Identification of GRP 78 (BiP) as a liver cell expressed receptor element for dengue virus serotype 2

Summary. This study sought to identify receptor elements for dengue virus serotype 2 on human liver cells (HepG2) using the viral overlay protein binding assay (VOPBA) technique and Mass Spectrometry fingerprinting. A single major and several minor virus binding bands were observed, and mass spectrometry identified a candidate binding protein for the major binding band as GRP 78 (BiP). GRP78 expression on the cell surface was confirmed, and antibodies directed against both the N and C-terminus of GRP 78 (BiP) altered both the binding of the virus to the cell surface as well as the infectivity profile of HepG2 cells in response to dengue serotype 2 infection. GRP 78 (BiP), which has previously been identified as a co-receptor protein for coxsackievirus A9, is the first non-Fc receptor protein identified for the dengue virus, although GRP78 probably functions as part of a receptor complex.     

Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand

Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.     

Binding of infectious pancreatic necrosis virus (IPNV) to membrane proteins from different fish cell lines

The early interactions between infectious pancreatic necrosis virus (IPNV) from Atlantic salmon and susceptible cell lines were studied using a virus overlay protein binding assay (VOPBA). Membrane preparations from four different cell lines, bluegill fry (BF)-2 cells, Chinook salmon embryo (CHSE)-214 cells, salmon head kidney (SHK)-1 cells and Atlantic salmon kidney (ASK) cells were separated by SDS-PAGE, blotted to nitrocellulose membranes and incubated with either a highly virulent IPNV field isolate or different recombinant IPNV strains exhibiting variations in virulence. Binding of virus to the respective membrane fractions was detected using either polyclonal or monoclonal antibodies. Independent of variations in virulence, IPNV bound in a specific manner to an approximately 220-kDa membrane protein from the salmonid cell lines CHSE-214, SHK-1 and ASK, whereas the size of the binding protein from the non-salmonid cell line BF-2 was approximately 190 kDa. Neutralization of IPNV prior to incubation with the blotted membrane fractions inhibited binding to the indicated proteins. Complete deglycosylation of the membrane proteins did not interfere with the binding of IPNV, suggesting that the interaction between virus and cells is not carbohydrate specific. The described binding proteins may represent putative cellular binding sites or receptors for IPNV.     

Identification of human hepatocyte protein(s), which binds specifically to the recombinant envelope-2/non-structural-1 protein of hepatitis C virus

Hepatitis C virus (HCV), which is the major pathogen responsible for human chronic liver disease, has special tropism for hepatocytes. Although, low-density lipoprotein receptor, CD81 and negatively charged glycosaminoglycans have been proposed as candidate receptors for HCV, no confirmed receptor(s) on the hepatocytes have been identified to date. It is also suggested that additional, yet unidentified, cellular proteins may be involved in the host-viral interaction. Therefore, this study was conducted with the main aim to identify hepatocyte protein(s) that may have affinity for the HCV structural protein, envelope-2/non-structural-1 (E2/NS1) protein. For the binding studies, hepatocytes were isolated from fresh normal human liver tissues. The hepatocyte proteins on the nitrocellulose paper were reacted with recombinant E2/NS1 protein and anti-E2 (rabbit). In another approach, to rule out the possibility of binding of rec-E2/NS1 with the hepatocyte cytoplasmic proteins, hepatocyte plasma membrane proteins were passed through CNBr-activated and recombinant E2/NS1 bound sepharose-4B column. The recombinant E2/NS1 binding hepatocyte plasma membrane protein(s) were eluted and were then analyzed. Altogether, our data suggest that E2/NS1 protein of HCV binds to two hepatocyte proteins of molecular weights 25-28 kDa and 59-60 kDa. These results indicate the possible role of the above proteins (25-28 kDa and 59-60 kDa) in the viral binding to the hepatocytes.     

Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris

The full-length envelope glycoprotein gene of dengue virus type 2 was cloned using an RT-PCR method from the infected C6/36 cells and inserted into pPICZaB vector. The recombinant plasmid was integrated into Pichia pastoris by electroporation and the expressed product was identified by SDS-PAGE and Western blotting. High-level secreted expression was performed by determining the Mut(+) phenotype and screening multi-copy integrants in the recombinant yeast cells. A recombinant protein with a molecular size of approximately 69 kDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed supernatant was able to bind with mouse polyclonal antibody or E-specific monoclonal antibody of dengue-2 virus. Purified E-poly (His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The results of Western blotting and solid-phase ELISA using dengue virus antibodies indicated that the purified recombinant E glycoprotein retained its antigenicity. High-level production of the recombinant E protein up to 100 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length envelope glycoprotein.     

Reovirus type 3 and [125I]-iodocyanopindolol bind to distinct domains on the beta-adrenergic like receptor

Antireceptor antibodies have been developed as a probe to study the cellular receptor for reovirus type 3. Using this probe, a glycoprotein with a molecular weight of 65-67 kilodaltons and a pI of 5.8-6.0 was isolated and identified as the reovirus receptor. This protein was also structurally similar to the affinity-purified beta-adrenergic receptor from calf lung. In this report, we employ [125I]-iodocyanopindolol, a high affinity beta-adrenergic antagonist, to further characterize this protein. We show that R1.1, a murine thymoma cell line, possesses about 2,000 receptors per cell with high affinity for ICYP (kD = 3.3 X 10(-11) M). Competitive inhibition studies suggest that the receptor is of the beta-2 subtype. Solubilized receptor proteins from R1.1 cells bound to the antireceptor antibody were further purified by SDS-PAGE and electroelution from the gel. Five percent of the proteins thus obtained could bind ICYP with high affinity (kD = 1.6 X 10(-10) M). This suggests that the purification procedure produced a collection of forms of this 65- to 67-kilodalton protein, some of which retained the conformation for binding the beta ligands. We also demonstrate that the isolated receptor protein was able to bind ICYP even when the virus binding site was occupied by the anti-idiotype, suggesting that reovirus type 3 and the beta ligands bind to distinct domains on the receptor protein.     

Cellular proteins bind to the 3' and 5' untranslated regions of dengue 2 virus genome

In vitro generated cloned full length dengue 2 virus untranslated regions (UTRs) were used in RNA gel mobility shift assays to examine cellular factors binding to the virus genomes. Cellular factors in lysates of Vero (monkey) and C6/36 (mosquito) cells bound specifically and non-specifically to the dengue 2 virus 3' UTR. Non-specific interaction with the 5' UTR, resulting in formation of at least 4 band shift complexes was noted with lysate of the C6/36 cells only. Pre-treating the cell lysates with proteinase K affected binding of cellular factors to the dengue 2 virus UTRs, suggesting that the cellular factors were proteins. These findings suggest that cellular proteins could interact with specific sites on the dengue virus genomes.     

重组汉滩病毒核蛋白的纯化及鉴定

目的：纯化重组表达的汉滩病毒核蛋白。方法：重组菌经IPTG诱导后，表达的目的蛋白为带有谷胱甘肽转硫酶（GST）标签的融合蛋白，并以包涵体形式存在。将包涵体变性、复性后，采用Glutathione Sepharose 4B亲和色谱对核蛋白进行纯化，并用夹心ELISA和Western blot检测纯化蛋白。结果：表达产物第一次过柱亲和层析后可去除杂蛋白，获得纯化的核蛋白与谷胱甘肽转硫酶的融合蛋白，再经凝血酶酶切，第二次过柱亲和层析后获得的穿过峰为核蛋白，洗脱峰为GST。纯化的融合蛋白和纯化的核蛋白均为SDS－PAGE单点纯，并具有良好的抗原活性。结论：Glutathione Sepharose 4B亲和色谱纯化重组汉滩病毒核蛋白是一种较为有效的方法。     

人血管内皮细胞上与2型登革病毒相互作用蛋白的筛选与鉴定

目的 建立高效病毒受体筛选方法,筛选并鉴定人血管内皮细胞膜上与2型登革病毒(DENV-2)相互作用的蛋白.方法 用纯化的DENV-2 E蛋白DⅢ区(EDⅢ)分子代替全病毒颗粒与转印至PVDF膜的人血管内皮ECV304细胞膜蛋白进行孵育,建立改良的VOPBA和2D-VOPBA技术筛选相互作用蛋白,筛选出的候选蛋白经质谱分...     

我国登革2型病毒43株包膜E蛋白MBP-B165抗原性的研究

目的 通过对我国登革2型病毒株包膜E蛋白包括B区在内的第267－432氨基酸编码基因片段的表达,研究MBP－B165蛋白的抗原性。方法 首先采用PCR方法扩增了编码B165蛋白的基因片段,并将其插入到pMal－C2核载体进行融合表达。采用蛋白印迹和ELISA法对表达产物进行特异性鉴定。用表达的融合蛋白MBP－B165免疫大白兔,产通过ELISA法检测兔免疫血清中登革2型病毒特异的抗体。结果 表达的融合蛋白MBP－B165可与我国登革2型病毒株抗体特异结合,而且与登革1、3和4型病毒参考株的多克隆抗体均具有较高的反应性。用表达蛋白免疫大白兔可产生针对我国登革2型病毒株E蛋白的特异抗体,并且该抗体与基他3个型病毒参考株有交叉反应。结论 我国登革2型病毒43株E蛋白包括B区在内的第267－432氨基酸序列具有一定的抗原性,而且具有黄病毒亚组特异的反应性表位,即4个型登革病毒的结构保守性表位。     

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype

The nucleotide sequence of the 5'-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5'-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5'-C-prM(M)-E-3' and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses.     

Identification and characterization of prohibitin as a receptor protein mediating DENV-2 entry into insect cells

Dengue is transmitted primarily by mosquitoes of the Aedes genus. Despite a number of studies, no insect dengue virus receptor protein has been clearly identified and characterized. Using a number of separation methodologies and virus overlay protein binding assays we identified a 35 kDa protein that segregated with susceptibility to dengue serotype 2 (DENV-2) infection in two mosquito species and two mosquito cell lines. Mass spectroscopy identified the protein to be prohibitin, a strongly conserved and ubiquitously expressed protein in eukaryotic cells. Antibody mediated inhibition of infection and siRNA mediated knockdown of prohibitin expression significantly reduced infection levels and subsequent virus production in both Aedes aegypti and Aedes albopictus cell lines. Confocal microscopy showed a significant degree of intracellular colocalization between prohibitin and DENV-2 E protein, and coimmunoprecipitation confirmed that prohibitin interacts with dengue E. Prohibitin is the first characterized insect cell expressed dengue virus receptor protein.     

Retrospective study of Western blot profiles in immune sera of natural dengue virus infections

The Western blot (WB) assay was used to determine dengue virus antibodies present in human immune sera arising from recent primary and secondary dengue virus infections in Singapore. Cell lysates of dengue-2 virus-infected C6/36 and Vero cells were used. Antibodies directed against structural proteins of dengue-2 virus including envelope (E, gp60/50), capsid-premembrane (C-PrM, gp35), and premembrane (PrM, gp20) were detected, with antibody against envelope protein being most dominant. Similar WB profiles were detected in both primary and secondary dengue virus infections. The reactivity rate of antibodies to dengue-2 virus proteins was higher in infected Vero cell lysate than in infected C6/36 cell lysate, with the exception of antibodies to nonstructural proteins of NS1 and NS3, which were detected predominantly in infected C6/36 cell lysate. More than 75% of "normal" individuals (with no complaint of recent dengue virus infection) examined had low levels of dengue virus antibodies, but all presented with similar WB profiles as patients with recent dengue virus infections. This finding reflects a high seroprevalence of dengue virus infections and the long lasting nature of E, C-PrM, and PrM antibodies. Results from this study indicate that in natural dengue virus infections, native E, C-PrM, and PrM antigens of dengue virus are immunogenic and elicit long-lasting antibodies.     

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (Mphi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to Mphi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of, various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca(2+)/Mg(2+) enhanced DV binding. This argues against involvement of beta(2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains.     

Antigenic cell associated dengue 2 virus proteins detected in vitro using dengue fever patients sera

At least three major antigenic dengue 2 virus proteins were recognized by pooled dengue fever patients' sera in infected Aedes albopictus (C6/36) mosquito cells. Dengue virus envelope (E), premembrane (PrM) and non-structural protein 1 (NS 1) dimer were detected beginning on day 3 postinfection in both the cell membrane and cytosolic fractions. Using the patients' sera, the presence of antigenic intermediate core protein (C)-PrM and NS1-non-structural protein 2a (NS2a) in the cytoplasmic fraction of dengue 2 virus infected cells was revealed. The presence of a approximately 92 and approximately 84 kDa NS 1 dimer in the membrane (NS 1m) and cytosolic (NS 1c) fractions of C6/36 cells, respectively, was also recognized. Using individual patient's serum, it was further confirmed that all patients' sera contained antibodies that specifically recognized E, NS 1 and PrM present in the dengue 2 virus-infected cell membrane fractions, suggesting that these glycosylated virus proteins were the main antigenic proteins recognized in vivo. Detection of dengue 2 virus C antibody in some patients further suggested that C could be antigenic if presented in vivo.     

Analysis of functional epitopes on the dengue 2 envelope (E) protein using monoclonal IgM antibodies

Summary Forty-two hybridomas secreting IgM antibody against dengue virus were derived from spleen cells of dengue 2 infected mice. Antibody from 27 of these recognised the E protein of this virus. Of the 22 antibodies which neutralised dengue 2, only two cross-reacted with other flaviviruses. These 22 antibodies recognised three discrete domains on dengue virions. Competitive binding studies with IgG monoclonal antibodies suggested that two of the three domains were recognised by both IgG and IgM antibodies and that there were two additional domains which may be recognised exclusively by either IgG or IgM antibodies. IgM antibodies reacting with domains recognised by IgG antibodies that enhanced infection of susceptible cells by dengue 2, had no enhancing properties. None of the IgM antibodies activated the serum complement system after reacting with dengue 2 virions.     

Involvement of ATP synthase β subunit in chikungunya virus entry into insect cells

Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase β subunit (ATPSβ). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPSβ. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPSβ showed a significant reduction in viral entry and virus production. These results suggest that ATPSβ is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells.     

Role of M protein aggregation in defective assembly of temperature-sensitive M protein mutants of vesicular stomatitis virus

The goal of these experiments was to determine the steps in virus assembly that are defective at the nonpermissive temperature in temperature-sensitive (ts) matrix (M) protein mutants of vesicular stomatitis virus. It has been proposed that mutations in M protein either reduce the binding affinity for nucleocapsids or lead to aggregation, reducing the amount of M protein available for virus assembly. Cytosolic or membrane-derived M proteins from wild-type VSV and two ts M protein mutant viruses, tsM301 and tsO23, as well as a revertant of tsO23 virus, O23R1, were analyzed for binding to nucleocapsid-M protein (NCM) complexes and for M protein aggregation. The experiments presented here showed that ts M proteins synthesized at the nonpermissive temperature were capable of binding to nucleocapsids and that aggregation of ts M proteins did not reduce the amount of soluble M protein below the amount required for assembly of the O23R1 virus. Instead, the most pronounced defect in ts M proteins was in the ability of membrane-derived M proteins to be solubilized in the presence of the detergent Triton X-100. It is proposed that this detergent-insoluble form of M protein interferes with a step necessary to initiate assembly of NCM complexes. A similar detergent, Triton X-114, caused aggregation of membrane-derived wild-type M protein, disproving an earlier proposal that membrane-derived M protein behaves like an integral membrane protein in the presence of Triton X-114. Aggregation of wild-type M protein in the presence of Triton X-100 could be induced by incubation at 37 degrees C with a high-molecular-weight fraction isolated from uninfected cells by sucrose gradient centrifugation. These results implicate host components in inducing M protein aggregation.     

Antibodies from dengue patient sera cross-react with endothelial cells and induce damage

Dengue virus infection causes a wide range of diseases from the mild febrile illness dengue fever to the life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and hemorrhagic syndrome are the clinical features associated with dengue infection, yet the mechanisms remain unclear. In this study, the cross-reactivity of dengue patient sera with endothelial cells was demonstrated. There were higher percentages of endothelial cells reactive with dengue hemorrhagic fever/dengue shock syndrome patient sera than those with dengue fever patient sera. The percentages of endothelial cells reactive with patient serum IgM were higher than those with IgG. Further studies showed that the endothelial cell binding activity was inhibited by pretreatment with dengue virus nonstructural protein 1 (NS1). The antibodies against NS1 produced after dengue virus infection may, at least in part, account for the cross-reactivity of patient sera with endothelial cells. Furthermore, dengue patient sera induced endothelial cell apoptosis via a caspase-dependent pathway that was also inhibited by NS1 pretreatment. In addition to apoptosis, patient sera caused cell lysis in the presence of complement, and DHF/DSS patient sera showed higher percentages of cytotoxicity than dengue fever patient sera. Thus, the generation of cross-reactive autoantibodies against endothelial cells would lead to their dysfunction, which may play a role in the pathogenesis of dengue virus infection.