The domain I-domain III linker plays an important role in the fusogenic conformational change of the alphavirus membrane fusion protein

The alphavirus Semliki Forest virus (SFV) infects cells through a low-pH-dependent membrane fusion reaction mediated by the virus fusion protein E1. Acidic pH initiates a series of E1 conformational changes that culminate in membrane fusion and include dissociation of the E1/E2 heterodimer, insertion of the E1 fusion loop into the target membrane, and refolding of E1 to a stable trimeric hairpin conformation. A highly conserved histidine (H3) on the E1 protein was previously shown to promote low-pH-dependent E1 refolding. An SFV mutant with an alanine substitution at this position (H3A) has a lower pH threshold and reduced efficiency of virus fusion and E1 trimer formation than wild-type SFV. Here we addressed the mechanism by which H3 promotes E1 refolding and membrane fusion. We identified E1 mutations that rescue the H3A defect. These revertants implicated a network of interactions that connect the domain I-domain III (DI-DIII) linker region with the E1 core trimer, including H3. In support of the importance of these interactions, mutation of residues in the network resulted in more acidic pH thresholds and reduced efficiencies of membrane fusion. In vitro studies of truncated E1 proteins demonstrated that the DI-DIII linker was required for production of a stable E1 core trimer on target membranes. Together, our results suggest a critical and previously unidentified role for the DI-DIII linker region during the low-pH-dependent refolding of E1 that drives membrane fusion.     

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.     

The intermediate domain defines broad nucleotide selectivity for protein folding in Chlamydophila GroEL1

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln(400) and Ile(404) in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.     

Interaction of heterochromatin protein 2 with HP1 defines a novel HP1-binding domain

Heterochromatin Protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster localized principally in the pericentric heterochromatin, telomeres, and fourth chromosome, all regions associated with HP1. Mutations in HP2 can suppress position effect variegation, indicating a role in gene silencing and heterochromatin formation [Shaffer, C. D. et al. (2002) Proc. Natl. Acad. Sci.U.S.A. 99, 14332-14337]. In vitro coimmunoprecipitation experiments with various peptides from HP2 have identified a single HP1-binding domain. Conserved domains in HP2, including those within the HP1-binding region, have been identified by recovering and sequencing Su(var)2-HP2 from D. willistoni and D. virilis, as well as examining available sequence data from D. pseudoobscura. A PxVxL motif, shown to be an HP1-binding domain in many HP1-interacting proteins, is observed but is not well-conserved in location and sequence and does not mediate HP2 binding to HP1. The sole HP1-binding domain is composed of two conserved regions of 12 and 16 amino acids separated by 19 amino acids. Site-directed mutagenesis within the two conserved regions has shown that the 16 amino acid domain is critical for HP1 binding. This constitutes a novel domain for HP1 interaction, providing a critical link for heterochromatin formation in Drosophila.     

The dynamic envelope of a fusion class II virus. E3 domain of glycoprotein E2 precursor in Semliki Forest virus provides a unique contact with the fusion protein E1

In alphaviruses, here represented by Semliki Forest virus, infection requires an acid-responsive spike configuration to facilitate membrane fusion. The creation of this relies on the chaperone function of glycoprotein E2 precursor (p62) and its maturation cleavage into the small external E3 and the membrane-anchored E2 glycoproteins. To reveal how the E3 domain of p62 exerts its control of spike functions, we determine the structure of a p62 cleavage-impaired mutant virus particle (SQL) by electron cryomicroscopy. A comparison with the earlier solved wild type virus structure reveals that the E3 domain of p62(SQL) forms a bulky side protrusion in the spike head region. This establishes a gripper over part of domain II of the fusion protein, with a cotter-like connection downward to a hydrophobic cluster in its central beta-sheet. This finding reevaluates the role of the precursor from being only a provider of a shield over the fusion loop to a structural playmate in formation of the fusogenic architecture.     

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.     

Retinol binding protein-albumin domain III fusion protein deactivates hepatic stellate cells

Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis.     

Interactions between the transmembrane segments of the alphavirus E1 and E2 proteins play a role in virus budding and fusion

The alphavirus envelope is built by heterodimers of the membrane proteins E1 and E2. The complex is formed as a p62E1 precursor in the endoplasmic reticulum. During transit to the plasma membrane (PM), it is cleaved into mature E1-E2 heterodimers, which are oligomerized into trimeric complexes, so-called spikes that bind both to each other and, at the PM, also to nucleocapsid (NC) structures under the membrane. These interactions drive the budding of new virus particles from the cell surface. The virus enters new cells by a low-pH-induced membrane fusion event where both inter- and intraheterodimer interactions are reorganized to establish a fusion-active membrane protein complex. There are no intact heterodimers left after fusion activation; instead, an E1 homotrimer remains in the cellular (or viral) membrane. We analyzed whether these transitions depend on interactions in the transmembrane (TM) region of the heterodimer. We observed a pattern of conserved glycines in the TM region of E1 and made two mutants where either the glycines only (SFV/E1(4L)) or the whole segment around the glycines (SFV/E1(11L)) was replaced by leucines. We found that both mutations decreased the stability of the heterodimer and increased the formation of the E1 homotrimer at a suboptimal fusion pH, while the fusion activity was decreased. This suggested that TM interactions play a role in virus assembly and entry and that anomalous or uncoordinated protein reorganizations take place in the mutants. In addition, the SFV/E1(11L) mutant was completely deficient in budding, which may reflect an inability to form multivalent NC interactions at the PM.     

Interactions of the cytoplasmic domain of Sindbis virus E2 with nucleocapsid cores promote alphavirus budding

Alphavirus budding from the plasma membrane occurs through the specific interaction of the nucleocapsid core with the cytoplasmic domain of the E2 glycoprotein (cdE2). Structural studies of the Sindbis virus capsid protein (CP) have suggested that these critical interactions are mediated by the binding of cdE2 into a hydrophobic pocket in the CP. Several molecular genetic studies have implicated amino acids Y400 and L402 in cdE2 as important for the budding of alphaviruses. In this study, we characterized the role of cdE2 residues in structural polyprotein processing, glycoprotein transport, and capsid interactions. Along with hydrophobic residues, charged residues in the N terminus of cdE2 were critical for the effective interaction of cores with cdE2, a process required for virus budding. Mutations in the C-terminal signal sequence region of cdE2 affected E2 protein transport to the plasma membrane, while nonbudding mutants that were defective in cdE2-CP interaction accumulated E2 on the plasma membrane. The interaction of cdE2 with cytoplasmic cores purified from infected cells and in vitro-assembled core-like particles suggests that cdE2 interacts with assembled cores to mediate budding. We hypothesize that these cdE2 interactions induce a change in the organization of the nucleocapsid core upon binding leading to particle budding and priming of the nucleocapsid cores for disassembly that is required for virus infection.     

Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

Background

Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored.

Methods

A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity.

Results

Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion.

Conclusions

Cells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.     

Mutational analysis of the beta-type platelet-derived growth factor receptor defines the site of interaction with the bovine papillomavirus type 1 E5 transforming protein.

The E5 polypeptide of bovine papillomavirus type 1 is a small membrane-bound protein which induces the transformation of immortalized fibroblasts, apparently via the formation of a ternary complex with the platelet-derived growth factor receptor (PDGFR) and the 16-kDa V-ATPase protein. This interaction seems to be mediated, at least in part, by their respective transmembrane domains. E5 also cooperates with transfected beta PDGFR to induce interleukin-3 (IL-3)-independent growth of a mouse myeloid precursor cell line (32D) which normally lacks expression of most known tyrosine kinase growth factor receptors. Cell proliferation induced by beta PDGFR and E5 is also highly specific, since the highly conserved alpha PDGFR and other related receptors did not physically or functionally interact with E5 in these cells. In the current study, analysis of chimeric alpha and beta PDGFRs confirmed that a short region encompassing the beta PDGFR transmembrane domain was sufficient for complex formation with E5, receptor autophosphorylation, and sustained proliferation of 32D cells in the absence of IL-3. Furthermore, a deletion mutant lacking the entire extracellular domain efficiently bound E5 and induced IL-3-independent growth. These data provide direct evidence that the interaction between E5 and the beta PDGFR involves amino acids 531 to 556 of the receptor transmembrane region and that this specific interaction is critical for activation of the PDGFR signaling complex.     

The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases

The Arabidopsis calcineurin B-like calcium sensor proteins (AtCBLs) interact with a group of serine-threonine protein kinases (AtCIPKs) in a calcium-dependent manner. Here we identify a 24 amino acid domain (NAF domain) unique to these kinases as being required and sufficient for interaction with all known AtCBLs. Mutation of conserved residues either abolished or significantly diminished the affinity of AtCIPK1 for AtCBL2. Comprehensive two-hybrid screens with various AtCBLs identified 15 CIPKs as potential targets of CBL proteins. Database analyses revealed additional kinases from Arabidopsis and other plant species harbouring the NAF interaction module. Several of these kinases have been implicated in various signalling pathways mediating responses to stress, hormones and environmental cues. Full-length CIPKs show preferential interaction with distinct CBLs in yeast and in vitro assays. Our findings suggest differential interaction affinity as one of the mechanisms generating the temporal and spatial specificity of calcium signals within plant cells and that different combinations of CBL-CIPK proteins contribute to the complex network that connects various extracellular signals to defined cellular responses.     

A specific domain of the Chikungunya virus E2 protein regulates particle formation in human cells: implications for alphavirus vaccine design

Virus-like particles (VLPs) can be generated from Chikungunya virus (CHIKV), but different strains yield variable quantities of particles. Here, we define the genetic basis for these differences and show that amino acid 234 in E2 substantially affects VLP production. This site is located within the acid-sensitive region (ASR) known to initiate a major conformational change in E1/E2. Selected other mutations in the ASR, or changes in pH, also increased VLP yield. These results demonstrate that the ASR of E2 plays an important role in regulating particle generation.     

Activation of the alphavirus spike protein is suppressed by bound E3

Alphaviruses are taken up into the endosome of the cell, where acidic conditions activate the spikes for membrane fusion. This involves dissociation of the three E2-E1 heterodimers of the spike and E1 interaction with the target membrane as a homotrimer. The biosynthesis of the heterodimer as a pH-resistant p62-E1 precursor appeared to solve the problem of premature activation in the late and acidic parts of the biosynthetic transport pathway in the cell. However, p62 cleavage into E2 and E3 by furin occurs before the spike has left the acidic compartments, accentuating the problem. In this work, we used a furin-resistant Semliki Forest virus (SFV) mutant, SFV(SQL), to study the role of E3 in spike activation. The cleavage was reconstituted with proteinase K in vitro using free virus or spikes on SFV(SQL)-infected cells. We found that E3 association with the spikes was pH dependent, requiring acidic conditions, and that the bound E3 suppressed spike activation. This was shown in an in vitro spike activation assay monitoring E1 trimer formation with liposomes and a fusion-from-within assay with infected cells. Furthermore, the wild type, SFV(wt), was found to bind significant amounts of E3, especially if produced in dense cultures, which lowered the pH of the culture medium. This E3 also suppressed spike activation. The results suggest that furin-cleaved E3 continues to protect the spike from premature activation in acidic compartments of the cell and that its release in the neutral extracellular space primes the spike for low-pH activation.     

Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli

Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1-106; S1(106)) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1(106) affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α(2)) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions.     

The immunoglobulin-like domain is involved in interaction of Neuregulin1 with ErbB

Neuregulin1 (NRG1) is a growth factor that signals through the interaction of the epidermal growth factor (EGF)-like domain with ErbB receptors. An immunoglobulin (Ig)-like domain is contained together with EGF-like domain in the ectodomain of some isoforms generated by alternative splicing, but its role in NRG1 signaling remained unclear. In the present study, we identified a novel isoform of NRG1 containing an Ig-like domain conserved among species from adult Xenopus laevis, which is predominantly expressed in the testis and brain. We generated recombinant proteins for the whole ectodomain and EGF-like domain alone of the isoform to compare their effects on cell proliferation, and phosphorylation of and their association with ErbB receptor, demonstrating that the ectodomain had approximately 10(3)-fold higher abilities than the EGF-like domain. Therefore, the Ig-like domain is probably essential for efficient interaction of an EGF-like domain with ErbB receptors.     

The colicin E3 outer membrane translocon: immunity protein release allows interaction of the cytotoxic domain with OmpF porin

The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.     

Self-association and mapping of the interaction domain of hepatitis E virus ORF3 protein

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information on the basic biology of the virus exists. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains. The C-terminal half of pORF3 is rich in proline residues and contains a putative src homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site. In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro. The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.