Charged Residues in the Transmembrane Domains of Hepatitis C Virus Glycoproteins Play a Major Role in the Processing, Subcellular Localization, and Assembly of These Envelope Proteins

ABSTRACT For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the familyFlaviviridae, these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope.

Download Full-text

Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.76.8.3720-3730.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3720-3730 ◽

Cited By ~ 143

Author(s):

Séverine Carrère-Kremer ◽

Claire Montpellier-Pala ◽

Laurence Cocquerel ◽

Czeslaw Wychowski ◽

François Penin ◽

...

Keyword(s):

Plasma Membrane ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Subcellular Localization ◽

Secretory Pathway ◽

Transmembrane Domain ◽

Signal Sequence ◽

C Terminus ◽

Membrane Spanning ◽

Polytopic Membrane Protein

ABSTRACT Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.

Download Full-text

Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2

Journal of General Virology ◽

10.1099/0022-1317-82-7-1629 ◽

2001 ◽

Vol 82 (7) ◽

pp. 1629-1635 ◽

Cited By ~ 33

Author(s):

Laurence Cocquerel ◽

Jean-Christophe Meunier ◽

Anne Op de Beeck ◽

Dorine Bonte ◽

Czeslaw Wychowski ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Transmembrane Domain ◽

Signal Sequence ◽

Envelope Proteins ◽

Membrane Insertion ◽

Virus Envelope ◽

Glycoprotein Complex ◽

The Stability ◽

Major Factors

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins, E1 and E2. These proteins contain a large N-terminal ectodomain, and are anchored into membranes by their C-terminal transmembrane domain (TMD). The TMDs of HCV envelope proteins are multifunctional. In addition to their role as membrane anchors, they possess a signal sequence function in their C-terminal half, and play a major role in subcellular localization and assembly of these envelope proteins. In this work, the expression of full-length E2 led to secretion of a proportion of this protein, which is likely to be due to inefficient membrane insertion of a fraction of E2 expressed alone. However, when E1 and E2 were coexpressed from the same polyprotein, E2 was not secreted and remained tightly associated with membranes, suggesting that an early interaction between the TMDs of HCV envelope proteins improves the stability of membrane insertion of E2. These results reinforce the hypothesis that the TMDs of E1 and E2 are major factors in the assembly of the HCV envelope glycoprotein complex.

Download Full-text

Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

Journal of Virology ◽

10.1128/jvi.78.6.2994-3002.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2994-3002 ◽

Cited By ~ 157

Author(s):

Anne Op De Beeck ◽

Cécile Voisset ◽

Birke Bartosch ◽

Yann Ciczora ◽

Laurence Cocquerel ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Conformational Changes ◽

Structural Changes ◽

Envelope Proteins ◽

Viral Envelope ◽

Envelope Glycoproteins ◽

Functional Envelope

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

Download Full-text

Preferential association of Hepatitis C virus with apolipoprotein B48-containing lipoproteins

Journal of General Virology ◽

10.1099/vir.0.82033-0 ◽

2006 ◽

Vol 87 (10) ◽

pp. 2983-2991 ◽

Cited By ~ 76

Author(s):

Olivier Diaz ◽

François Delers ◽

Marianne Maynard ◽

Sylvie Demignot ◽

Fabien Zoulim ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Density Lipoprotein ◽

Viral Envelope ◽

Density Fractions ◽

The Family ◽

Apolipoprotein B48 ◽

Fat Meal ◽

Preferential Association

Hepatitis C virus (HCV) in cell culture has a density comparable to that of other members of the family Flaviviridae, whereas in vivo infectious particles are found partially in low-density fractions, associated with triacylglycerol (TG)-rich lipoproteins (TRLs). In the blood of infected patients, HCV circulates as heterogeneous particles, among which are lipo-viroparticles (LVPs), globular particles rich in TG and containing viral capsid and RNA. The dual viral and lipoprotein nature of LVPs was addressed further with respect to apolipoprotein composition and post-prandial dynamic lipid changes. The TRLs exchangeable apoE, -CII and -CIII, but not the high-density lipoprotein apoA-II, were present on LVPs, as well as the viral envelope proteins. apoB100 and-B48, the two isoforms of the non-exchangeable apoB, were represented equally on LVPs, despite the fact that apoB48 was barely detectable in the plasma of these fasting patients. This indicates that a significant fraction of plasma HCV was associated with apoB48-containing LVPs. Furthermore, LVPs were enriched dramatically and rapidly in triglycerides after a fat meal. As apoB48 is synthesized exclusively by the intestine, these data highlight the preferential association of HCV with chylomicrons, the intestine-derived TRLs. These data raise the question of the contribution of the intestine to the viral load and suggest that the virus could take advantage of TRL assembly and secretion for its own production and of TRL fate to be delivered to the liver.

Download Full-text

Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer

Journal of General Virology ◽

10.1099/vir.0.81140-0 ◽

2005 ◽

Vol 86 (10) ◽

pp. 2793-2798 ◽

Cited By ~ 36

Author(s):

Yann Ciczora ◽

Nathalie Callens ◽

Claire Montpellier ◽

Birke Bartosch ◽

François-Loïc Cosset ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Transmembrane Domain ◽

Envelope Glycoproteins ◽

Retention Function ◽

Virus Glycoprotein ◽

Er Retention ◽

Charged Residues ◽

Tm Domain

The envelope glycoproteins of Hepatitis C virus (HCV), E1 and E2, form a heterodimer that is retained in the endoplasmic reticulum (ER). The transmembrane (TM) domains play a major role in E1E2 heterodimerization and in ER retention. Two fully conserved charged residues in the middle of the TM domain of E2 (Asp and Arg) are crucial for these functions. Replacement of the Asp residue by a Leu impaired E1E2 heterodimerization, whereas the Arg-to-Leu mutation had a milder effect. Both Asp and Arg residues were shown to contribute to the ER retention function of E2. In addition, the entry function of HCV envelope glycoproteins was affected by these mutations. Together, these data indicate that the charged residues present in the TM domain of E2 play a major role in the biogenesis and the entry function of the E1E2 heterodimer. However, the Asp and Arg residues do not contribute equally to these functions.

Download Full-text

Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Journal of Virology ◽

10.1128/jvi.78.17.9257-9269.2004 ◽

2004 ◽

Vol 78 (17) ◽

pp. 9257-9269 ◽

Cited By ~ 55

Author(s):

Kevin C. Klein ◽

Stephen J. Polyak ◽

Jaisri R. Lingappa

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

De Novo ◽

Signal Sequence ◽

Buoyant Density ◽

Capsid Assembly ◽

Culture Systems ◽

Capsid Formation ◽

Cell Culture Systems

ABSTRACT The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of ≈100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.

Download Full-text

A Retention Signal Necessary and Sufficient for Endoplasmic Reticulum Localization Maps to the Transmembrane Domain of Hepatitis C Virus Glycoprotein E2

Journal of Virology ◽

10.1128/jvi.72.3.2183-2191.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 2183-2191 ◽

Cited By ~ 166

Author(s):

Laurence Cocquerel ◽

Jean-Christophe Meunier ◽

André Pillez ◽

Czeslaw Wychowski ◽

Jean Dubuisson

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Transmembrane Domain ◽

Intracellular Localization ◽

Native Form ◽

Glycosyl Phosphatidylinositol ◽

Er Retention ◽

Er Targeting ◽

Necessary And Sufficient

ABSTRACT The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which is retained in the endoplasmic reticulum (ER). To identify whether E1 and/or E2 contains an ER-targeting signal potentially involved in ER retention of the E1-E2 complex, these proteins were expressed alone and their intracellular localization was studied. Due to misfolding of E1 in the absence of E2, no conclusion on the localization of its native form could be drawn from the expression of E1 alone. E2 expressed in the absence of E1 was shown to be retained in the ER similarly to E1-E2 complex. Chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4) were constructed to identify the sequence responsible for its ER retention. The transmembrane domain (TMD) of E2 (C-terminal 29 amino acids) was shown to be sufficient for retention of the ectodomain of CD4 in the ER compartment. Replacement of the E2 TMD by the anchor signal of CD4 or a glycosyl phosphatidylinositol (GPI) moiety led to its expression on the cell surface. In addition, replacement of the E2 TMD by the anchor signal of CD4 or a GPI moiety abolished the formation of E1-E2 complexes. Together, these results suggest that, besides having a role as a membrane anchor, the TMD of E2 is involved in both complex formation and intracellular localization.

Download Full-text

Blocking Hepatitis C Virus Infection with Recombinant Form of Envelope Protein 2 Ectodomain

Journal of Virology ◽

10.1128/jvi.00800-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11078-11089 ◽

Cited By ~ 43

Author(s):

Jillian Whidby ◽

Guaniri Mateu ◽

Hannah Scarborough ◽

Borries Demeler ◽

Arash Grakoui ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Expression System ◽

Developed Countries ◽

Viral Envelope ◽

Hcv Infection ◽

Size Exclusion ◽

Amino Terminal ◽

Endosomal Acidification

ABSTRACT More than 120 million people worldwide are chronically infected with hepatitis C virus (HCV), making HCV infection the leading cause of liver transplantation in developed countries. Treatment is limited, and efficacy depends upon the infecting strain and the initial viral load. The HCV envelope glycoproteins (E1 and E2) are involved in receptor binding, virus-cell fusion, and entry into the host cell. HCV infection proceeds by endosomal acidification, suggesting that fusion of the viral envelope with cellular membranes is a pH-triggered event. E2 consists of an amino-terminal ectodomain, an amphipathic helix that forms a stem region, and a carboxy-terminal membrane-associating segment. We have devised a novel expression system for the production of a secreted form of E2 ectodomain (eE2) from mammalian cells and performed a comprehensive biochemical and biophysical characterization. eE2 is properly folded, as determined by binding to human CD81, blocking of infection of cell culture-derived HCV, and recognition by antibodies from patients chronically infected with different genotypes of HCV. The glycosylation pattern, number of disulfide bonds, oligomerization state, and secondary structure of eE2 have been characterized using mass spectrometry, size exclusion chromatography, circular dichroism, and analytical ultracentrifugation. These results advance the understanding of E2 and may assist in the design of an HCV vaccine and entry inhibitor.

Download Full-text