Rubella virus E2 signal peptide is required for perinuclear localization of capsid protein and virus assembly

The rubella virus (RV) structural proteins capsid, E2, and E1 are synthesized as a polyprotein precursor. The signal peptide that initiates translocation of E2 into the lumen of the endoplasmic reticulum remains attached to the carboxy terminus of the capsid protein after cleavage by signal peptidase. Among togaviruses, this feature is unique to RV. The E2 signal peptide has previously been shown to function as a membrane anchor for the capsid protein. In the present study, we demonstrate that this domain is required for RV glycoprotein-dependent localization of the capsid protein to the juxtanuclear region and subsequent virus assembly at the Golgi complex.     

The Alphavirus E3 Glycoprotein Functions in a Clade-Specific Manner

The 80 trimeric, glycoprotein spikes that cover the surface of alphavirus particles are required for mediating viral entry into a host cell. Spike assembly is a regulated process that requires interactions between five structural proteins, E3, E2, 6K and its translational frameshift product TF, and E1. E3 is a small, ∼65-amino-acid glycoprotein that has two known functions: E3 serves as the signal sequence for translocation of the E3-E2-6K-E1 polyprotein into the endoplasmic reticulum (ER), and cleavage of E3 from E2 is essential for virus maturation. Nonetheless, when E3 is replaced with an ER signal sequence, spikes do not form and infectious particles are not assembled, suggesting an additional role(s) for E3 in the viral life cycle. To further investigate the role of E3 in spike assembly, we made chimeric viruses in which E3 from one alphavirus species is replaced with E3 from another species. Our results demonstrate that when E3 is interchanged between alphavirus species that belong to the same virus clade, viral titers and particle morphologies and compositions are similar to what are observed for the parental virus. In contrast, for chimeras in which E3 is derived from a different clade than the parental virus, we observed reduced titers and the formation of particles with atypical morphologies and protein compositions. We further characterized the E3 chimeras using a combination of structure-function and revertant analyses. This work revealed two specific interactions between E3 and its cognate E2 glycoprotein that are important for regulating spike assembly.     

Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions

During infection of sac- cells by murine coronavirus MHV A59 the intracellular sites at which progeny virions bud correlate with the distribution of the viral glycoprotein E1. Budding is first detectable by electron microscopy at 6 to 7 hours post infection in small, smooth, perinuclear vesicles and tubules in a region transitional between the rough endoplasmic reticulum and the Golgi apparatus. At later times the rough endoplasmic reticulum becomes the major site of budding and accumulation of progeny virus particles. Indirect immunofluorescence microscopy shows that E1 is confined at 6 hours post infection to the perinuclear region while at later times it also accumulates in the endoplasmic reticulum. At 6 hours post infection the second viral glycoprotein, E2, is distributed throughout the endoplasmic reticulum and is not restricted to the site at which budding begins. Core protein, the third protein in virions, can be detected 2 hours before E1 is detectable and budding begins, and at 6 hours post infection it is distributed throughout the cytosol. We conclude that the time and the site at which the maturation of progeny virions occurs is determined by the accumulation of glycoprotein E1 in intracellular membranes. Only rarely do progeny virions bud directly into the cisternae of the Golgi apparatus but at least some already budded virions are transported to the Golgi apparatus where they occur in structures some of which also contain TPPase, a trans Golgi marker.     

Formation of native hepatitis C virus glycoprotein complexes.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.     

A new determinant of endoplasmic reticulum localization is contained in the juxtamembrane region of the ectodomain of hepatitis C virus glycoprotein E1

Hepatitis C virus glycoproteins E1 and E2 do not reach the plasma membrane of the cell but accumulate intracellularly, mostly in the endoplasmic reticulum. Previous studies based on transient expression assays have shown that the transmembrane domains of both glycoproteins are sufficient to localize reporter proteins in the endoplasmic reticulum and that other localization signals may be contained in the ectodomain of E1 protein. To identify such signals we generated chimeric proteins between E1 and two reporter proteins, the human CD8 glycoprotein and the human alkaline phosphatase, and analyzed their subcellular localization in stable as well as transient transfectants. Our results showed that (i) an independent localization determinant for the endoplasmic reticulum is present in the juxtamembrane region of the ectodomain of E1 protein and (ii) the localization dictated by this determinant is either due to direct retention or to a recycling mechanism from the intermediate compartment/cis-Golgi complex region, which is clearly different from those previously described for other retrieval signals. These results show for the first time in mammalian cells that the localization in the endoplasmic reticulum of transmembrane protein can be determined by specific targeting signals acting in the lumen of the compartment.     

Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity

Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.     

Inhibition of translocation of beta -lactamase into the yeast endoplasmic reticulum by covalently bound benzylpenicillin.

We found recently that beta-lactamase folds in the yeast cytosol to a native-like, catalytically active, and trypsin-resistant conformation, and is thereafter translocated into the ER and secreted to the medium. Previously, it was thought that pre-folded proteins cannot be translocated. Here we have studied in living yeast cells whether beta-lactamase, a tight globule in authentic form, must be unfolded for ER translocation. A beta-lactamase mutant (E166A) binds irreversibly benzylpenicillin via Ser(70) in the active site. We fused E166A to the C terminus of a yeast-derived polypeptide having a post-translational signal peptide. In the presence of benzylpenicillin, the E166A fusion protein was not translocated into the endoplasmic reticulum, whereas translocation of the unmutated variant was not affected. The benzylpenicillin-bound protein adhered to the endoplasmic reticulum membrane, where it prevented translocation of BiP, carboxypeptidase Y, and secretory proteins. Although the 321-amino acid-long N-terminal fusion partner adopts no regular secondary structure and should have no constraints for pore penetration, the benzylpenicillin-bound protein remained fully exposed to the cytosol, maintaining its signal peptide. Our data suggest that the beta-lactamase portion must unfold for translocation, that the unfolding machinery is cytosolic, and that unfolding of the remote C-terminal beta-lactamase is required for initiation of pore penetration.     

Oligomerization of hepatitis C virus core protein is crucial for interaction with the cytoplasmic domain of E1 envelope protein

Hepatitis C virus (HCV) contains two membrane-associated envelope glycoproteins, E1 and E2, which assemble as a heterodimer in the endoplasmic reticulum (ER). In this study, predictive algorithms and genetic analyses of deletion mutants and glycosylation site variants of the E1 glycoprotein were used to suggest that the glycoprotein can adopt two topologies in the ER membrane: the conventional type I membrane topology and a polytopic topology in which the protein spans the ER membrane twice with an intervening cytoplasmic loop (amino acid residues 288 to 360). We also demonstrate that the E1 glycoprotein is able to associate with the HCV core protein, but only upon oligomerization of the core protein in the presence of tRNA to form capsid-like structures. Yeast two-hybrid and immunoprecipitation analyses reveal that oligomerization of the core protein is promoted by amino acid residues 72 to 91 in the core. Furthermore, the association between the E1 glycoprotein and the assembled core can be recapitulated using a fusion protein containing the putative cytoplasmic loop of the E1 glycoprotein. This fusion protein is also able to compete with the intact E1 glycoprotein for binding to the core. Mutagenesis of the cytoplasmic loop of E1 was used to define a region of four amino acids (residues 312 to 315) that is important for interaction with the assembled HCV core. Taken together, our studies suggest that interaction between the self-oligomerized HCV core and the E1 glycoprotein is mediated through the cytoplasmic loop present in a polytopic form of the E1 glycoprotein.     

Functional Analysis of Cell Surface-Expressed Hepatitis C Virus E2 Glycoprotein

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.     

Molecular basis for the transport of cytochrome P450 2E1 to the plasma membrane

Endoplasmic reticulum-resident cytochrome P450 enzymes that face the cytosol are present on the plasma membrane of hepatocytes, but the molecular origin for their transport to this compartment has until now remained unknown. The molecular basis for the transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two NH(2)-terminal CYP2E1 mutants were constructed: N(++)2E1, which carried two positive charges in the NH(2) terminus, and 2C-2E1, in which the transmembrane domain of CYP2E1 was replaced with that of CYP2C1, which was previously described to cause retention of CYP2C1 in the endoplasmic reticulum, as well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent microscopy and cell surface biotinylation experiments revealed that all CYP2E1 variants were present on the extracellular side of the plasma membrane. The VSV-G epitope on CYP2E1 was detected on the outside of the plasma membrane using VSV-G-specific antibodies, indicating that the large COOH-terminal part of CYP2E1 is indeed exposed on the outside of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and 2E1-VSV-G on the cell surface were found to be about 2% of total cellular enzyme, whereas twice this amount of N(++)2E1 was recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells revealed that a small fraction of CYP2E1 and all variant proteins was found to be located in the lumen of the endoplasmic reticulum (type II orientation), whereas the majority of the proteins were in the expected cytosolic or type I orientation. It is concluded that the NH(2)-terminal transmembrane domain of CYP2E1 plays a critical role in directing the protein to the cell surface and that topological inversion of a small fraction of CYP2E1 in the endoplasmic reticulum directs the protein to the plasma membrane.     

Suppression of a sec63 mutation identifies a novel component of the yeast endoplasmic reticulum translocation apparatus.

Mutations in the SEC63 gene are associated with defects in protein translocation into the endoplasmic reticulum (ER) as well as in nuclear protein localization in Saccharomyces cerevisiae. To identify proteins that might interact and/or function with SEC63p, we cloned a high copy suppressor (HSS1) of the temperature-sensitive lethal phenotype of the sec63-101 mutant. HSS1 is an allele-specific sec63 suppressor that encodes an integral ER membrane glycoprotein of 206 amino acids with the N-terminus in the ER lumen and C-terminal region in the cytoplasm. Haploid strains disrupted for HSS1 are temperature-sensitive for growth and accumulate precursor forms of Kar2p and invertase. The HSS1 null allele is synthetically lethal in combination with mutations affecting ER translocation. We propose that HSS1p is important for ER translocation and interacts with previously identified components of the yeast translocation apparatus. HSS1 is identical to SEC66, which encodes a glycoprotein complexed with SEC62p and SEC63p.     

The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum

Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex.     

Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) are released from the polyprotein by signal peptidase-mediated cleavage and interact to form a heterodimer. Since properly folded subunits are usually required for specific recognition and stable oligomer formation, the rate of stable E1E2 complex formation, which is low, may be limited by the rate of HCV E1 and/or E2 folding. In this study, the folding of the HCV E1 and E2 glycoproteins was monitored by observing the kinetics of intramolecular disulfide bond formation. The association/dissociation of E1 and E2 with calnexin was also examined, since this molecular chaperone appears to play a major role in quality control via retention of incompletely folded or misfolded proteins in the endoplasmic reticulum. Our results indicate that the disulfide-dependent folding of E2 occurs rapidly and appears to be complete upon cleavage of the precursor E2-NS2. In contrast, folding of E1 is slow (> 1 h), suggesting that this step may be rate limiting for E1E2 oligomerization. Both HCV glycoproteins associated rapidly with calnexin, but dissociation was slow, consistent with the slow folding and assembly of E1E2 glycoprotein complexes. These results suggest a role for prolonged association with calnexin in the folding and assembly of HCV glycoprotein heterodimer complexes.     

Post-translational glycosylation of coronavirus glycoprotein E1: inhibition by monensin.

The intracellular sites of biosynthesis of the structural proteins of murine hepatitis virus A59 have been analyzed using cell fractionation techniques. The nucleocapsid protein N is synthesized on free polysomes, whereas the envelope glycoproteins E1 and E2 are translated on the rough endoplasmic reticulum (RER). Glycoprotein E2 present in the RER contains N-glycosidically linked oligosaccharides of the mannose-rich type, supporting the concept that glycosylation of this protein is initiated at the co-translational level. In contrast, O-glycosylation of E1 occurs after transfer of the protein to smooth intracellular membranes. Monensin does not interfere with virus budding from the membranes of the endoplasmic reticulum, but it inhibits virus release and fusion of infected cells. The oligosaccharide side chains of E2 obtained under these conditions are resistant to endoglycosidase H and lack fucose suggesting that transport of this glycoprotein is inhibited between the trans Golgi cisternae and the cell surface. Glycoprotein E1 synthesized in the presence of monensin is completely carbohydrate-free. This observation suggests that the intracellular transport of this glycoprotein is also blocked by monensin.     

Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes

The E2 glycoprotein of the alphavirus Sindbis is a typical type I membrane protein with a single membrane spanning domain and a cytoplasmic tail (endo domain) containing 33 amino acids. The carboxyl terminal domain of the tail has been implicated as (a) attachment site for nucleocapsid protein, and (b) signal sequence for integration of the other alpha-virus membrane proteins 6K and E1. These two functions require that the carboxyl terminus be exposed in the cell cytoplasm (a) and exposed in the lumen of the endoplasmic reticulum (b). We have investigated the orientation of this glycoprotein domain with respect to cell membranes by substituting a tyrosine for the normally occurring serine, four amino acids upstream of the carboxyl terminus. Using radioiodination of this tyrosine as an indication of the exposure of the glycoprotein tail, we have provided evidence that this domain is initially translocated into a membrane and is returned to the cytoplasm after export from the ER. This is the first demonstration of such a transient translocation of a single domain of an integral membrane protein and this rearrangement explains some important aspects of alphavirus assembly.     

Displacement of SERCA from SR lipid caveolae-related domains by Bcl-2: a possible mechanism for SERCA inactivation

Bcl-2 exerts its anti-apoptotic effect in part through the regulation of Ca2+ homeostasis at the level of the endoplasmic reticulum. Earlier, we demonstrated that a truncated form of Bcl-2, Bcl-2delta21, interacts with and destabilizes the skeletal muscle sarco/endoplasmic reticulum Ca-ATPase (SERCA) [Dremina, E. S., Sharov, V. S., Kumar, K., Zaidi, A., Michaelis, E. K., and Sch?neich, C. (2004) Biochem. J. 383, 361-370]. Here we show that (i) the transmembrane (TM) domain of Bcl-2 accelerates SERCA inactivation, (ii) both Bcl-2delta21 and full-length Bcl-2 selectively interact with SERCA1, and (iii) the inactivation of SERCA is accompanied by a translocation of SERCA from caveolae-related domains (CRD) of the sarcoplasmic reticulum (SR). In rat skeletal muscle SR, intact SERCA1 was detected only in the CRD fractions of a sucrose density gradient. Co-incubation of SR with either Bcl-2delta21 or full-length Bcl-2 resulted in both the appearance of Bcl-2delta21 or Bcl-2 in the fractions containing SERCA1 and translocation of SERCA1 from CRD fractions; the latter effect correlated with the loss of the Ca-ATPase activity of the protein.     

Retention of a co-translational translocated mutant protein of carboxypeptidase Y of Saccharomyces cerevisiae in endoplasmic reticulum

Abstract Co-translational translocation of Saccharomyces cerevisiae vacuolar glycoprotein carboxypeptidase Y (CpY) was highly efficient when studied with an in vivo and in vitro homologous system, comparison of limited proteolytic cleavage of immunoprecipitated translational products of CpY and subcellular localisation of a mutant CpY. The efficient segregation of CpY mRNA in highly purified fractions of rough microsomes was characterised. CpY₁ mutant showed retention of core glycosylated material (proCpY₁) in the rough and smooth endoplasmic reticulum fractions. It is suggested that the presence of structures that are incompatible with intercompartmental transport of vacuolar protein leads to retention of the mutated CpY by the endoplasmic reticulum.     

The N-terminal (pre-S2) domain of a hepatitis B virus surface glycoprotein is translocated across membranes by downstream signal sequences.

The coding region for the hepatitis B virus surface antigens contains three in-phase ATG codons which direct the synthesis of three related polypeptides. The 24-kilodalton major surface (or S) glycoprotein is initiated at the most distal ATG and is a transmembrane protein whose translocation across the bilayer is mediated by at least two uncleaved signal sequences. The product of the next upstream ATG is the 31-kilodalton pre-S2 protein, which contains 55 additional amino acids attached to the N terminus of the S protein. This pre-S2-specific domain is translocated into the endoplasmic reticulum. Using a coupled in vitro translation-translocation system, we showed that (i) the pre-S2 domain itself lacks functional signal sequence activity, (ii) its translocation across the endoplasmic reticulum membrane is mediated by downstream signals within the S domain, and (iii) the N-terminal signal sequence of the S protein can translocate upstream protein domains in the absence of other signals. The hepatitis B virus pre-S2 protein is an example of a natural protein which displays upstream domain translocation, a phenomenon whose existence was originally inferred from the behavior of synthetic fusion proteins in vitro.     

YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism.

The role of S. cerevisiae YDJ1 protein (YDJ1p) in polypeptide translocation across membranes has been examined. A conditional ydj1 mutant strain (ydj1-151TS) is defective for import of several polypeptides into mitochondria and alpha factor into the endoplasmic reticulum at 37 degrees C. These defects are suppressed by E. coli dnaJ or overexpression of S. cerevisiae SIS1 proteins. A different ydj1 mutant, which cannot be farnesylated (ydj1-S406), displays similar transport defects to the ydj1-151 strain. Furthermore, the ability of purified ydj1-151p to stimulate the ATPase activity of hsp70SSA1 was greatly diminished compared with the wild-type protein. Together, these data suggest that YDJ1p functions in polypeptide translocation in a conserved manner, probably acting at organelle membranes and in association with hsp70 proteins.