High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor BI

Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3-5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum.     

Multiple colonizations from Madagascar and converged acquisition of dioecy in the Mascarene Dombeyoideae (Malvaceae) as inferred from chloroplast and nuclear DNA sequence analyses

Background and Aims

In the Mascarenes, a young oceanic archipelago composed of three main islands, the Dombeyoideae (Malvaceae) have diversified extensively with a high endemism rate. With the exception of the genus Trochetia, Mascarene Dombeyoideae are described as dioecious whereas Malagasy and African species are considered to be monocline, species with individuals bearing hermaphrodite/perfect flowers. In this study, the phylogenetic relationships were reconstructed to clarify the taxonomy, understand the phylogeographic pattern of relationships and infer the evolution of the breeding systems for the Mascarenes Dombeyoideae.

Methods

Parsimony and Bayesian analysis of four DNA markers (ITS, rpl16 intron and two intergenic spacers trnQ-rsp16 and psbM-trnD) was used. The molecular matrix comprised 2985 characters and 48 taxa. The Bayesian phylogeny was used to infer phylogeographical hypotheses and the evolution of breeding systems.

Key Results

Parsimony and Bayesian trees produced similar results. The Dombeyoideae from the Mascarenes are polyphyletic and distributed among four clades. Species of Dombeya, Trochetia and Ruizia are nested in the same clade, which implies the paraphyly of Dombeya. Additionally, it is shown that each of the four clades has an independent Malagasy origin. Two adaptive radiation events have occurred within two endemic lineages of the Mascarenes. The polyphyly of the Mascarene Dombeyoideae suggests at least three independent acquisitions of dioecy.

Conclusions

This molecular phylogeny highlights the taxonomic issues within the Dombeyoideae. Indeed, the limits and distinctions of the genera Dombeya, Trochetia and Ruizia should be reconsidered. The close phylogeographic relationships between the flora of the Mascarenes and Madagascar are confirmed. Despite their independent origins and a distinct evolutionary history, each endemic clade has developed a different breeding systems (dioecy) compared with the Malagasy Dombeyoideae. Sex separation appears as an evolutionary convergence and may be the consequence of selective pressures particular to insular environments.     

Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus that causes serious liver diseases in humans (31). More than 170 million people worldwide are seropositive for HCV and at risk for developing cirrhosis and hepatocellular carcinoma (50). HCV is a small, enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (31). Its genome encodes a single polyprotein precursor of about 3,000-amino-acid residues that is cleaved co- and posttranslationally by cellular and viral proteases to yield at least 10 mature products (31). The two envelope glycoproteins, E1 and E2, are released from the polyprotein by signal peptidase cleavages. These two proteins assemble as noncovalent heterodimers, which are retained mainly in the endoplasmic reticulum (ER) (36), and they are found as large disulfide-linked oligomers on the surfaces of HCV particles (46). HCV glycoproteins are involved in the entry process, and since they are present on the surfaces of viral particles, these proteins are the targets of neutralizing antibodies (4, 21).E1 and E2 generally contain 4 and 11 N-glycosylation sites, respectively, all of which have been shown to be modified by glycans (19). Despite variability in HCV envelope glycoprotein sequences, the four glycosylation sites of E1 and nine of E2 are highly conserved, suggesting that the glycans associated with these proteins play an essential role in the HCV life cycle (22). Using retroviral particles pseudotyped with genotype 1a (H strain) HCV envelope glycoproteins (HCVpp), recent studies have determined the potential roles played by these glycans in protein folding, HCV entry, and protection against neutralization (14, 19, 22). Indeed, the lack of glycan E1N1, E1N4, E2N8, or E2N10 strongly affects the incorporation of HCV glycoproteins into HCVpp, suggesting that these glycans are important for correct protein folding (19). Furthermore, mutation of glycosylation sites E2N2 or E2N4 alters HCVpp infectivity despite normal incorporation into pseudotyped particles, suggesting a role for the corresponding glycans in viral entry, at least in this model system (19). Finally, glycans at positions E2N1, E2N6, and E2N11 were shown to reduce the sensitivity of HCVpp to antibody neutralization as well as access of the CD81 coreceptor to its binding site on E2, suggesting that glycans also contribute to HCV evasion of the humoral immune response (14, 22).It has recently been proposed that targeting glycans could be a promising approach to inhibiting viral infection (1). Indeed, HCV, as well as several other viruses with highly glycosylated envelope proteins, can be inhibited by carbohydrate binding agents such as cyanovirin-N and pradimicin A (1, 7, 23). Furthermore, resistance against drugs that target glycans is likely to develop and will probably result in mutations at some glycosylation sites (3, 52). However, since glycans associated with viral envelope proteins play an important role in the viral life cycle, adaptation of viruses to the selective pressure of carbohydrate-binding agents will most likely come at a replicative cost to the virus (2).Although the role of HCV glycans has been studied using mutant recombinant HCV envelope glycoproteins incorporated into HCVpp, these particles do not recapitulate all the functions of HCV envelope proteins. Cell culture-derived virus (HCVcc) (32, 49, 55) assembles in an ER-derived compartment in association with very low density lipoproteins (17, 26), whereas HCVpp are assembled in a post-Golgi compartment and are not associated with lipoproteins (44). Importantly, this leads to differences between HCVpp and HCVcc in the oligomerization of the envelope glycoproteins (46). It is also important to note that the carbohydrate composition of viral glycoproteins can differ when the same virus is grown in different cell lines (13). Thus, HCVpp that are produced in 293T cells are not the most appropriate model for glycosylation studies, since HCV tropism is restricted to the liver. Furthermore, differences in envelope protein glycosylation have been observed between HCVpp and HCVcc particles (46). Differences in some HCV envelope protein functions were also observed when the HCVpp and HCVcc systems were compared (28, 29, 42, 43). The development of the HCVcc system provides, therefore, the opportunity to characterize the role of E1/E2-associated glycans in the context of authentic infectious virions. Here, we analyzed the role of E1/E2 glycans by introducing point mutations at N-linked glycosylation sites in the context of the HCVcc system. The effects of these mutations on virus replication, particle secretion, infectivity, and sensitivity to neutralizing antibodies were investigated. Our results demonstrate that several glycans play an important role in HCVcc assembly and/or infectivity and reduce access of neutralizing antibodies to their epitopes.     

Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor

Several cell surface molecules have been proposed as receptor candidates, mediating cell entry of hepatitis C virus (HCV) on the basis of their physical association with virions or with soluble HCV E2 glycoproteins. However, due to the lack of infectious HCV particles, evidence that these receptor candidates support infection was missing. Using our recently described infectious HCV pseudotype particles (HCVpp) that display functional E1E2 glycoprotein complexes, here we show that HCV is a pH-dependent virus, implying that its receptor component(s) mediate virion internalization by endocytosis. Expression of the CD81 tetraspanin in non-permissive CD81-negative hepato-carcinoma cells was sufficient to restore susceptibility to HCVpp infection, confirming its critical role as a cell attachment factor. As a cell surface molecule likely to mediate endosomal trafficking, we demonstrate that the human scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein-internalization molecule that we previously proposed as a novel HCV receptor candidate due to its affinity with E2 glycoproteins, is required for infection of CD81-expressing hepatic cells. By receptor competition assays, we found that SR-B1 antibodies that blocked binding of soluble E2 could prevent HCVpp infectivity. Furthermore, we establish that the hyper-variable region 1 of the HCV E2 glycoprotein is a critical determinant mediating entry in SR-B1-positive cells. Finally, by correlating expression of HCV receptors and infectivity, we suggest that, besides CD81 and SR-B1, additional hepatocyte-specific co-factor(s) are necessary for HCV entry.     

Molecular systematics of the fern genus Hymenophyllum s.l. (Hymenophyllaceae) based on chloroplastic coding and noncoding regions

We investigated the phylogenetic relationships of the filmy fern genus Hymenophyllum s.l. using the rbcL and rps4 genes and the intergenic spacer rps4-trnS. Because of variation in length of the noncoding marker, we tested and compared three methods for integrating indels. They proved to be useful for estimating a phylogeny of the genus. The rps4-trnS marker, with coded indels integrated, produced better resolution than analysis of either rps4 or rbcL, and combining the three data sets allowed us to obtain a well resolved and strongly supported topology. We interpret our data as showing support for the classical bigeneric system for the family, and call into question several classifications proposed in the past century. The segregate genera Cardiomanes, Hymenoglossum, Serpyllopsis, and Rosenstockia are embedded within Hymenophyllum s.l. Although the deepest relationships within the genus remain uncertain, two subgenera described by Morton do have some support: (1) Sphaerocionium, in which the problematic section Microtrichomanes is embedded; and (2) a diverse Hymenophyllum, including species that were placed originally in Serpyllopsis, Rosenstockia, Hemicyatheon, and Craspedophyllum by Copeland. Subgenus Mecodium appears to be polyphyletic; nevertheless, a subgroup within Mecodium is strongly supported. Several unexpected associations gain support from cytological data and certain morphological characters not previously used to distinguish species groups within Hymenophyllum s.l.     

Molecular phylogeny of the fern genus Elaphoglossum (Elaphoglossaceae) based on chloroplast non-coding DNA sequences: contributions of species from the Indian Ocean area

We performed a phylogenetic analysis of the fern genus Elaphoglossum using two non-coding chloroplast spacers: trnL-trnF and rps4-trnS. The sampling includes 123 species, of which 80 have not been previously sequenced, and for the first time includes species from Africa and the Indian Ocean area. The results of this expanded study largely agree with an earlier molecular study based on a smaller group of neotropical species and with the morphology-based classification of Mickel and Atehortua. We found, however, that some infrageneric groups such as section Elaphoglossum are not monophyletic. Besides section Elaphoglossum pro parte, we recognize six sections: two new monospecific, unnamed sections, and the previously established sections Lepidoglossa, Squamipedia, Amygdalifolia, and "Subulate-scaled clade." We divide the subulate-scaled clade into subsection Setosa (hydathodes present) and Polytrichia (hydathodes absent), and section Elaphoglossum is divided into subsections Platyglossa and Pachyglossa, two groups that do not appear to be supported by any single morphological character. In general, however, the main clades are supported by morphology. Finally, we discuss the species of the Indian Ocean region and their affinities with the neotropical ones. Out of the 11 species pairs postulated by Moran and Smith on the basis of morphology, two are well supported (E. eximium-E. aubertii; E. piloselloides-E. spatulatum) and three are not supported (E. ciliatum-E. humbertii; E. muscosum-E. poolii; E. paleaceum-E. deckenii), and two remain unresolved (E. erinaceum-E. hybridum; E. glabellum-E. acrostichoides) because our molecular markers were not variable enough. Four species pairs could not be tested because specimens were lacking. Unsupported species pairs are best interpreted as morphological convergences. Two additional species pairs are proposed: E. cuspidatum-E. succisaefolium; E. doanense-E. hornei. Placement of the species from the Indian Ocean suggests that at least 13 long-distance dispersal events occurred between the Neotropics and the Indian Ocean-Africa.     

Incomplete humoral immunity against hepatitis C virus is linked with distinct recognition of putative multiple receptors by E2 envelope glycoprotein

Little is known about the role of the humoral immune response to hepatitis C virus (HCV). This study provides molecular evidence for the mechanism by which neutralizing Abs from the sera of chronic HCV patients have lower inhibitory activities against the binding of HCV E2 envelope protein to human hepatoma cell lines than to a lymphoma cell line. E2 binds to several putative receptors, specifically human CD81; human scavenger receptor, class B, type 1; and heparan sulfate. We have shown that E2 binds to target cells via these receptors in a noncompetitive manner. Thus, incomplete inhibition of one of the receptors leads to only a partial E2 blockade and, possibly, evasion of the host immune response. We demonstrated that the difference in and reduction of inhibition was closely related to impaired blockade of E2 binding to scavenger receptor, class B, type 1, and heparan sulfate. We have also shown that soluble E2 protein binds to multiple soluble receptors via separate binding domains on E2, providing further evidence for the distinct recognition of multiple cellular receptors by E2. This report suggests a novel finding that biased humoral immune responses to HCV E2 might provide an alternative mechanism for viral escape without the involvement of mutation. Additionally, our data give crucial consideration to the development of HCV vaccines that stimulate protective humoral immune responses.     

Characterization of functional hepatitis C virus envelope glycoproteins

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.     

Modulation of 1alpha,25-dihydroxyvitamin D3-membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1alpha,25-(OH)2D3

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.