The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell. The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell. Signal peptide peptidase (SPP) 5The abbreviations used are: SPP, signal peptide peptidase; ER, endoplasmic reticulum; HCV, hepatitis C virus; SFV, Semliki forest virus; WT, wild-type; PBS, phosphate-buffered saline; HIV, human immunodeficiency virus; nOG, octyl β-d-glucopyranoside.5The abbreviations used are: SPP, signal peptide peptidase; ER, endoplasmic reticulum; HCV, hepatitis C virus; SFV, Semliki forest virus; WT, wild-type; PBS, phosphate-buffered saline; HIV, human immunodeficiency virus; nOG, octyl β-d-glucopyranoside. belongs to a novel family of polytopic membrane-associated aspartyl proteases that catalyze cleavage of peptide bonds within the hydrophobic environment of a lipid bilayer (for review see Ref. 1Martoglio B. Golde T.E. Hum. Mol. Genet. 2003; 12: R201-R206Crossref PubMed Scopus (92) Google Scholar). SPP is highly conserved in higher eucaryotes from plants to humans, including insects (2Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (456) Google Scholar, 3Casso D.J. Tanda S. Biehs B. Martoglio B. Kornberg T.B. Genetics. 2005; 170: 139-148Crossref PubMed Scopus (40) Google Scholar). This membrane protein with nine proposed transmembrane domains exerts its action in the endoplasmic reticulum (ER), where it resides by virtue of a classical ER retrieval signal (3Casso D.J. Tanda S. Biehs B. Martoglio B. Kornberg T.B. Genetics. 2005; 170: 139-148Crossref PubMed Scopus (40) Google Scholar, 4Urny J. Hermans-Borgmeyer I. Gercken G. Schaller H.C. Gene Expr. Patterns. 2003; 3: 685-691Crossref PubMed Scopus (24) Google Scholar, 5Friedmann E. Lemberg M.K. Weihofen A. Dev K.K. Dengler U. Rovelli G. Martoglio B. J. Biol. Chem. 2004; 279: 50790-50798Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The SPP substrates identified so far have in common a type II (N terminus facing the cytosol and C terminus facing the lumen) orientation but do not unequivocally point to a common role for SPP activity. In humans, SPP carries out the intramembrane cleavage of signal peptides of polymorphic major histocompatibility complex class I molecules after they have been cleaved off by signal peptidase; the short N-terminal signal peptide remnants then serve as epitopes presented at the cell surface by nonpolymorphic human lymphocyte antigen-E molecules for immune surveillance (6Lemberg M.K. Bland F.A. Weihofen A. Braud V.M. Martoglio B. J. Immunol. 2001; 167: 6441-6446Crossref PubMed Scopus (152) Google Scholar). Thus, SPP activity may promote the liberation from the ER lipid bilayer of some bioactive signal peptide fragments. Another role of SPP activity may be to protect cells from the toxicity of some signal peptides, e.g. the signal peptide of eosinophil cationic protein (7Wu C.-M. Chang M.D.-T. Biochem. Biophys. Res. Commun. 2004; 322: 585-592Crossref PubMed Scopus (20) Google Scholar). Finally, the substrate spectrum of SPP may not be restricted to signal peptides but extend to transmembrane domains of polytopic proteins (8Moliaka Y.K. Grigorenko A. Madera D. Rogaev E.I. FEBS Lett. 2004; 557: 185-192Crossref PubMed Scopus (27) Google Scholar). The recent finding that a misassembled transmembrane domain of a polytopic protein interacted with SPP has suggested a role for SPP in the ER quality control of membrane proteins (9Crawshaw S.G. Martoglio B. Meacock S.L. High S. Biochem. J. 2004; 384: 9-17Crossref PubMed Scopus (29) Google Scholar). Beside cellular substrates, an internal signal sequence of the polyprotein of hepatitis C virus (HCV) was shown to interact with and be cleaved by SPP (10McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (391) Google Scholar, 11Okamoto K. Moriishi K. Miyamura T. Matsuura Y. J. Virol. 2004; 78: 6370-6380Crossref PubMed Scopus (88) Google Scholar). This makes SPP a potential target for therapeutic intervention, but a clear understanding of the role of SPP in the HCV life cycle is needed before this approach can be envisaged. HCV was discovered by molecular cloning, but its life cycle has remained speculative due to difficulties encountered in reproducing infection in tissue culture cells (12Lindenbach B.D. Rice C.M. Knipe D.M. Howley P.M. Fields Virology. 4th. 1. Lippincott/Williams & Wilkins, Philadelphia2001: 991-1041Google Scholar). Entry of this enveloped virus into target cells most likely involves virion internalization by endocytosis followed by envelope-mediated fusion upon endosomal acidification (13Tscherne D.M. Jones C.T. Evans M.J. Lindenbach B.D. McKeating J.A. Rice C.M. J. Virol. 2006; 80: 1734-1741Crossref PubMed Scopus (324) Google Scholar). The viral capsid must then be disassembled to begin translation of the positive strand RNA genome, but no data are available regarding the uncoating process. Flanked by 5′- and 3′-noncoding regions, the single long open reading frame of the HCV genome encodes a polyprotein of about 3,000 amino acids. During and after translation, this polyprotein undergoes a series of proteolytic cleavages by host and viral proteases, which generate the structural and nonstructural proteins, respectively. The nonstructural proteins are sufficient for replication of the viral genome but are not believed to be contained in virus particles, whereas the structural components (including core protein and two envelope glycoproteins, E1 and E2) assemble with the viral genome to form progeny virions. That HCV morphogenesis most certainly proceeds through budding of nascent particles into the ER lumen is supported by several arguments as follows: (i) the analogy with other viruses of the Flaviviridae family (12Lindenbach B.D. Rice C.M. Knipe D.M. Howley P.M. Fields Virology. 4th. 1. Lippincott/Williams & Wilkins, Philadelphia2001: 991-1041Google Scholar, 14Mackenzie J.M. Westaway E.G. J. Virol. 2001; 75: 10787-10799Crossref PubMed Scopus (257) Google Scholar); (ii) the rare available EM images of liver tissue from HCV-infected patients or chimpanzees showing putative particles in ER-related cisternae of hepatocytes (15Shimizu Y.K. Feinstone S.M. Kohara M. Purcell R.H. Yoshikura H. Hepatology. 1996; 23: 205-209Crossref PubMed Google Scholar, 16Bosman C. Valli M.B. Bertolini L. Serafino A. Boldrini R. Marcellini M. Carloni G. Res. Virol. 1998; 149: 311-314Crossref PubMed Scopus (21) Google Scholar); (iii) the orientation of HCV core and envelope proteins with respect to the ER membrane; and (iv) the accumulation of E1 and E2 envelope glycoproteins in the ER due to ER retention signals (17Op De Beeck A. Dubuisson J. Rev. Med. Virol. 2003; 13: 233-241Crossref PubMed Scopus (35) Google Scholar). HCV core protein is the most N-terminal component of the viral polyprotein and terminates with a signal peptide (Fig. 1A) (18McLauchlan J. J. Viral Hepat. 2000; 7: 2-14Crossref PubMed Scopus (264) Google Scholar). This signal peptide directs the nascent polypeptide chain to the ER membrane and induces translocation of the downstream E1 region into the ER lumen, while leaving the core protein region on the cytosolic side. Cleavage by signal peptidase at the luminal side of the ER separates E1 from the so-called immature form of core protein, containing 191 residues, which migrates in SDS-PAGE with an apparent molecular mass of 23 kDa (p23). This complete form of HCV core protein is anchored in the ER lipid bilayer by the C-terminal signal peptide (10McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (391) Google Scholar). Intramembrane cleavage catalyzed by SPP generates the so-called mature form of core protein, which migrates in SDS-PAGE with an apparent molecular mass of 21 kDa (p21). The precise position of the SPP cleavage site has been debated (10McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (391) Google Scholar, 19Hüssy P. Langen H. Mous J. Jacobsen H. Virology. 1996; 224: 93-104Crossref PubMed Scopus (127) Google Scholar, 20Liu Q. Tackney C. Bhat R.A. Prince A.M. Zhang P. J. 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This was demonstrated not only in a variety of tissue culture cells, but also in liver biopsies from HCV-infected patients and chimpanzees. A fraction of HCV core protein was also shown to localize to the outer membrane of mitochondria in several cell lines (28Okuda M. Li K. Beard M.R. Showalter L.A. Scholle F. Lemon S.M. Weinman S.A. Gastroenterology. 2002; 122: 366-375Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar, 29Schwer B. Ren S. Pietschmann T. Kartenbeck J. Kaehlcke K. Bartenschlager R. Yen T.S.B. Ott M. J. Virol. 2004; 78: 7958-7968Crossref PubMed Scopus (130) Google Scholar). When SPP-catalyzed cleavage to generate p21 was inhibited, however, none of these subcellular locations was reached, and the unprocessed p23 was retained in the ER membrane (10McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (391) Google Scholar, 29Schwer B. Ren S. Pietschmann T. Kartenbeck J. Kaehlcke K. Bartenschlager R. Yen T.S.B. Ott M. J. Virol. 2004; 78: 7958-7968Crossref PubMed Scopus (130) Google Scholar). Because core protein in sera from HCV-infected patients has an identical molecular mass to p21, this form is considered to constitute the HCV virion capsid (18McLauchlan J. J. Viral Hepat. 2000; 7: 2-14Crossref PubMed Scopus (264) Google Scholar, 21Yasui K. Wakita T. Tsukiyama-Kohara K. Funahashi S.-I. Ichikawa M. Kajita T. Moradpour D. Wands J.R. Kohara M. J. Virol. 1998; 72: 6048-6055Crossref PubMed Google Scholar). It might be inferred from this observation that cleavage of core protein by SPP is a prerequisite for HCV particle morphogenesis. As a capsid protein is expected to reside at the virus budding site, however, the propensity of the processed p21 to move to other sites seems paradoxical and may be related to at least some of the many other reported functions of the HCV core protein (18McLauchlan J. J. Viral Hepat. 2000; 7: 2-14Crossref PubMed Scopus (264) Google Scholar). By contrast, p23 is not free for trafficking from the ER to other organelles within the cell (10McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (391) Google Scholar, 29Schwer B. Ren S. Pietschmann T. Kartenbeck J. Kaehlcke K. Bartenschlager R. Yen T.S.B. Ott M. J. Virol. 2004; 78: 7958-7968Crossref PubMed Scopus (130) Google Scholar) and may therefore be prone to virus particle budding. This would imply that HCV morphogenesis can occur when SPP-catalyzed cleavage to generate the p21 form of HCV core protein is abolished. The aim of this study was to decide between these two opposite hypotheses regarding the role of SPP in the HCV life cycle. Two independent approaches, one pharmacological and the other genetic, were used to inhibit SPP-catalyzed cleavage of core protein in two complementary experimental systems that allow the study of HCV morphogenesis in a cellular context. The first uses a recombinant Semliki forest virus (SFV) replicon to express HCV structural proteins in mammalian BHK-21 cells (30Blanchard E. Brand D. Trassard S. Goudeau A. Roingeard P. J. Virol. 2002; 76: 4073-4079Crossref PubMed Scopus (91) Google Scholar, 31Blanchard E. Hourioux C. Brand D. Ait-Goughoulte M. Moreau A. Trassard S. Sizaret P.-Y. Dubois F. Roingeard P. J. Virol. 2003; 77: 10131-10138Crossref PubMed Scopus (54) Google Scholar). Because HCV budding is abortive in this system, it does not allow the isolation of virus-like particles but instead appears as a choice model for EM visualization of budding events in situ in the ER. In the second system, expression of HCV structural proteins is achieved in insect Sf9 cells by means of a recombinant baculovirus vector, which leads to the recovery of enveloped virus-like particles (32Baumert T.F. Ito S. Wong D.T. Liang T.J. J. Virol. 1998; 72: 3827-3836Crossref PubMed Google Scholar, 33Baumert T.F. Vergalla J. Satoi J. 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These findings are consistent with a model in which p23 is the form of HCV core protein that is committed to virus particle morphogenesis, and cleavage by SPP occurs during and/or after virus budding in the ER to predispose the capsid to subsequent disassembly during virus uncoating in a new target cell. Plasmid Constructs—The pFastBac-HCV1a construct was generated by inserting into the multiple cloning site of the vector pFastBac™1 (Invitrogen) the BamHI-BglII fragment of the pGmAc-5′NTRm-NS2Δ plasmid (35Girard C. Ravallec M. Mariller M. Bossy J.-P. Cahour A. López-Ferber M. Devauchelle G. Inchauspé G. Duonor-Cérutti M. J. Gen. Virol. 2004; 85: 3659-3670Crossref PubMed Scopus (6) Google Scholar), a generous gift from M. Duonor-Cérutti and G. Devauchelle (CNRS UMR 5160, Saint-Christol-lès-Alès, France). This fragment contains the portion of the cDNA of the HCV-H strain (genotype 1a) coding for all HCV structural proteins and the first 5 amino acids of NS2 protein, downstream of the complete 5′-noncoding region modified by the mutation of its five AUG codons (35Girard C. Ravallec M. Mariller M. Bossy J.-P. Cahour A. López-Ferber M. Devauchelle G. Inchauspé G. Duonor-Cérutti M. J. Gen. Virol. 2004; 85: 3659-3670Crossref PubMed Scopus (6) Google Scholar). For expression in BHK-21 cells, this fragment was also inserted into the unique BamHI site of the vector pSFV1 (36Liljeström P. Garoff H. Bio/Technology. 1991; 9: 1356-1361Crossref PubMed Scopus (738) Google Scholar), yielding the pSFV-HCV1a construct. The plasmid pFastBac-HCV1b (originally designated as pFastBacHCV.S in Ref. 32Baumert T.F. Ito S. Wong D.T. Liang T.J. J. Virol. 1998; 72: 3827-3836Crossref PubMed Google Scholar), which contains the portion of the cDNA of the HCV-J strain (genotype 1b) coding for all HCV structural proteins and the first 21 amino acids of NS2 protein downstream of the 71 last nucleotides of the 5′-noncoding region, was a kind gift from T. J. Liang (NIDDK, National Institutes of Health, Bethesda). For expression in BHK-21 cells, the 2432-bp NruI-NdeI fragment of this plasmid was subcloned into the pSFV1 vector modified by the insertion of restriction sites for NruI and NdeI immediately downstream of the BamHI site, yielding the pSFV-HCV1b construct. Site-directed Mutagenesis—Oligonucleotide-directed mutagenesis by the Kunkel method (37Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar) was used to introduce mutations into the sequence encoding the signal peptide at the core-E1 junction. To do this, the 2534-bp HindIII-BglII fragment of pGmAc-5′NTRm-NS2Δ and the 1702-bp EcoRI-NotI fragment of pFastBac-HCV1b, which contained the sequences to be mutated, were subcloned into the vectors pGEM-3Zf(+) and pGEM-11Zf(+) (Promega), respectively. Mutagenesis was designed both to introduce an SpeI restriction site and to replace residues Ala180, Ser183, and Cys184 of HCV polyprotein by Val, Leu, and Val, respectively (Fig. 1A). The mutated fragments were then inserted in place of the homologous wild-type (WT) fragments into the pFastBac-HCV1a and pFastBac-HCV1b plasmids. For expression in mammalian cells, they were also inserted in place of the homologous WT fragments into the pSFV-HCV1a and pSFV-HCV1b plasmids. The presence of the desired mutations was checked both by restriction map analysis and DNA sequencing. Mammalian Cell Culture and Transfection—BHK-21 cells were grown in Glasgow minimal essential medium (Invitrogen) supplemented with 10 mm HEPES (pH 7.3), 10% tryptose phosphate broth (Sigma), and 5% fetal calf serum (PAA Laboratories) at 37 °C in a humidified 5% CO2 atmosphere. For transfection, the WT and mutated forms of pSFV-HCV1a or pSFV-HCV1b plasmids were linearized at the SpeI and SphI site, respectively, and served as templates for in vitro transcription by the Sp6 RNA polymerase. Typical reactions were performed for 2 h at 37 °C in a mixture containing 40 mm Tris-HCl (pH 7.9), 10 mm NaCl, 6 mm MgCl2, 2 mm spermidine, 10 mm dithiothreitol, 1 mm each of ATP, CTP, and UTP, 0.5 mm GTP, 1 mm of the cap analog m7G(5′)ppp(5′)G (Invitrogen), 1.6 units/μl recombinant RNasin® ribonuclease inhibitor (Promega), and 0.6 units/μl Sp6 RNA polymerase (Promega). For the negative control, recombinant RNA coding for β-galactosidase (SFV-lacZ) was synthesized from the SpeI-linearized pSFV3-lacZ plasmid (36Liljeström P. Garoff H. Bio/Technology. 1991; 9: 1356-1361Crossref PubMed Scopus (738) Google Scholar). BHK-21 cells in mid-log phase were detached with trypsin, suspended in phosphate-buffered saline (PBS), mixed with the capped transcripts in an electroporation cuvette (0.4-cm gap), and pulsed twice at 0.83 kV, 25 microfarads, in a Gene Pulser® II electroporator (Bio-Rad). Immediately after electroporation, cells were plated and cultured for 20 h in growth medium, containing 20 μm (Z-LL)2-ketone (Calbiochem) where indicated. Insect Cell Culture and Recombinant Baculovirus Generation—Spodoptera frugiperda Sf9 cells were grown in monolayer cultures at 28 °C in Sf-900 II SFM medium (Invitrogen) supplemented with 5% fetal calf serum (PAA Laboratories). The WT and mutated forms of pFastBac-HCV1a and pFastBac-HCV1b were used to generate recombinant baculoviruses by using the Bac-to-Bac® Baculovirus Expression System (Invitrogen) according to the manufacturer's protocols. For the negative control, the plasmid pFastBac™1-Gus (Invitrogen), which contains the cDNA coding for β-glucuronidase, was used to generate the recombinant baculovirus BV-gus. Recombinant baculoviruses harvested from the supernatant of transfected Sf9 cells were amplified by subsequent rounds of Sf9 cell infection until a high titer was achieved. For all expression experiments, Sf9 cells in mid-log phase were infected with the appropriate recombinant baculovirus at a multiplicity of infection of 5 and cultured for 3–4 days. Where indicated, (Z-LL)2-ketone was added every 12 h to the growth medium at a final concentration of 20 μm. Isolation of HCV-like Particles and Capsids—Recombinant baculovirus-infected Sf9 cells were washed three times in PBS and collected by centrifugation. Cells were ruptured by three freeze-thaw cycles, using dry ice plus ethanol for freezing and a 37 °C water bath for thawing, and suspended in TNE buffer (50 mm Tris-HCl (pH 7.4), 100 mm NaCl, 0.5 mm EDTA) containing 0.2 mg/ml of the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (ICN Biomedicals) plus RNasin® (complete TNE buffer). Homogenization further proceeded with 10 passages through a 23-gauge needle. After low speed centrifugation, the supernatant was pelleted over a 2-ml cushion of 20% (w/w) sucrose in complete TNE, at 77,000 × g for 90 min at 4 °C. The pellet was allowed to dissolve in complete TNE buffer for 2 h at 4°C, before loading on a sucrose gradient made in complete TNE. To partially purify enveloped HCV-like particles, this consisted of an 8-ml continuous 30–65% (w/w) sucrose gradient overlaid with a 1.5-ml layer of 15% (w/w) sucrose, itself covered with a 0.5-ml layer of 10% (w/w) sucrose. Isopycnic ultracentrifugation was performed at 150,000 × g for 20 h at 4 °C, followed by fractionation from the bottom of the tube into 18 fractions of 0.6 ml. The density of each fraction was determined by measuring the refractive index of a 10-μl aliquot with an Abbe refractometer (Atago) at a constant temperature of 20 °C. To isolate capsids of HCV-like particles, a protocol was adapted from the "spin-thru" method described for the purification of mature human immunodeficiency virus (HIV) capsids (38Kewalramani V.N. Emerman M. Virology. 1996; 218: 159-168Crossref PubMed Scopus (45) Google Scholar). Briefly, isopycnic ultracentrifugation was performed as described above except that the 15% sucrose layer contained 60 mm octyl β-d-glucopyranoside (nOG) (Sigma). In this method, the 10% sucrose layer serves as a barrier to minimize mixing of the virus and detergent interfaces prior to centrifugation, and the enveloped particles pass through the detergent interface at the quickest possible speed, thus minimizing the time that particles are incubated in detergent. Western Blotting—For analysis of HCV structural protein expression, recombinant SFV RNA-transfected BHK-21 cells or recombinant baculovirus-infected Sf9 cells were lysed in reducing sample buffer. Cell lysates or fractions collected from sucrose gradients were electrophoresed in SDS-15% polyacrylamide gels under reducing conditions, and subsequently transferred onto nitrocellulose membranes (PROTRAN®, Schleicher & Schuell). Membranes were blocked with PBS containing 0.05% Tween 20 and 5% skimmed milk and probed overnight at 4 °C with monoclonal antibodies against HCV core protein (1856, Virostat), E1 glycoprotein (A4 (39Dubuisson J. Hsu H.H. Cheung R.C. Greenberg H.B. Russell D.G. Rice C.M. J. Virol. 1994; 68: 6147-6160Crossref PubMed Google Scholar), kindly provided by J. Dubuisson, CNRS UPR 2511, Institut Pasteur de Lille, Lille, France), or E2 glycoprotein (AP33 (34Clayton R.F. Owsianka A. Aitken J. Graham S. Bhella D. Patel A.H. J. Virol. 2002; 76: 7672-7682Crossref PubMed Scopus (130) Google Scholar), kindly provided by A. H. Patel, MRC Virology Unit, Institute of Virology, Glasgow, UK), diluted 1:10,000, 1:750, and 1:500 in blocking buffer, respectively. Excess antibody was removed with four washes, and the second-step antibody (horseradish peroxidase-linked mouse-specific sheep antibody; Amersham Biosciences) diluted 1:2,000 in blocking buffer was allowed to bind for 1 h. After several washes, protein bands were visualized using membrane incubation in SuperSignal® West Pico Chemiluminescence Substrate (Pierce) and exposure to BioMax Light films (Eastman Kodak). Blots used for multiple hybridizations were stripped of antibodies by washing for 30 min at 55 °C in stripping buffer (125 mm Tris-HCl (pH 6.7), 2% SDS, 100 mm β-mercaptoethanol), and reprobed as described above. Capsid Stability Assays—Freshly prepared capsids were tested for stability under different conditions. After isopycnic ultracentrifugation using the spin-thru method described above, the fractions with densities ranging from 1.23 to 1.26 g/ml were pooled and diluted 1:10 in TNE buffer, in high salt TNE buffer (TNE containing 1 m instead of 100 mm NaCl), or in low pH TNE buffer (TNE adjusted to pH 5.5). Following incubation at 37 °C for various times, samples were split in half. The total proteins contained in one-half were precipitated by adding trichloroacetic acid to a final concentration of 15% in the presence of 0.1 mg/ml bovine serum albumin; the precipitate was spun down at 13,000 × g in a microcentrifuge, washed with acetone to remove the trichloroacetic acid, and resuspended in reducing sample buffer for Western blotting detection of total HCV core protein. The other half was subjected to ultracentrifugation at 77,000 × g for 90 min at 4 °C, and the resulting pellet was resuspended in reducing sample buffer for Western blotting detection of pelletable HCV core protein. The NIH ImageJ version 1.33 software was used for quantitative evaluation of the relative intensities of the bands. For each incubation time, the capsid stability index was calculated as follows: 100 × (intensity of the band of pelletable core protein at the given time/intensity of the band of pelletable core protein at time 0)/(intensity of the band of total core protein at the given time/intensity of the band of total core protein at time 0). EM Analyses—For in situ visualization of HCV-like particle budding, recombinant SFV RNA-transfected BHK-21 cells were fixed for 1 h with 3% glutaraldehyde in phosphate buffer (0.1 m NaH2PO4, 0.1 m Na2HPO4, pH 7.2), and post-fixed for 2 h with 1% OsO4 in 0.1 m cacodylate buffer. Fixed cells were washed in water for 5 min, then in 0.1 m cacodylate buffer for 15 min, and finally in 0.2 m cacodylate buffer for 30 min. They were transferred to 30% methanol for 10 min, st
(1) Background: What is the role of serum CMV PCR in the diagnosis of recent primary infection (PI) in pregnant women when IgG avidity is uninformative? (2) Methods: Retrospective cohort study to compare serum versus whole blood CMV PCR. (a) Qualitative assessment: CMV PCR was performed on 123 serum samples and 74 whole blood samples collected from 132 pregnant women with recent CMV PI. PCR positivity rate was used to calculate sensitivity in serum and whole blood. (b) Quantitative assessment: CMV PCR was performed on 72 paired samples of serum and whole blood collected on the same day from 57 patients. (3) Results: In pregnant women, PCR positivity rate was 89% for serum samples versus 100% in whole blood in the case of very recent PI (<15 days), but only 27% in serum versus 68% in whole blood for PI occurring from 6 weeks to 3 months before. Comparing CMV viral loads between serum and whole blood, we determined the limit of CMV DNA detection in serum as 3 log copies/mL (whole blood equivalent). (4) Conclusions: Serum CMV PCR is reliable in confirming PI in cases when only IgM is detected. It is therefore a valuable tool in introducing valaciclovir treatment as early as possible to prevent mother-to-child CMV transmission.
Varicella-zoster primary infection in a pregnant woman is an uncommon occurrence in France, but can cause concern for patients and for clinicians. Varicella (chickenpox) is usually benign, but can have serious consequences for the mother (especially in the third trimester of pregnancy), for the fetus (risk of fetal varicella syndrome particularly if maternal primary infection occurs before 20 weeks of gestation), and for the newborn (risk of congenital neonatal varicella if the mother is infected in the peripartum period). Several diagnostic tools, prophylaxis measures and antiviral treatments are currently available and help in the management of varicella during pregnancy.
L’infection a cytomegalovirus (CMV) est la plus frequente des infections virales transmises au cours de la grossesse : 1 % des enfants naissent avec une infection congenitale a CMV. C’est essentiellement au decours d’une primo-infection a CMV en cours de grossesse qu’existe un risque (d’environ 10 %) d’atteinte fœtale multiviscerale touchant en particulier le systeme nerveux central, et dont les sequelles sont lourdes (surdite, chorioretinite, deficits moteurs, crises convulsives, retard mental, microcephalie). La frequence et la gravite potentielle de l’infection justifient le diagnostic virologique antenatal de l’infection materno-fœtale, mais l’absence de parametres predictifs stricts de gravite rend sa prise en charge tres delicate. Des donnees nouvelles sur la physiopathologie de l’infection placentaire et sa possible prevention, ainsi que l’identification d’eventuels parametres virologiques pronostiques devraient permettre d’eviter l’infection fœtale grave, en attendant la mise au point d’un vaccin, ou de traitements efficaces.
