E2 Polypeptide Research Articles

Conformational Flexibility in the CD81-Binding Site of the Hepatitis C Virus Glycoprotein E2.

Numerous antibodies have been described that potently neutralize a broad range of hepatitis C virus (HCV) isolates and the majority of these antibodies target the binding site for the cellular receptor CD81 within the major HCV glycoprotein E2. A detailed understanding of the major antigenic determinants is crucial for the design of an efficient vaccine that elicits high levels of such antibodies. In the past 6 years, structural studies have shed additional light on the way the host’s humoral immune system recognizes neutralization epitopes within the HCV glycoproteins. One of the most striking findings from these studies is that the same segments of the E2 polypeptide chain induce antibodies targeting distinct antigen conformations. This was demonstrated by several crystal structures of identical polypeptide segments bound to different antibodies, highlighting an unanticipated intrinsic structural flexibility that allows binding of antibodies with distinct paratope shapes following an “induced-fit” mechanism. This unprecedented flexibility extends to the entire binding site for the cellular receptor CD81, underlining the importance of dynamic analyses to understand (1) the interplay between HCV and the humoral immune system and (2) the relevance of this structural flexibility for virus entry. This review summarizes the current understanding how neutralizing antibodies target structurally flexible epitopes. We focus on differences and common features of the reported structures and discuss the implications of the observed structural flexibility for the viral replication cycle, the full scope of the interplay between the virus and the host immune system and—most importantly—informed vaccine design.

Lettuce-produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination.

SummaryThe hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV‐related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium‐mediated transient expression technology. The wild‐type dimer (E1E2) and a variant without an N‐glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N‐glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell‐expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti‐HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N‐glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti‐HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low‐cost oral vaccine development.

Evaluation of chikungunya virus infection in children from India during 2009-2010: A cross sectional observational study.

Chikungunya virus, a small (about 60-70 nm diameter), spherical, enveloped, positive, single stranded RNA virus is transmitted by Aedes mosquitoes. After a short period of incubation (3-5 days) symptoms like fever with joint pains and others start appearing. After a gap of 20 years, this virus re-emerged during 2006-2008 in India causing a major outbreak of CHIKV in India. This study was conducted subsequent to the major outbreak in order to evaluate the proportion of chikungunya virus infection in children with suggestive symptoms at three geographical locations of India. Lineage of circulating strains and changes in the E1 structural polypeptide were also determined. Blood samples were collected (in Sodium citrate vacutainer tubes) during 1st June 2009 to 31st May 2010 from children (age 0 ≤ 18 years) suspected to have chikungunya infection, that is, those who presented with sudden onset of fever and/or joint pain, myalgia, and headache from three regions of India, All India Institute of Medical Sciences (AIIMS) in New Delhi, Karnataka Institute of Medical Sciences (KIMS) in Hubli and Sawai Mansingh Medical College (SMS) in Jaipur. Detection of CHIKV antibodies in all acute-phase patient plasma samples was done by IgM ELISA and for samples within ≤5 days of fever, a one-step RT-PCR was carried out on a block thermo-cycler targeting 294 bp region of E1 gene that codes for the viral envelope protein. Comparison of nucleotide and amino acid sequences from few positive samples of two regions was done with African S-27 reference strain using BioEdit. A phylogenetic tree was constructed using MEGA 6 by using the Maximum Likelihood method based on the Kimura 2-parameter model. Out of the 723 acute phase samples tested from three geographical locations of India, Chikungunya virus infection was detected in 249/723 (34.44%) subjects by either IgM Elisa (180/723) or RT-PCR (69/412). RT-PCR was employed in samples collected from children with ≤5 days of fever. Maximum positive cases were from KIMS center, Hubli. Seasonally, positivity varied with number of enrolled cases at KIMS and SMS. Joint pain was significantly associated with CHIKV positivity (P = 0.0156). Presence/absence of certain clinical features varied with age (P < 0.05). Sequence analysis revealed four amino acid changes. Phylogenetic analysis with partial sequences of E1 gene from KIMS (n = 12) and SMS (n = 5) showed that the study isolates clustered with Indian Ocean Lineage strains (IOL) of East, Central and South African (ECSA) type. Evaluation of chikungunya virus infection in children from India during 2009-2010 showed high proportion of CHIKV infection in Southern region of India compared to Northern region. The circulating CHIKV strains were of Indian Ocean Lineage (IOL) group within the East, Central, and South African (ECSA) genotype. However few amino acid changes were observed in E1 polypeptide with reference to African strain S-27 (AF369024). Further studies are needed to know the implications of these changes in vector-pathogen compatibility and host-pathogen interactivity. As a whole, this study highlighted the proportion of CHIKV cases, lineage of causative strain and evolutionary pattern of circulating strain in terms of amino acid changes in the structural protein.

Why are the 2-oxoacid dehydrogenase complexes so large? Generation of an active trimeric complex.

The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures.

Salt-Induced Changes in the Subunit Structure of theBacillus stearothermophilusLipoate Acetyltransferase

The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C(A)) and a quasi-stable complex (C(Q)). C(A) was larger than C(Q), but smaller than intact E2. C(A) and C(Q), were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2.

Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding

The E2 proteins of several papillomaviruses link the viral genome to mitotic chromosomes to ensure retention and the efficient partitioning of genomes into daughter cells following cell division. Bovine papillomavirus type 1 E2 binds to chromosomes in a complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking or mutated in this domain do not interact with Brd4. However, we found that the C-terminal DNA binding/dimerization domain of E2 is also required for efficient binding to Brd4. Mutations that eliminated the DNA binding function of E2 had no effect on the ability of E2 to interact with Brd4, but an E2 protein with a mutation that disrupted C-terminal dimerization bound Brd4 with greatly reduced efficiency. Furthermore, E2 proteins in which the C-terminal domains were replaced with the dimeric DNA binding domain of EBNA-1 or Gal4 bound efficiently to the Brd4 protein, but the replacement of the E2 C-terminal domain with a monomeric red fluorescent protein did not rescue efficient Brd4 binding. Thus, E2 bound to Brd4 most efficiently as a dimer. To prove this finding further, the E2 DNA binding domain was replaced with an FKBP12-derived domain in which dimerization was regulated by a bivalent ligand. This fusion protein bound Brd4 efficiently only in the presence of the ligand, confirming that a dimer of E2 was required. Correspondingly, E2 proteins that could dimerize were able to bind to mitotic chromosomes much more efficiently than monomeric E2 polypeptides.

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.

Multiple Display of Peptides and Proteins on a Macromolecular Scaffold Derived from a Multienzyme Complex

The acyltransferase components (E2) from the family of 2-oxo acid dehydrogenase multienzyme complexes form large protein scaffolds, to which multiple copies of peripheral enzymes bind tightly but non-covalently. Sixty copies of the E2 polypeptide from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus assemble to form a pentagonal dodecahedral scaffold with icosahedral symmetry. This protein scaffold can be modified to present foreign peptides and proteins on its surface. We show that it is possible to display two epitopes (MAL1 and MAL2) from the circumsporozoite CS proteins of Plasmodium falciparum and Plasmodium berghei, respectively , and a green fluorescent protein (EGFP), on the E2 surface. Immunization with an E2 scaffold displaying the MAL1 epitope elicited MAL1-specific antibodies in rabbits. EGFP (25 kDa) displayed as an N-terminal fusion in each of the 60 copies of the E2 chain folded into its active form, as judged by its fluorescence and detection in localized foci in Escherichia coli cells in vivo. Simultaneous display of a hexahistidine affinity tag, the MAL1 epitope and the green fluorescent protein, all on the same E2 scaffold, could be achieved by reversible denaturation and reassembly of mixtures of appropriately modified E2 chains. This new methodology offers several important advantages over other current display technologies, not least in the size of insert that can be accommodated and the multiplicity of display that can be achieved.

Protein-protein interaction revealed by NMR T2 relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

T 2 relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase component, a heterotetramer E1(α 2β 2), is responsible for the first committed and irreversible catalytic step. The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain. The interactions between the free lipoyl domain (9 kDa) and free E1α (41 kDa), E1β (35 kDa) and intact E1α 2β 2 (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uniformly 15N-labeled lipoyl domain and unlabeled E1 components. Major contact points on the lipoyl domain were identified from changes in the backbone 15N spin-spin relaxation time in the presence and absence of E1(α 2β 2) or its individual E1α or E1β components. Although the E1α subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1α and E1β. A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1. None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain. This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine β-turn region, as indicated by chemical shift data. Interaction with E1 and subsequent reductive acetylation are not necessarily coupled.

Effect of Saccharomyces boulardii on cAMP- and Ca2+ -dependent Cl- secretion in T84 cells.

Several reports have confirmed that the cooperative interaction between cAMP- and Ca2+-mediated transduction pathways may contribute to the stimulatory or inhibitory regulation of Cl- secretion in intestinal epithelium. Saccharomyces boulardii has been shown to inhibit cholera toxin-induced secretion in rat jejunum. We have identified a 120-kDa protein in medium conditioned by Saccharomyces boulardii that reduces cholera toxin-induced cAMP in intestinal cells. The present study evaluated the effect of medium conditioned by Saccharomyces boulardii on cAMP- and Ca2+-mediated Cl- secretion in T84 cells. Experiments performed with cAMP agonists revealed that 1 hr of preincubation of cells with medium conditioned by Saccharomyces boulardii was necessary to elicit a 40-50% reduction in receptor (cholera toxin, prostaglandin E2, and vasoactive intestinal polypeptide) and nonreceptor (forskolin) mediated cAMP synthesis and 125I efflux. Secretion induced by carbachol was inhibited when cells were pretreated for 1 hr with medium conditioned by Saccharomyces boulardii despite the absence of inhibition of Ins (1,4,5)P3. From this study we conclude that Saccharomyces boulardii exerts an inhibitory effect in vitro on Cl- secretion mediated through both cAMP- and Ca2+-mediated signaling pathways.

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro.

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.

Transient viral DNA replication and repression of viral transcription are supported by the C-terminal domain of the bovine papillomavirus type 1 E1 protein.

The bovine papillomavirus type 1 E1 protein is important for viral DNA replication and transcriptional repression. It has been proposed that the full-length E1 protein consists of a small N-terminal and a larger C-terminal domain. In this study, it is shown that an E1 polypeptide containing residues 132 to 605 (which represents the C-terminal domain) is able to support transient viral DNA replication, although at a level lower than that supported by the wild-type protein. This domain can also repress E2-mediated transactivation from the P89 promoter as well as the wild-type E1 protein can.

Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells

The structural part of the hepatitis C virus (HCV) genome encodes a capsid protein, C and two envelope glycoproteins, E1 and E2, released from the virus polyprotein precursor by signalase(s) cleavage(s). The processing of E1 was investigated by infecting simian cells with recombinant vaccinia viruses expressing parts of the HCV structural proteins. When the predicted E1 sequence was expressed alone (amino acid residues 174-370 of the polyprotein) or with the capsid protein gene (residues 1-370). it showed an apparent molecular mass of 35 kDa as measured by SDS-PAGE analysis. However, when E1 was expressed as part of a truncated C-E1-truncated E2 polypeptide (residues 132-383), the processed E1 product had the expected apparent molecular mass of 31 kDa, suggesting that flanking sequences are necessary for the generation of the mature 31 kDa El form. The N-terminal sequence of the two E1 forms was found to be the same. Analysis of the glycosylation pattern showed that, in both species, only four of the five potential N-linked glycosylation sites were recognized, indicating that glycosylation was not involved in the molecular mass difference. We showed that expression of E1 with or without the hydrophobic stretch of amino acids residues 371-383, defined as the E2 signal sequence, may be responsible for the difference in electrophoretic mobility of the two E1 species. In vitro translation assays and site-directed mutagenesis experiments suggest that this sequence remains part of the 31 kDa E1 mature protein.

Co-operative interaction between the initiator E1 and the transcriptional activator E2 is required for replicator specific DNA replication of bovine papillomavirus in vivo and in vitro.

The E1 polypeptide from bovine papillomavirus binds to the origin of replication (ori) and possesses the activities attributed to initiator proteins. E1 is also the only viral protein required for replication in a cell-free replication system. Replication in vivo, however, absolutely requires in addition the viral transcription factor E2. We demonstrate that the basis for this distinction between in vitro and in vivo requirements is the limited sequence specificity of the E1 protein. E1 and E2, which bind the ori individually with low sequence specificity, together bind with greatly increased sequence specificity. This combinatorial effect provides a function for the involvement of transcriptional activation domains in replication and suggests common mechanisms of action for transcription factors in both transcription and replication. It also provides a possible explanation for the differential specificity that is observed for auxiliary transcription factors in vivo.

Regulation of the Activin-Binding Protein Follistatin in Cultured Human Luteinizing Granulosa Cells: Characterization of the Effects of Follicle Stimulating Hormone, Prostaglandin E2, and Different Growth Factors1

Regulation of the activin-binding protein follistatin (FS) by recombinant human (rh) FSH, prostaglandin E2 (PGE2), and several polypeptide growth factors was examined in cultures of human granulosa-luteal (GL) cells obtained from in-vitro fertilization patients at oocyte retrieval. Northern and dot blot hybridization analyses demonstrated that both rhFSH and PGE2 caused stimulatory effects on FS mRNA levels in a culture stage-, time-, and concentration-dependent manner. An 8-h stimulation with rhFSH (100 ng/ml) significantly increased FS mRNA levels on Days 5 and 7 of culture and PGE2 (10(-6)M) on Days 2, 4, and 7. The stimulatory effect of rhFSH and PGE2 on FS mRNA levels were rapid and transient. Maximal inductions occurred 8 h after stimulation, whereas weak or no stimulatory effects were seen at 24 or 48 h. PGF2 alpha did not affect FS mRNA levels at any time point studied. Treatment of the cells with the protein synthesis inhibitor cycloheximide prior to rhFSH stimulation did not inhibit the rapid induction of FS mRNAs, but it prevented the decline at 24 h. Both rhFSH and PGE2 clearly also increased the levels of secreted FS proteins are detected by immunoprecipitation studies with a specific antibody. The effects of the polypeptide growth factors epidermal growth factor (EGF); transforming growth factor beta 1 (TGF beta 1), and activin A on FS mRNA levels were also examined. TGF beta 1 and activin A had no effect on basal FS expression at any concentration or time point studied. An 8-h stimulation with EGF increased FS mRNA levels, but the effect was weaker than those caused by rhFSH and PGE2. We conclude that rhFSH and PGE2 induce FS mRNA and protein in human cultured GL cells. EGF is able to induce FS mRNA to a lesser extent than are rhFSH and PGE2, whereas PGF2 alpha, TGF beta 1, and activin A do not affect basal FS mRNA levels in human cultured GL cells. This study together with our previous report on the stimulatory effect of hCG on FS levels suggest that in the luteal phase of the human menstrual cycle, FS expression in granulosa cells is likely to be positively controlled by luteotropic factors such as gonadotropins and PGE2. Consequently, elevated FS levels may support the survival of the human CL since FS is known to prevent the antisteroidogenic effects of activin in human GL cells.

Binding properties of the components of branched-chain 2-oxo acid dehydrogenase complex on ELISA.

Binding characteristics among three catalytic components of rat liver branched-chain 2-oxo acid dehydrogenase complex (BCKADH) were investigated by ELISA. Dihydrolipoamide dehydrogenase (E3) was bound to solid-phase dihydrolipoamide acyltransferase (E2). The binding curve was hyperbolic giving a calculated half-maximal binding concentration of 167 ng/ml for E3. Specificity of the binding of E3 to E2 was certified by a competition experiment measuring binding of biotin-labeled E3 in the presence of unlabeled E3. The decarboxylase component (E1), which is the other catalytic component of the complex, prevented the E3 binding to E2. The specific binding between E2 and E3 was verified in the opposite direction with immobilized E3. E2 also bound to solid-phase E1 in a specific manner, and addition of E3 prevented the E2 binding to E1. No binding between E1 and E3 was observed. Thus, E1 and E3 prevented each other's binding to E2, suggesting that E1 and E3 may recognize overlapped binding sites on the E2 polypeptide or that they may, at least in part, sterically interact with each other on their binding to E2. The reconstitution experiment of the complex also supported such a mutually exclusive binding of E1 and E3 to the E2 core.

Analysis of Hepatitis C Virus Capsid, E1, and E2/NS1 Proteins Expressed in Insect Cells

The baculovirus/insect cell expression system was used to express the capsid protein and glycoproteins (e1 and e2/NS1) of the hepatitis C virus (HCV). Each polypeptide domain was expressed individually using two different constructs varying at their carboxy termini in order to retain or delete hydrophobic domains that may be involved in membrane association. The capsid proteins were transported to the nucleus where they formed a single large crystal-like inclusion. The capsid proteins were phosphorylated in insect cells. The e1 and e2 polypeptides were present in both the soluble and insoluble cellular fractions. Deletion of a hydrophobic domain in the carboxy terminus of e2 resulted in the polypeptide becoming soluble but not secreted. Deletion of the carboxy terminus of e1 had no effect on solubility. Both e1 and e2 were glycosylated, with variable glycosylation of e1 giving rise to a series of polypeptides varying in apparent molecular weight Co-infection of insect cells with viruses expressing e1 and e2 resulted in a complex that permitted the coimmunoprecipitation of e1 with antibodies to e2 end vice versa. Immunofluorescence staining of insect cells expressing e1 and e2 indicated that reactivity to e2 was more prevalent in anti-HCV positive human sera.

Cooperative DNA binding of the bovine papillomavirus E2 transcriptional activator is antagonized by truncated E2 polypeptides

Cooperative DNA binding of the bovine papillomavirus type 1 (BPV-1) E2 transcriptional activator (E2-TA) is thought to play a role in the transcriptional synergism of multiple E2-responsive DNA elements (J. Ham, N. Dostatni, J.-M. Gauthier, and M. Yaniv, Trends Biochem. Sci. 16:440-444, 1991). Binding-equilibrium considerations show that such involvement is unlikely, thereby suggesting that the E2-TA cooperative capacity may have evolved to play other, different roles. The role of cooperative interactions in the antagonistic activity of BPV-1-positive and BPV-1-negative E2 regulatory proteins was investigated by an in vitro quantitative gel shift assay. Viral repressor E2-TR, a truncated peptide encompassing the activator DNA-binding domain, possesses a small but measurable cooperative capacity. Furthermore, the minimal E2 DNA-binding domain interacts with the activator in a positive, heterocooperative manner. As a result, the in vitro competition of full-length and truncated E2 peptides appears to be (macroscopically) noncooperative. This heterocooperative effect is probably dominant in latently infected G0-G1 cells, in which repressor E2-TR is 10- to 20-fold more abundant than the activator. The data are discussed considering the possible role of homo- and heterocooperative DNA binding in E2-conditional gene expression.

The bovine papillomavirus origin of replication requires a binding site for the E2 transcriptional activator.

The bovine papillomavirus type I transcriptional activator E2 is essential for replication of bovine papillomavirus DNA, yet most of the high-affinity binding sites for E2 are dispensable. Here we demonstrate an absolute requirement for a binding site for the E2 polypeptide as a cis-acting replication element, establishing that site-specific binding of E2 to the origin is a prerequisite for bovine papillomavirus replication in vivo. The position and distance of the E2 binding site relative to the other origin of replication components are flexible, but function at a distance requires high-affinity E2 binding sites. Thus, low-affinity binding sites function only when located close to the origin of replication, while activity at greater distances requires multimerized high-affinity E2 binding sites. The requirement for E2, although different in some respects, shows distinct similarities to what has been termed replication enhancers and may provide insight into the function of this class of DNA replication element.

Recognition of synthetic peptides with sequences of rubella virus E1 polypeptide by antibodies and T lymphocytes.

Antigenicity of rubella virus E1 polypeptide was analyzed using synthetic peptides with predicted amino acid sequences. Overlapping solid-phase bound peptides were used to define antibody binding domains and a panel of free peptides to study T-cell responsiveness. Several antibody-binding areas including those earlier described to contain major neutralizing epitopes were recognized by human sera positive for rubella antibodies. T-cell lines specific for rubella virus were established from 14 rubella immune subjects. All cell lines responded to rubella virion-derived antigen but only eight (57%) responded to one or more of the synthetic peptides. Individual patterns of peptide recognition were found but peptide 8 representing amino acids 402-422 was most often stimulatory to T-cells lines, either alone (3 subjects) or in combination with peptide 3 (amino acids 245-269) or 3 and 4 (amino acids 269-287). The response was HLA restricted but no single DR specificity for this restriction was identified.