MRC Virology Unit, Division of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, UK1
Author for correspondence: John McLauchlan. Fax +44 141 337 2236. e-mail j.mclauchlan{at}vir.gla.ac.uk
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Abstract |
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Introduction |
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Data from a number of studies have identified two major core species, p23 and p21 (Santolini et al., 1994 ; Hussy et al., 1996
; Moradpour et al., 1996
; Liu et al., 1997
; Yasui et al., 1998
). p23 is a 191 amino acid product which contains the signal sequence that directs E1 to the endoplasmic reticulum (ER). Cleavage of the polyprotein occurs between residues 191 and 192 to generate the N-terminal end of E1 (Hijikita et al., 1991
). By contrast, the maturation process for producing p21 has not been precisely identified although the C terminus of p21 lies close to amino acid 174. In tissue culture cells, p21 is the predominant form of core detected and is the major species found in viral particles from infected sera, suggesting that it is the mature form of the protein (Yasui et al., 1998
). A third core product, p16, has been identified in studies with the HCV-1 strain of the virus and probably results from cleavage at around residue 151 (Lo et al., 1994
, 1995
). The production of p16 is dependent on a lysine residue at amino acid position 9 and the absence of E1 sequences linked in cis to the 3' end of the core coding region.
Functional analyses described in a number of reports have shown that core protein can influence a variety of intracellular processes. In tissue culture systems, core modulates apoptosis (Chen et al., 1997 ; Ruggieri et al., 1997
; Ray et al., 1998a
; Zhu et al., 1998
; Marusawa et al., 1999
), signalling pathways involved in apoptotic events (Chen et al., 1997
; Shrivastava et al., 1998
; Zhu et al., 1998
; Marusawa et al., 1999
; You et al., 1999
) and expression levels directed from various viral and cellular promoters (Ray et al., 1995
, 1997
, 1998b
; Chang et al., 1998
). Moreover, two lines of transgenic mice that express the protein only in the liver develop steatosis and thereafter hepatocellular carcinoma (Moriya et al., 1997
, 1998
). These disease states are prevalent in infected human individuals also (Scheuer et al., 1992
; Lefkowitch et al., 1993
), and thus it has been suggested that core protein may have a role in their development. From immunolocalization studies, core protein has a complex distribution. Most of the protein is cytoplasmic where it is found both attached to the ER and at the surface of lipid droplets (Moradpour et al., 1996
; Barba et al., 1997
; Yasui et al., 1998
). However, a minor proportion is present in the nucleus also (Yasui et al., 1998
). Although several studies have analysed the effects of alterations to core coding sequences on its intracellular distribution (Lo et al., 1995
; Suzuki et al., 1995
; Moradpour et al., 1996
; Liu et al., 1997
; Marusawa et al., 1999
), no systematic examination has been performed to identify elements that affect protein distribution.
Here, we have examined the sequences that constitute core and propose that there are three distinct regions within the protein. The role of each of these regions in directing core to lipid droplets has been studied by mutational analysis. In addition, a subset of sequences within a region that was necessary for lipid droplet association and is unique to core had a role in maturation and stability of the protein. Maturation was studied also in cells depleted of lipid to test whether lipid droplet association was required for cleavage and protein stability.
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Methods |
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Immunological reagents.
Monoclonal antibody JM122 was raised in mice against a fusion protein, purified from bacteria, which was composed of the N-terminal 118 amino acid residues of core protein encoded by HCV strain Glasgow linked to a histidine tag. Antisera R308 was raised in rabbits against a branched peptide, ([A/P]KPQRKTKRNT[I/N]RRPQDVKFPGG)8K7A, consisting of amino acid residues 5 to 27 of core protein (Fig. 1a). The two degenerate sites at positions 1 and 12 were introduced to obtain antisera which would be reactive against core proteins from other isolates. Monoclonal antibody ALP98, specific for HCV glycoprotein E2, has been described previously (Patel et al., 1999
); the anti-ADRP antibody, AP125, was obtained from Cymbus Biotechnology.
In vitro transcription and electroporation of SFV RNA into cells.
RNA was transcribed in vitro from recombinant pSFV constructs linearized with SpeI and BHK cells were electroporated with transcripts as described in Patel et al. (1999) . Huh7 and HepG2 cells were prepared for electroporation in the same manner as BHK cells but were electroporated at 360 V with a capacitance of 950 µF.
Preparation of cell extracts, polyacrylamide gel electrophoresis and Western blot analysis.
To prepare extracts, electroporated cells were harvested by removing the growth medium and washing the cell monolayers with PBS. Cells were scraped into PBS and pelleted by centrifugation at 100 g for 5 min at 4 °C. The cell pellet was solubilized in sample buffer (160 mM Tris, pH 6·7, 2% SDS, 700 mM -mercaptoethanol, 10% glycerol, 0·004% bromophenol blue). Alternatively, sample buffer was added directly to cells that had been washed with PBS. Cells were solubilized at a concentration of approximately 4x106 cell equivalents per ml sample buffer. Samples were heated to 100 °C for 5 min to fully denature proteins and nucleic acids. Gel electrophoresis was performed on 12·5% polyacrylamide gels cross-linked with 2·5% (w/w) N,N'-methylene bisacrylamide.
For Western blot analysis, proteins separated on polyacrylamide gels were transferred to nitrocellulose membrane. After blocking with 3% gelatine, 4 mM TrisHCl, pH 7·4, 100 mM NaCl, membranes were incubated with monoclonal antibodies (diluted to 1/500) in 1% gelatine, 4 mM TrisHCl, pH 7·4, 100 mM NaCl, 0·05% Tween 20. After washing, bound antibody was detected using a horseradish peroxidase-conjugated secondary antibody followed by enhanced chemiluminescence (Amersham).
Indirect immunofluorescence and staining of lipids.
Cells on 13 mm coverslips were fixed for 30 min in either methanol at -20 °C or 4% paraformaldehyde, 0·1% Triton X-100 (prepared in PBS) at 4 °C. After washing with PBS and blocking with PBS1% NCS, cells were incubated with primary antibody (diluted in PBSNCS at 1/200 for JM122 antibody, 1/1000 for R308 antisera) for 2 h at room temperature. Cells were washed extensively with PBSNCS and then incubated with conjugated secondary antibody (either anti-mouse or anti-rabbit IgG raised in goat) for 2 h at room temperature. Cells were washed extensively in solutions of PBSNCS followed by PBS and finally H2O before mounting on slides using Citifluor. Samples were analysed using a Zeiss LSM confocal microscope.
After incubation with both antibodies and washing, lipid droplets were stained in paraformaldehyde-fixed cells by briefly rinsing coverslips in 60% propan-2-ol followed by incubation with 0·5 ml 60% propan-2-ol containing oil red O (final concentration approximately 0·6%) for 1·5 to 2 min at room temperature. Coverslips were briefly rinsed with 60% propan-2-ol, washed with PBS and H2O and mounted as described above. The oil red O staining solution was prepared from a saturated stock of approximately 1% oil red O (Sigma) dissolved in propan-2-ol. Before staining, the stock was diluted with H2O and then filtered.
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Results |
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Characterization of sequences within core protein necessary for lipid droplet association
Expression of core protein from HCV strain Glasgow in BHK-21 C13 cells from an SFV construct, pSFV/1195, revealed that a high proportion of the protein was associated with globular vesicles. By combining indirect immunofluorescence for detection of core protein with a lipid-specific stain, oil red O, we identified the globular structures as lipid droplets that are storage compartments containing triacylglycerols and cholesterol esters (Londos et al., 1999 ; Murphy & Vance, 1999
). Representative images of core associated with lipid droplets are shown in Fig. 2
(a
c
) and 5
(a, panel i) at 18 h after electroporation. In cells propagated in normal growth media, core was invariably detected at the surface of lipid droplets and this distribution was apparent from early times (9 h) after electroporation (data not shown). This localization confirms previous findings from electron microscopy (EM) studies that core protein is present at the surface of such intracellular structures (Moradpour et al., 1996
; Barba et al., 1997
). The sizes of lipid droplets in BHK cells are highly variable (approx. 0·43·0 µm in diameter; Fig. 2d
). In BHK cells expressing core protein, lipid droplets were rarely greater than 1·4 µm in diameter (Fig. 2ac
), suggesting that association of core with these structures reduces the upper limits of their sizes and hence their size heterogeneity. From Western blot analysis, 95% of the core protein expressed in cells from pSFV/1195 corresponded to p21 (see Fig. 3a
, b
, lane 1) and presumably is therefore the predominant species associated with droplets.
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Further analysis with variants containing internal deletions showed that lipid droplet association was not abolished by removing various segments spanning residues 2 and 118 (Fig. 2kr
). The most notable features observed with these variants were firstly that there was an increase in the relative sizes of some lipid droplets in a proportion of BHK cells as compared to the sizes found with wild-type core protein. An example is shown for mutant
243 (panels k and l) and there was apparent fusion of droplets also with mutant
943 (panels m and n). These phenomena were reproducible in BHK cells but to varying degrees over several experiments. Secondly, removal of residues 80 to 118 did reduce but did not abolish association of core with lipid droplets (Fig. 2q
, r
). Thirdly, in Huh7 cells, protein levels of the mutant lacking residues 49 to 75 were barely detectable, thus precluding conclusions on the behaviour of this mutant in those cells (data not shown). Overall, localization of core protein at the surface of lipid droplets was not eliminated with the domain 1 variants in either BHK or Huh7 cells. By contrast, lipid droplet association was not detected for proteins which lacked various regions between residues 125 and 166 (Fig. 2s
z
). Indeed, the only sequences dispensable in this portion of core protein lay between amino acids 155 and 161 (panels w and x). Identical behaviour for each of these mutants was observed in Huh7 cells (data not shown). From these data we conclude that domain 2 contains elements that are essential for lipid droplet association.
Sequences within core required for lipid droplet association also facilitate maturation of precursor protein
During our investigations into the residues required for lipid droplet association, we analysed the intracellular levels of protein made by the core constructs that had been processed at the p21 maturation site. With mutants in domain 2, we found that deletion of residues between 125 and 144 dramatically reduced the level of mature product to only about 25% of the abundance of unprocessed precursor protein (Fig. 3a, lane 4). To further define the sequences in this region which affected maturation, two other constructs were made and these showed that deletion of residues from 125 to 134 reduced maturation efficiency to a greater extent than removal of residues from 135 to 144 (~30% as compared to 50%, Fig. 3a
, lanes 6 and 7). The removal of other sequences between residues 145 and 166 also gave reduced amounts of mature protein but the reductions were less pronounced (Fig. 3a
, lanes 810). Furthermore, the levels of mature protein from one mutant which lacked residues 125 to 144 and 161 to 166 were extremely low (approximately 10%; lane 11), suggesting that more than one element may be important in domain 2 for protein maturation. Analysis of a number of these mutants in Huh7 (Fig. 3b
) and HepG2 cells (data not shown) also indicated that residues between amino acids 125 and 144 had the most dramatic effect on maturation of core protein.
Identification of sequences that modulate the stability of mature core protein
From analysis of the effect of sequences on levels of p21 produced, we observed that the amount of core protein detected in extracts from cells electroporated with pSFV/1124,145169 RNA was lower in a number of experiments than that made in cells electroporated with pSFV/1169 RNA (Fig. 3a, compare lanes 2 and 5). This could suggest that either translation is inhibited as a result of the removal of these sequences or that the protein produced was unstable and proteolytically degraded. This prompted us to examine the effect of removing amino acids 125 to 144 from the coding sequences of the HCV structural proteins to determine whether protein levels of the glycoproteins as well as core were affected by these residues. Thus, a plasmid called pSFV/C
125144E1E2 (see Methods) was constructed that contains sequences encoding residues 1 to 124 and 145 to 191 of core protein linked to those for E1 and E2. Given that removal of residues 125 to 144 apparently reduced the amount of p21 derived from p23 (Fig. 3a
, compare lanes 1 and 4; Fig. 3b
, compare lanes 1 and 2), this construct would address also whether generation of p21 was affected by these sequences on linked expression with E1 and E2. For comparative purposes, levels of HCV structural proteins were determined from pSFV/CE1E2Gla RNA, which expresses unmodified core, E1 and E2 from strain Glasgow.
Western blot analysis showed that p21 and E2 proteins were detected in extracts from BHK, Huh7 and HepG2 cells electroporated with pSFV/CE1E2Gla RNA (Fig. 4a, panels i and ii, lanes 1, 3 and 5). The amount of E2 made by pSFV/C
125144E1E2 in each cell type was similar to that for pSFV/CE1E2Gla (Fig. 4a
, panel ii, lanes 2, 4 and 6); however, no core protein was detected (panel i, lanes 2, 4 and 6). Similar relative patterns of expression for core and envelope glycoproteins were observed visually in BHK cells by indirect immunofluorescence (Fig. 4c
, panels i, ii, iv and v); in pSFV/C
125144E1E2-electroporated cells, glycoprotein was readily identified but there was almost no fluorescent signal for core protein. This indicates that translation of the structural proteins is not inhibited by deletion of residues 125 to 144. To examine whether protein stability could account for the different levels of core protein detected, electroporated BHK cells were seeded in duplicate and one set of cultures was treated with the protease inhibitor MG132. Compared with untreated cells, there was no significant effect on the amount of E2 detected in MG132-treated samples (Fig. 4b
, panel ii, lanes 14). Comparable levels of p21 were found also in treated and untreated samples from pSFV/CE1E2Gla-electroporated cells (Fig. 4b
, panel i, lanes 3 and 4). In pSFV/C
125144E1E2-electroporated cells, core protein could be detected in MG132-treated but not untreated cells (Fig. 4b
, panel i, lanes 1 and 2). Indirect immunofluorescence analysis of pSFV/CE1E2Gla-electroporated cells also revealed that considerably more core protein could be found in MG132-treated than untreated cells (Fig. 4c
, panels ii and iii). Thus, residues between 125 and 144 are critical for the stability of p21. Finally, the size of core protein detected in the pSFV/C
125144E1E2-electroporated cells treated with MG132 corresponded to that of a product which had been processed to give a mature protein with a C terminus at approximately residue 172 (Fig. 4b
, panel i, lane 2; data not shown). This is in contrast to the data obtained with construct pSFV/
125144 in which the HCV coding region is terminated at residue 195, and lower relative levels of processed to unprocessed product were detected (Fig. 3a
, lane 4; Fig. 3 b
, lane 2). This would suggest that the reduced amounts of mature product made by pSFV/
125144 are a consequence of the removal of residues 125 to 144 as well as the absence of any substantial E1 and E2 coding sequences.
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Electroporation of cells with RNA from pSFV/1195 and pSFV/1153 showed that the core proteins made from both constructs in cells grown in normal medium had distributions consistent with those shown in Fig. 2 (Fig. 5a
, panels i and iii). However, in cells grown in media containing delipidated media, core protein made by pSFV/1195 remained in the cytoplasm and had a similar localization to the truncated protein produced from pSFV/1153 (panels ii and iii). From Western blot analysis, the efficiency of cleavage of precursor molecules from pSFV/1195 to generate p21 was similar in cells grown either under normal conditions or in lipid-deficient serum (Fig. 5b
, lanes 1 and 4). Moreover, growth conditions had no effect on the abundance of the protein. Hence, we conclude that association with lipid droplets is not required for maturation of core protein and reduced amounts of lipid droplets in cells do not significantly affect protein stability.
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Discussion |
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In our experiments, processing of core was not required for lipid droplet association since variants truncated at residues 173 and 169 had identical distributions to the processed p21 form of the protein; these results were reproducible in both liver- (Huh7 cells) and non-liver-derived (BHK cells) cell lines. Our results differ from those of Liu et al. (1997) who presented evidence that processing of core is linked to intracellular localization and that protein truncated at residue 173 accumulated in the nucleus. The difference may be a consequence of the different cell types and HCV strains used in the two studies. We also note that the truncated construct used in their study had an additional isoleucine residue immediately following residue 173 which is not present in the natural HCV sequence. It is possible that this additional residue could influence the intracellular localization of their truncated protein. In agreement with our data, Sabile et al. (1999)
also have shown that truncation of the core coding region to residue 173 does not induce nuclear localization of the protein. Further truncation of core protein to residue 153 did abolish lipid droplet association, a result which was consistent in both Huh7 and BHK cells. However, in BHK cells, the truncated protein had both nuclear and cytoplasmic distributions whereas only nuclear localization was observed in Huh7 cells (unpublished data). Other reports have shown that proteins truncated at residues 151 to 153 have a nuclear localization (Lo et al., 1995
; Suzuki et al., 1995
; Marusawa et al., 1999
; Sabile et al., 1999
). The cytoplasmic distribution of the protein truncated at residue 153 in BHK cells may reflect differences in nuclear/cytoplasmic trafficking events or other intracellular processes between these cells and other cell types. Nevertheless, there was a clear correlation between the behaviour of the core mutants in BHK and Huh7 cells, indicating that the processes involved in directing core to lipid droplets are similar in cell types of different lineages.
The lipid droplets to which p21 is directed are storage compartments for triacylglycerols and cholesterol esters (Londos et al., 1999 ; Murphy & Vance, 1999
). In mammalian cells, very few proteins that bind to lipid droplets have been identified and the sequences required to direct proteins to these storage compartments are not known. However, a family of plant proteins, called oleosins, found at the surface of oil bodies, have been studied in greater detail (reviewed in Huang, 1992
; Murphy & Vance, 1999
). Oleosins are considered to play an important structural role in maintaining stability and preventing coalescence of these storage structures (LePrince et al., 1997
). They are small proteins that consist of three domains, a central hydrophobic segment of about 70 amino acids which is bounded by N- and C-terminal amphipathic segments (Huang, 1992
). Targeting studies have established that the N-terminal and central hydrophobic domains are necessary for association with oil bodies but the C-terminal region may be dispensable (van Rooijen & Moloney, 1995
). The N-terminal region has positively charged residues that are presumed to aid neutralization of the negative charges on phospholipids at the oil body surface while the central hydrophobic domain appears to interact with the lipid matrix. A similar series of interactions may occur between core protein and lipid droplets. Thus, domain 2 of core protein may form hydrophobic interactions with lipid droplets while the basic residues in domain 1 could contact with phospholipid at the droplet surface. Such surface contacts need not be sequence-specific. This would be consistent with our findings since there are no specific elements in domain 1 that apparently are essential for lipid droplet association. However, this region is not entirely dispensable (data not shown) and does contribute to efficient localization to droplets. Moreover, removal of portions of domain 1 does alter the morphology and size of droplets detected.
A protein that is truncated at residue 195 and lacks residues 125 to 144 is inefficiently processed to yield a mature core species. Previous studies have indicated that maturation of core is carried out by host cell proteases which are located at the ER (Santolini et al., 1994 ; Hussy et al., 1996
). Hence, the reduced levels of cleaved protein made by the above construct could result from inefficient trafficking of the ribosome/nascent polypeptide complex to the rough ER. It may be significant that the data were obtained with constructs in which the HCV coding region was truncated at residue 195 and therefore core was not expressed as part of a longer polyprotein. Therefore, another possibility is that the combined absence of residues 125 to 144 as well as any extended sequence downstream from the core/E1 cleavage site may not permit stable interaction of the nascent proteinribosome complex with components at the ER that are required for processing. In the context of expression as part of a longer polyprotein that contains E1 and E2, removal of residues 125 to 144 does not appear to affect maturation of core but does have a dramatic effect on stability of the mature protein. Based on our studies with MG132, an inhibitor of protein degradation (Lee & Goldberg, 1996
), mature core protein lacking this stretch of amino acids is degraded by post-translational processes and it is likely that proteolysis is performed by the proteasome. Since this region of core is required also for lipid droplet association, it may be that, following maturation, inability of the protein to associate with the appropriate cellular components targets the protein for proteolysis. Previously described control mechanisms with other cellular proteins show similar characteristics. For example, apolipoprotein B (apoB) is assembled with cholesterol and triacylglycerol into lipoprotein particles which are secreted by hepatocytes. Intracellular levels of apoB are regulated by the availability of lipid and excess protein is degraded by the ubiquitin/proteasome pathway (Chen et al., 1998
, and references therein). In addition, the levels of a lipid droplet binding protein, ADRP, are regulated by post-transcriptional events in which the abundance of the protein is controlled by the apparent availability of a lipophilic surface (Brasaemle et al., 1997
). Experiments are in progress to elucidate further the role of residues 125 to 144 in the interactions of core protein with cellular processes.
Our results with cells grown in lipid-deficient serum, and which therefore do not accumulate lipid droplets, indicate that the distribution of core is different from that in cells maintained in normal serum. Under these conditions, cleavage of core is efficient and the abundance of the protein is not substantially diminished. Thus, core does not require association with lipid droplets for stability. A number of reports have shown both punctate and reticular localizations for core (Lo et al., 1995 ; Suzuki et al., 1995
; Moradpour et al., 1996
; Barba et al., 1997
; Yasui et al., 1999
), and from EM studies the protein is present at the surface of lipid droplets and on ER membranes (Moradpour et al., 1996
; Barba et al., 1997
). In the absence of lipid droplets, core assumes a reticular cytoplasmic distribution in BHK cells and we suggest that this represents association with ER membranes. Therefore, the different extents to which core is found attached either to lipid droplets or membranes may be dependent on the presence of lipid droplets in the various cell types used for analysis. The processes involved in lipid droplet formation are not well understood but it is thought that they bud from ER membranes (Murphy & Vance, 1999
). Core may bind to a common lipid or protein component present both on lipid droplets and ER membranes. One example could be the apoAII protein that associates with core, an interaction that requires residues 160 to 173 contained within domain 2 (Sabile et al., 1999
).
In conclusion, the HCV core protein contains a domain which is not present in related proteins from pesti- and flaviviruses. This region of the protein plays a vital role in the localization of core to lipid droplets and has a motif which is important for stability of the mature protein. It also may impart other properties to core protein which are relevant to virion morphogenesis and viral pathogenesis and thus provide unique features to the life-cycle of HCV.
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Acknowledgments |
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Received 1 December 1999;
accepted 20 April 2000.