ORFs 1a and 1b encode non-structural proteins towards the 5' end of the genome. ORF 1b is translated as an ORF 1a/1b fusion protein via a -1 ribosome frame shifting event following translation of ORF 1a (Marczinke et al., 1994 ; Lewis & Matsui, 1996 ). ORF 1b (59 kDa) encodes the viral RNA-dependent RNA polymerase (Lewis et al., 1994 ), whereas ORF 1a (101 kDa) encodes a variety of proteins including a serine protease (Jiang et al., 1993 ). Both ORF products are thought to mature by proteolysis, but their processing pathways are unknown. Early analysis also revealed the presence of a bipartite nuclear addressing signal (Robbins et al., 1991 ) in the carboxyl half of the ORF 1a polyprotein (Willcocks et al., 1994 ). In human astrovirus (HAst)-1 strain A2/88 Newcastle, this is encoded between bases 2081–2131, downstream of the serine protease. This addressing signal is completely conserved in all astroviruses sequenced (Carter, 1994 ). Nuclear addressing signals occur in 56% of proteins present in the cell nucleus, but only 4·2% of those that are not (Dingwall & Laskey, 1986 ). Thus, although suggestive of nuclear transport, the mere presence of this signal does not in itself justify the conclusion that such proteins must be transported to the nucleus. Nuclear involvement is unusual in the non-enveloped RNA viruses and, if such transport were to occur, it would constitute yet another characteristic astrovirus feature. Some indications of nuclear involvement have already been obtained: nuclear fluorescence has been noted early in HAst infection (A. D. T. Barrett & W. D. Cubitt, personal communication) and was also observed during bovine astrovirus infection (Woode & Bridger, 1978 ;Aroonprasert et al., 1989). However, this has been attributed to structural protein accumulation in this compartment as the sera used were raised to purified virus. Further, similar results were also obtained using monoclonal antibody specific for virion protein (W. D. Cubitt, personal communication).

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 643–940; 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 (NAR–baculovirus) 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 NAR–baculovirus 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 Tris–HCl, 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.



View larger version (130K):
[in this window]
[in a new window]
 
Fig. 1. Nuclear/cytoplasmic fractionation of mock- and recombinant NAR–baculovirus-infected Sf21 cells. Track 1, molecular mass markers (kDa); tracks 2–4, recombinant NAR–baculovirus-infected Sf21 cells; tracks 5–7, mock-infected Sf21 cells; tracks 2 and 5, total cell extract; tracks 3 and 6, cytoplasmic fraction; tracks 4 and 7, nuclear fraction. The recombinant protein expressed is indicated (arrow).

 
Recombinant baculovirus expressing ORF 1b induced a 59 kDa protein in infected cells. This corresponds well with the size expected from the region cloned.

Recombinant proteins were purified from infected cells. These were washed at 4 °C in 10 mM Tris–HCl, 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 Tris–HCl, 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 nickel–NTA 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 1b–baculovirus gave lower yields than NAR–baculovirus. 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.



View larger version (82K):
[in this window]
[in a new window]
 
Fig. 2. Western blot analysis of HAst-1-infected CaCo-2 cells, stained with (a) anti-ORF 1a (nuclear addressing region) antiserum or (b) anti-ORF 1b antiserum. In both panels: track 1, mock-infected CaCo-2 cells; tracks 2–4, HAst-1-infected CaCo-2 cells. Samples were harvested at the following times. (a) track 2, 18 h; track 3, 24 h; track 4, 48 h. (b) Track 2, 24 h; track 3, 48 h. Astrovirus-specific bands referred to in the text are indicated by arrows and identified by their molecular mass as determined by gel electrophoresis (markers not shown).

 
Fig. 2(a) indicates that extensive cleavage takes place in ORF 1a and a variety of protein products of different sizes were observed. However, the sera used here can only recognize products containing regions of ORF 1a extending from the presumed protease to the end of the ORF (i.e. overlapping residues 643–940). Products containing the nuclear targeting motif are not specifically identified. Small amounts of a high molecular mass protein were stained from 18 h p.i. (maximum at 24 h p.i.). This could correspond to the read through product generated when ORFs 1a and 1b are fused together via the ribosome frameshift mechanism (predicted molecular mass 160 kDa). A 75 kDa protein was also observed from 18 to 48 h; this was present in larger amounts than the high molecular mass product and appeared to decrease at later times p.i. The full-size ORF 1a product without frameshift has an expected molecular mass of 101 kDa. However, such a band was never observed, suggesting that some 26 kDa, not detectable with this serum, may be rapidly cleaved from the N terminus of this protein. Three smaller bands were also detected in these gels: 34, 20 and 8 kDa. The smallest of these bands was further resolved into two and also sized more accurately (6·5 and 5·5 kDa) by analysis on 15% gels (data not shown). All these bands remained stable throughout infection and may therefore represent end-products of proteolysis from the region probed by this serum.

In contrast, analysis using sera raised to the ORF 1b–baculovirus 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 NAR–baculovirus 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 18–96 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-NAR–baculovirus antiserum, anti-ORF 1b–baculovirus 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 glycerol–PBS 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 24–72 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 NAR–baculovirus 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.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 3. Localization of astrovirus-specific proteins within infected CaCo-2 cells investigated by immunofluorescence. All figures presented at 48 h p.i. (a) Mock-infected CaCo-2 cells stained with Giemsa to reveal the nuclei. (b)–(d) Astrovirus-infected cells; (e) mock-infected CaCo-2 cells; panels stained with (b) anti-astrovirus structural protein antiserum, (c, e) anti-ORF 1a (nuclear addressing region) antiserum; (d) anti-ORF 1b antiserum.

 
These data suggest that although astrovirus structural proteins may be found in the nucleus, some non-structural proteins derived from astrovirus ORF 1a appear to accumulate within that compartment throughout infection (18–96 h p.i.). The reasons for this are unclear and we are currently examining possible interactions between virus and host proteins in this compartment.


   Acknowledgments
 
This work was supported by a grant from the Medical Research Council. We thank Peter Scobie-Trumper for carrying out the animal work.


   References
Top
Abstract
Main text
References
 
Aroonprasert, D., Fagerland, J. A., Kelso, N. E., Zheng, S. & Woode, G. N. (1989). Cultivation and partial characterisation of bovine astrovirus. Veterinary Microbiology 19, 113-125.[Medline]

Carter, M. J. (1994). Genomic organisation and expression of astroviruses and caliciviruses. Archives of Virology Suppl. 9, 429–439.

Carter, M. J. & Willcocks, M. M. (1996). The molecular biology of astroviruses. Archives of Virology Suppl. 12, 277–286.

Dingwall, C. & Laskey, R. A. (1986). Protein import into the cell nucleus. Annual Review of Cell Biology 2, 367-390.

Hart, G. W., Holt, G. D. & Hattiwanger, R. S. (1988). Nuclear and cytoplasmic glycosylation: novel saccharide linkages in unexpected places. Trends in Biochemical Science 13, 380-384.

Jiang, B., Monroe, S. S., Koonin, E. V., Stine, S. E. & Glass, R. I. (1993). RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frame-shifting signal that directs the viral replicase synthesis. Proceedings of the National Academy of Sciences, USA 90, 10539-10543.[Abstract]

Lewis, T. L. & Matsui, S. M. (1996). Astrovirus ribosomal frameshifting in an infection-transfection transient expression system. Journal of Virology 70, 2869-2875.[Abstract]

Lewis, T. L., Greenberg, H. B., Herrmann, J. E., Smith, L. S. & Matsui, S. M. (1994). Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein. Journal of Virology 68, 77-83.[Abstract]

Marczinke, B., Bloys, A. J., Brown, T. D. K., Willcocks, M. M., Carter, M. J. & Brierley, I. (1994). The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting. Journal of Virology 68, 5588-5595.[Abstract]

Monroe, S. S., Carter, M. J., Herrmann, J. E., Kurtz, J. B. & Matsui, S. M. (1995). The Astroviridae. In Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses, pp. 364-367. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo & M. D. Summers. Vienna & New York: Springer-Verlag.

Penman, S. (1966). RNA metabolism in HeLa cell nucleus. Journal of Molecular Biology 17, 117-130.[Medline]

Robbins, J., Dilworth, S. M., Laskey, R. A. & Dingwall, C. (1991). Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequences. Cell 64, 615-623.[Medline]

Willcocks, M. M. & Carter, M. J. (1993). Identification and sequence determination of the capsid protein gene of human astrovirus serotype 1. FEMS Microbiology Letters 114, 1-8.[Medline]

Willcocks, M. M., Carter, M. J., Laidler, F. R. & Madeley, C. R. (1990). Growth and characterisation of human faecal astrovirus in a continuous cell line. Archives of Virology 113, 73-82.[Medline]

Willcocks, M. M., Brown, T. D. K., Madeley, C. R. & Carter, M. J. (1994). The complete sequence of a human astrovirus. Journal of General Virology 75, 1785-1788.[Abstract]

Woode, G. N. & Bridger, J. C. (1978). Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves. Journal of Medical Microbiology 11, 441-452.[Abstract]

Received 26 April 1999; accepted 3 August 1999.