| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
Correspondence
John McLauchlan
j.mclauchlan{at}vir.gla.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
A supplementary figure showing the stability of corewt and coreASC/VLV proteins that lack aa 125–144 is available in JGV Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
a). Proteolysis is directed by signal peptidase (SP), a cellular activity, thereby generating the N and C termini of glycoproteins E1 and E2. For core, a second cellular enzyme called signal peptide peptidase (SPP) cuts within the signal peptide between core and E1 (SigPcore–E1) to give the mature protein (Fig. 1a; Hüssy et al., 1996; McLauchlan et al., 2002). Mutations in SigPcore–E1 from HCV strain Glasgow, a genotype 1a strain, revealed that amino acids Ala180, Ser183 and Cys184 were necessary for efficient SPP processing (Lemberg & Martoglio, 2002; McLauchlan et al., 2002). However, Okamoto et al. (2004) reported that these residues did not affect SPP cleavage and that the critical amino acids lay upstream of SigPcore–E1 in strains J1 and H77, which are genotype 1b and 1a strains, respectively. Here, we examine the basis of these differences.
|
, the HCV proteins were expressed using vector pcDNA3.1, whereas we employed the Semliki Forest virus (SFV) vector system (McLauchlan et al., 2002). To compare these systems, DNA fragments containing strain J1 sequences were amplified from the relevant pcDNA3.1 plasmids by using two primers (5'-GGGCAGATCTGCCGCCACCATGAGCACAAATCCTAAACCCCAAAGA-3' and 5'-CAACAGATGGCTGGCAACTAGAAGGC-3'). The resultant PCR products contained the coding region for aa 1–382 of strain J1, followed by 17 residues that incorporated an HA tag. After digestion with BglII/XbaI, the PCR products were introduced into pSFV1 to generate two vectors, pSFV/C[wt]-E1-HA and pSFV/C[ASC/VLV]-E1-HA (Fig. 1a
). pSFV/C[ASC/VLV]-E1-HA contained mutations in SigPcore–E1 identical to those in pSFV/CspmtE1E2, the construct that expressed the structural proteins of HCV strain Glasgow (Fig. 1b; McLauchlan et al., 2002); core made by these constructs will be referred to as coreASC/VLV and corespmt to discriminate between the strain J1 and Glasgow products, respectively. For completeness, we also included a construct, pSFV/C[IF/AL]-E1-HA, that contains mutations at Ile176 and Phe177 (Fig. 1b). Alteration of these residues was reported to impair SPP processing in strain J1 (Okamoto et al., 2004).
The relative mobilities of wild-type (wt) and mutant core made from the pcDNA3.1 constructs in BHK cells were almost indistinguishable following electrophoresis on 12·5 % polyacrylamide gels using a Tris/glycine buffering system (Laemmli, 1970), although coreASC/VLV did migrate slightly above corewt (Fig. 1c, lanes 2–4). Core made from the equivalent SFV constructs showed an identical pattern of electrophoretic mobilities in either BHK or HuH-7 cells (Fig. 1d, upper and lower panels, lanes 1–3). Core expressed by the pcDNA plasmids had reduced mobility compared with the species produced by the pSFV constructs, which resulted from a 22 aa extension at the N terminus of core (Okamoto et al., 2004) that was removed in the pSFV constructs (Fig. 1c, compare lanes 1 and 2). With both expression systems, we found that Core-IF/AL-E1-HA and Core-ASC/VLV-E1-HA constructs generated additional species, which have a similar apparent molecular mass to the predicted size of core–E1 precursor protein [marked by an asterisk in Fig. 1(c, d)]. The abundance of this precursor was reduced in HuH-7 cells (Fig. 1d, lower panel, lane 2). Moreover, both the pcDNA and pSFV constructs for Core-IF/AL-E1-HA consistently gave low levels of core in BHK cells (Fig. 1c, lane 4; Fig. 1d, upper panel, compare lanes 2 and 3), although the mutant protein detected did co-migrate with coreASC/VLV. In HuH-7 cells, core protein from the SFV construct could not be detected (Fig. 1d, lower panel, lane 3).
For constructs that express a polyprotein, which consists of core and E1, but does not include E2, we have observed core–E1 precursor proteins under conditions where processing at SigPcore–E1 is impaired by mutations in core (R. G. Hope, S. Boulant & J. McLauchlan, unpublished data). This phenomenon is likely to arise from reduced efficiency in translocation at the endoplasmic reticulum (ER) membrane, which impairs cleavage at SigPcore–E1 by cellular signalases. Any defect in translocation efficiency that gives rise to such precursors is reduced considerably by extending the length of the HCV polyprotein to include E2. To increase the polyprotein length made by the three pSFV constructs expressing strain J1 sequences, we inserted a BamHI/XbaI fragment from pSFV/CE1E2gla to create pSFV/C[wt]-E1E2, pSFV/C[IF/AL]-E1E2 and pSFV/C[ASC/VLV]-E1E2. In each plasmid, amino acid codons 1–340 were derived from strain J1, whilst codons 341–829 were from strain Glasgow (Fig. 1a). This approach reduced the amounts of the core–E1 products, but did not improve separation of coreASC/VLV from corewt (Fig. 1d, upper and lower panels, lanes 4 and 5). In parallel, BHK and HuH-7 cells were electroporated with RNA from strain Glasgow constructs pSFV/CE1E2gla and pSFV/CspmtE1E2. Again, the mutant protein (corespmt) migrated marginally above wt core (Fig. 1d, upper and lower panels, lanes 7 and 8). Only very low levels of core were made by pSFV/C[IF/AL]-E1E2, which correlates with the data obtained with construct pSFV/C[IF/AL]-E1-HA, but again, the detected protein co-migrated with coreASC/VLV (Fig. 1d, upper panel, compare lanes 5 and 6). The basis for the low levels of coreIF/AL was not studied rigorously; however, pSFV/C[IF/AL]-E1E2 does produce quantities of E2 similar to those from the other pSFV constructs (Fig. 1e, upper and lower panels, compare lane 3 with lanes 1, 2, 4 and 5). Therefore, mutations at residues Ile176 and Phe177 not only impair SPP processing, but also decrease the stability of core. Due to differences in abundance of coreIF/AL compared with corewt and coreASC/VLV, particularly in HuH-7 cells, this mutant was not included in further experiments.
Based on the inability to distinguish corewt from coreASC/VLV on polyacrylamide gels, Okamoto et al. (2004) concluded that these residues were not important for SPP processing. For strain Glasgow, the difficulty with discriminating corewt (which has been cleaved by SPP and ends putatively at about aa 179) from corespmt (which terminates at the SP site at aa 191) by gel electrophoresis using a Tris/glycine buffer system has been highlighted previously (McLauchlan et al., 2002; Lemberg & Martoglio, 2003). Separation of the two forms was possible on multiphasic Tris/Bicine polyacrylamide gels. Paradoxically, corespmt had greater mobility than corewt in this gel system, despite the longer length of the mutant protein (McLauchlan et al., 2002; Lemberg & Martoglio, 2003). To examine in greater detail the core species made by pSFV/C[wt]-E1E2 and pSFV/C[ASC/VLV]-E1E2, RNA from these plasmids was electroporated into cells that were either treated or not treated with (Z-LL)2 ketone, an inhibitor of SPP (Weihofen et al., 2000, 2003), and extracts were analysed in both Tris/glycine and Tris/Bicine gel systems. In Tris/glycine gels, a single product corresponding to SPP-cleaved core was found for the wt form of the protein in untreated cells (Fig. 2a, lane 1); for cells treated with (Z-LL)2 ketone, two species that represented SP- and SPP-cleaved core were detected (upper and lower bands respectively in Fig. 2a, lane 2). From quantification of the bands in lane 2 (Fig. 2a), SPP proteolysis was reduced by about 60 % by the inhibitor. For pSFV/C[ASC/VLV]-E1E2, only a single species was detected either in the presence or absence of (Z-LL)2 ketone, which migrated more closely to the SP-cleaved wt core protein than to the SPP-cleaved form (Fig. 2a, compare lane 2 with lanes 3 and 4). The improved resolution of core species made by pSFV/C[wt]-E1E2 and pSFV/C[ASC/VLV]-E1E2 compared with the data presented in Fig. 1 was achieved by increasing the time taken to resolve the samples on gels. The inability to detect two species made by pSFV/C[ASC/VLV]-E1E2 in the presence of a specific SPP inhibitor indicated that the core protein produced from this plasmid is generated solely by SP. On Tris/Bicine gels, it was not possible to separate SPP- and SP-cleaved forms of corewt (Fig. 2b, lane 2). However, the migration of coreASC/VLV was greater than that of corewt (Fig. 2b, compare lanes 1 and 2 with lanes 3 and 4). This phenomenon was reproducible in three separate experiments and is identical to the migratory properties for proteins made from strain Glasgow, where the longer, SP-cleaved corespmt product migrates faster than the shorter, SPP-cleaved corewt species (compare Figs 2c and d; McLauchlan et al., 2002; Lemberg & Martoglio, 2003). Hence, the results indicate that core protein made from pSFV/C[ASC/VLV]-E1E2 is generated by SP and not by SPP. From the data obtained on the two gel systems, we conclude that mutation of Ala180, Ser183 and Cys184 in SigPcore–E1 in strain J1 does impair SPP processing, in agreement with our findings for the critical role that these amino acids play in SPP cleavage at SigPcore–E1 from HCV strain Glasgow (McLauchlan et al., 2002).
|
; McLauchlan et al., 2002; Weihofen et al., 2003). Hence, the intracellular localizations of wt and mutant forms of core from strain J1 were examined. In BHK cells transfected with pcDNA/Flag-Core-E1-HA, core was present at the surface of lipid droplets (Fig. 3a–c). By contrast, coreASC/VLV was distributed throughout the cytoplasm and was not directed to lipid droplets (Fig. 3d–f). In a small proportion of cells (approx. 5–10 %), there was some staining for coreASC/VLV around lipid droplets, which would be consistent with a minor fraction of mutant protein that is processed by SPP. To demonstrate further that coreASC/VLV was not targeted to lipid droplets, HuH-7 cells were electroporated with RNA transcribed from the pSFV plasmids. Again, corewt was localized to the surface of lipid droplets, whereas coreASC/VLV was distributed throughout the cytoplasm in a reticular pattern, consistent with localization at the ER membrane (Fig. 3g–i). Identical patterns were found in BHK cells expressing core from the SFV plasmids (data not shown). In methanol-fixed cells, coreASC/VLV co-localized with E2, indicating that the mutant protein was present at the ER membrane (data not shown). These data for coreASC/VLV agree with the results for corespmt produced by strain Glasgow (McLauchlan et al., 2002) and demonstrate that the requirement for SPP cleavage to enable transfer of core from the ER membrane to lipid droplets is not a strain-specific characteristic.
|
). By contrast, introduction of mutations into SigPcore–E1, which impairs SPP cleavage, prevents degradation due to retention of core at the ER membrane. Analysis of the corresponding mutants in strain J1 gave results identical to those obtained for strain Glasgow (see Supplementary Figure, available in JGV Online), demonstrating further the similarity in the properties of core made by the two strains.
Contrary to the conclusions by Okamoto et al. (2004), our results demonstrate that amino acids Ala180, Ser183 and Cys184 in SigPcore–E1 for strain J1 are necessary for efficient processing by SPP. These data agree with the sequence requirements for SPP cleavage in strain Glasgow (McLauchlan et al., 2002). We conclude that the disparity in the two published reports was not a consequence of the different expression systems employed or sequence differences elsewhere in the core protein, but the inability to separate coreASC/VLV from corewt reliably on polyacrylamide gels by using a Tris/glycine buffering system. These species could be resolved consistently on gels by using a Tris/Bicine buffering system, although the SP-cleaved form of coreASC/VLV had greater mobility than wt core processed by SPP. It is likely that higher hydrophobicity of coreASC/VLV compared with corewt, which could enable increased binding of SDS to the mutant protein, accounts for its increased migration (Lemberg & Martoglio, 2003). In an analogous situation, two A
peptides (A1–40 and A1–42), which are major components in amyloid deposits, do not separate according to the normal relationship between mass and electrophoretic mobility in Tris/Bicine-buffered gels (Klafki et al., 1996; Kawooya et al., 2003).
Two other characteristics of coreASC/VLV were identical to those of corespmt produced by strain Glasgow. Firstly, coreASC/VLV failed to target lipid droplets and remained at the ER membrane. These mutations in SigPcore–E1 do not apparently affect ER targeting of core in the context of a longer polyprotein, as processing of the glycoproteins in the ER lumen is not altered and core retains a reticular-staining pattern, consistent with association at the ER membrane. Secondly, removal of aa 125–144 led to degradation of SPP-cleaved corewt, whereas coreASC/VLV lacking this segment was stable. From these independent assays, we conclude that these amino acids, previously identified in SigPcore–E1 for strain Glasgow, are also critical for efficient SPP processing of core from strain J1. Comparative studies revealed that SigPcore–E1 is completely conserved in 85 % of 450 HCV sequences deposited in public databases. The only changes identified were at either aa 178 (Leu to Ile in 2 % of sequences) or 187 (Val to Ile in 13 % of sequences). Therefore, amino acids at positions 180, 183 and 184 are invariant in the HCV polyprotein. Extrapolating from our results with two HCV strains from different genotypes, we propose that these amino acids in SigPcore–E1 play a fundamental role in SPP cleavage for all strains of HCV.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Hope, R. G. & McLauchlan, J. (2000). Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein. J Gen Virol 81, 1913–1925.
Hope, R. G., Murphy, D. J. & McLauchlan, J. (2002). The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins. J Biol Chem 277, 4261–4270.
Hüssy, P., Langen, H., Mous, J. & Jacobsen, H. (1996). Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 224, 93–104.[CrossRef][Medline]
Kawooya, J. K., Emmons, T. L., Gonzalez-DeWhitt, P. A., Camp, M. C. & D'Andrea, S. C. (2003). Electrophoretic mobility of Alzheimer's amyloid- peptides in urea–sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Anal Biochem 323, 103–113.[Medline]
Klafki, H.-W., Wiltfang, J. & Staufenbiel, M. (1996). Electrophoretic separation of A4 peptides (1–40) and (1–42). Anal Biochem 237, 24–29.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lemberg, M. K. & Martoglio, B. (2002). Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis. Mol Cell 10, 735–744.[CrossRef][Medline]
Lemberg, M. K. & Martoglio, B. (2003). Analysis of polypeptides by sodium dodecyl sulfate–polyacrylamide gel electrophoresis alongside in vitro-generated reference peptides. Anal Biochem 319, 327–331.[CrossRef][Medline]
McLauchlan, J., Lemberg, M. K., Hope, G. & Martoglio, B. (2002). Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 21, 3980–3988.[CrossRef][Medline]
Okamoto, K., Moriishi, K., Miyamura, T. & Matsuura, Y. (2004). Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein. J Virol 78, 6370–6380.
Weihofen, A., Lemberg, M. K., Ploegh, H. L., Bogyo, M. & Martoglio, B. (2000). Release of signal peptide fragments into the cytosol requires cleavage in the transmembrane region by a protease activity that is specifically blocked by a novel cysteine protease inhibitor. J Biol Chem 275, 30951–30956.
Weihofen, A., Lemberg, M. K., Friedmann, E., Rueeger, H., Schmitz, A., Paganetti, P., Rovelli, G. & Martoglio, B. (2003). Targeting presenilin-type aspartic protease signal peptide peptidase with 
-secretase inhibitors. J Biol Chem 278, 16528–16533.
Received 22 July 2005;
accepted 16 November 2005.
This article has been cited by other articles:
|
|
 |
|
  K. Okamoto, Y. Mori, Y. Komoda, T. Okamoto, M. Okochi, M. Takeda, T. Suzuki, K. Moriishi, and Y. Matsuura Intramembrane Processing by Signal Peptide Peptidase Regulates the Membrane Localization of Hepatitis C Virus Core Protein and Viral Propagation J. Virol., September 1, 2008; 82(17): 8349 - 8361. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Targett-Adams, G. Hope, S. Boulant, and J. McLauchlan Maturation of Hepatitis C Virus Core Protein by Signal Peptide Peptidase Is Required for Virus Production J. Biol. Chem., June 13, 2008; 283(24): 16850 - 16859. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Murray, C. T. Jones, J. Tassello, and C. M. Rice Alanine Scanning of the Hepatitis C Virus Core Protein Reveals Numerous Residues Essential for Production of Infectious Virus J. Virol., October 1, 2007; 81(19): 10220 - 10231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C Hourioux, R Patient, A Morin, E Blanchard, A Moreau, S Trassard, B Giraudeau, and P Roingeard The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model Gut, September 1, 2007; 56(9): 1302 - 1308. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Boulant, P. Targett-Adams, and J. McLauchlan Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus J. Gen. Virol., August 1, 2007; 88(8): 2204 - 2213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Targett-Adams, T. Schaller, G. Hope, R. E. Lanford, S. M. Lemon, A. Martin, and J. McLauchlan Signal Peptide Peptidase Cleavage of GB Virus B Core Protein Is Required for Productive Infection in Vivo J. Biol. Chem., September 29, 2006; 281(39): 29221 - 29227. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
marks the location of the HA tag. (b) Amino acid sequence of SigPcore–E1 for wt and mutant proteins. The wt sequence is identical in strains Glasgow and J1. (c) Expression of wt and mutant core proteins from strain J1. BHK cells were transfected with pcDNA/Flag-Core-E1-HA (lane 2), pcDNA/Flag-Core-ASC/VLV-E1-HA (lane 3) or pcDNA/Flag-Core-IF/AL-E1-HA (lane 4) for 15 h at 37 °C. Western blot analysis was performed with R308, a core antiserum (Hope & McLauchlan, 2000
