Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-␤-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.Dengue (DEN) virus, the most important arthropod-borne human pathogen, represents a serious public health threat. DEN virus is transmitted to humans by the bite of the domestic mosquito, Aedes aegypti, and circulates in nature as four distinct serological types (DEN-1 to -4). DEN virus has been recognized in over 100 countries, and 2.5 billion people live in areas where DEN virus is endemic (16). The clinical manifestations of DEN virus infection range in severity from a simple self-limited febrile illness known as dengue fever to a hemorrhagic fever (DHF) and potentially fatal hemorrhagic shock syndrome. Each year, more than 50 million cases of dengue fever and several hundred thousand cases of DHF occur. During the past 8 years the incidence of dengue has grown in areas of endemicity, particularly in the American region. A specific treatment or vaccine is not yet available.DEN virus is an enveloped virus that belongs to the Flaviviridae family. Mature virions are icosahedral, 50 nm in diameter, and contain a single-strand and positive-polarity RNA as genome of about 10.7 kb (21). The DEN virus genome encodes three structural proteins (envelope glycoprotein, E; membrane, M; and capsid, C) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). E protein is the major structural protein exposed on the surface of the particle, and it arrays in homodimers, parallel to viral surface. Recently, the structure of DEN virus E protein has been determined by X-ray crystallography (24). Each monomer consists of three domains: the structurally central amino-terminal domain I that organizes the structure; the dimerization domain II that contains the hydrophobic fusion peptide essential for virus-cell fusion; and finally the carboxy-terminal immunoglobulin (Ig)-like domain III, which has been proposed to function as the binding site for cellular re...

SummaryIn order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen‐presenting cells. These self‐adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII‐PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII‐PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII‐PIGS also induced a significant cellular immune response, IFN‐γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof‐of‐principle study shows that the potent antibody Fc‐mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad‐ranging immune response.

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The signaling lymphocytic activation molecule (SLAM; CD150) is the immune cell receptor for measles virus (MV). To assess the importance of the SLAM-MV interactions for virus spread and pathogenesis, we generated a wild-type IC-B MV selectively unable to recognize human SLAM (SLAM-blind). This virus differs from the fully virulent wild-type IC-B strain by a single arginine-to-alanine substitution at amino acid 533 of the attachment protein hemagglutinin and infects cells through SLAM about 40 times less efficiently than the isogenic wild-type strain. Ex vivo, this virus infects primary lymphocytes at low levels regardless of SLAM expression. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with the SLAM-blind virus, no clinical symptoms were documented. Only one monkey had low-level viremia early after infection, whereas all the hosts in the control group had high viremia levels. Despite minimal, if any, viremia, all six hosts generated neutralizing antibody titers close to those of the control monkeys while MV-directed cellular immunity reached levels at least as high as in wild-type-infected monkeys. These findings prove formally that efficient SLAM recognition is necessary for MV virulence and pathogenesis. They also suggest that the selectively SLAM-blind wild-type MV can be developed into a vaccine vector.Measles virus (MV) is an enveloped virus with a negativesense RNA genome (2). It is still a major cause of death in children of developing countries, mainly due to opportunistic secondary infections facilitated by MV-induced immune suppression (12, 29). Transient but severe immune suppression is explained at least in part by the rapid spread of MV infection in immune cells (6,37,41). MV targets immune cells through its hemagglutinin (H) that binds cellular receptors and triggers the other glycoprotein F to fuse cellular membranes (22).Two MV receptors have been identified. The first one was the membrane cofactor protein (MCP; CD46), a ubiquitously expressed regulator of complement activation sustaining infection by the MV vaccine strain (8, 21) but not by wild-type (WT) strains (24). Wild-type MV strains, as well as the vaccine strain, enter cells through the signaling lymphocytic activation molecule (SLAM; CD150) (10,15,35). SLAM is an immune cellspecific protein expressed on the surface of thymocytes, activated lymphocytes, mature dendritic cells, and activated macrophages (4, 31). The existence of another receptor on cells derived from human lung and bladder epithelium has been inferred (18,33). While this epithelial receptor (EpR) has not been identified yet, it appears to be a basolateral protein expressed by cells forming tight junctions (18).We are characterizing the mechanisms by which MV spreads in its host and the pathogenic consequences of the interactions with different receptors. We previously showed that an MV unable to recognize EpR remains virulent in rhesus monkeys but cannot cross the epithelium and is not shed (18). This result is consistent with the model ...

Measles remains a leading cause of death worldwide among children because it suppresses immune function. The measles virus (MV) P gene encodes three proteins (P, V, and C) that interfere with innate immunity, controlling STAT1, STAT2, mda5, and perhaps other key regulators of immune function. We identified here three residues in the shared domain of the P and V proteins-tyrosine 110, valine 112, and histidine 115-that function to retain STAT1 in the cytoplasm and inhibit interferon transcription. This information was used to generate a recombinant measles virus unable to antagonize STAT1 function (STAT1-blind MV) differing only in these three residues from a wild-type strain of well-defined virulence. This virus was used to assess the relevance of P and V interactions with STAT1 for virulence in primates. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with STAT1-blind MV, viremia was short-lived, and the skin rash and other clinical signs observed with wild-type MV were absent. The STAT1-blind virus less efficiently controlled the inflammatory response, as measured by enhanced transcription of interleukin-6 and tumor necrosis factor alpha in peripheral blood mononuclear cells from infected hosts. Importantly, neutralizing antibody titers and MV-specific T-cell responses were equivalent in hosts infected with either virus. These findings indicate that efficient MV interactions with STAT1 are required to sustain virulence in a natural host by controlling the inflammatory response against the virus. They also suggest that selectively STAT1-blind MV may have utility as vectors for targeted oncolysis and vaccination.Innate immunity, and in particular the interferon (IFN) system, protects the host from viral infections. However, viruses have evolved multiple complementary strategies to evade or control the type I (␣/␤) IFN responses. They can interfere with gene expression and/or protein synthesis, minimize IFN induction by specifically blocking IFN induction cascades, inhibit IFN signaling, block the action of IFN-induced antiviral proteins, or have a replication strategy not sensitive to IFN action (19,21,24).The IFN-␣/␤ signaling pathway is well characterized: secreted IFN binds to its receptor, activating the tyrosine kinases JAK1 and Tyk2, which in turn phosphorylate the signal transducers and activators of transcription STAT1 and STAT2. Phosphorylated STAT1 and STAT2 form a stable heterodimer that interacts with the DNA-binding protein IRF-9. The IRF-9/STAT1/STAT2 heterotrimer, named IFN-stimulated gene factor 3 (ISGF3), translocates to the nucleus, and binds the IFN-stimulated response element (ISRE) in target promoters, resulting in transcriptional activation of multiple genes that establishes an antiviral state in infected and surrounding noninfected cells (11,12).Innate immunity control strategies can be remarkably sophisticated even for RNA viruses that have small genomes and a limited coding capacity. For example, the P gene of measles virus (MV), the enveloped nonsegmented...

Dengue virus (DENV) is transmitted to humans by mosquitoes of the genus Aedes. Although several molecules have been described as part of DENV receptor complex in mosquito cells, none of them have been identified. Our group characterized two glycoproteins (40 and 45 kD) as part of the DENV receptor complex in C6/36 cells. Because identification of the mosquito cell receptor has been unsuccessful and some cell receptors described for DENV in mammalian cells are heat-shock proteins (HSPs), the role of HSPs in DENV binding and infection in C6/36 cells was evaluated. Our results indicate that gp45 and a 74-kD molecule (p74), which interact with DENV envelope protein, are immunologically related to HSP90. Although p74 is induced by heat shock, gp45 apparently is not. However, these proteins are relocated to the cell surface after heat-shock treatment, causing an increase in virus binding without any effect on virus yield.

The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a singlestranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5 and 3 ends. The 3UTR in the minus-strand RNA [3UTR (؊)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3UTR (؊) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3UTR (؊) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3 UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5 portion of the 3UTR (؊). Complex I, which was the main ribonucleoprotein complex formed with the 3UTR (؊) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.Dengue virus (DEN), a mosquito-borne member of the Flaviviridae family, contains a single-stranded, positive-polarity RNA as a genome. The genomic RNA has a long open reading frame that encodes for a polyprotein that is processed during and after translation (reviewed in reference 8). This unique open reading frame is flanked by two untranslated regions located at the 5Ј and 3Ј ends (5ЈUTR and 3ЈUTR). In the 5Ј end, viral RNA contains a type I cap structure, and in the 3Ј end, it lacks the poly(A) track. The 5ЈUTR and 3ЈUTR of dengue 4 virus (DEN4) are 101 and 384 nucleotides (nt) in length, respectively. Both regions have been predicted to form stable stem-loop structures that are highly conserved among flaviviruses (5,7,26,37). The presence of conserved secondary structures within both UTRs suggests that they might contain cis-acting elements involved in translation and replication (17, 33). Positive-polarity RNA viruses use the positive-strand genomic 3ЈUTR [3ЈUTR (ϩ)] as a promoter for negativestrand RNA synthesis (Ϫ). In a similar manner, the 3ЈUTR present in the minus-polarity RNA could, like the 3ЈUTR (ϩ), contain promoter sequences for the transcription of positivepolarity RNAs. Besides, it has been revealed that DEN 5ЈUTR (ϩ) and 3ЈUTR (ϩ) contain several conserved elements which are involved in viral viability and replication (7,25,45). The presence of both regions is required for in vitro replication of DEN, supporting t...

Hepatitis C virus (HCV) is the prototype member of the Hepacivirus genus within the family Flaviviridae. The virus is transmitted almost exclusively by the parenteral route, and acute infections, which are frequently subclinical, progress to chronicity in about 70% of cases. Persistent HCV carriers may develop liver cirrhosis, hepatocellular carcinoma, and end-stage liver disease. Despite an efficient preventive campaign based on the identification of HCV-infected blood donors, the prevalence of the virus among American young adults has not declined (35). Recent advances have improved treatment options for infections with certain viral genotypes, although limitations in efficacy remain and a preventive vaccine is not available.HCV is an enveloped virus, with a positive-sense, singlestranded RNA genome. The genome is translated into a polyprotein which is proteolytically processed into 10 individual proteins (reviewed in reference 64). The structural proteins, core protein (C) and two envelope glycoproteins (E1 and E2), form the physical viral particle; C functions to encapsidate the RNA genome, while E1 and E2 mediate virus attachment and entry into host cells. E1 and E2 are highly glycosylated type I transmembrane proteins with an N-terminal ectodomain. Residues within the transmembrane domains are important for heterodimerization and dimer retention in the endoplasmic reticulum (ER) (22,46). Replacement of the E1 or E2 transmembrane domains can direct transport of the corresponding chimeric proteins to the plasma membrane (1, 10). E2 also encompasses well-conserved antibody neutralization determinants, which are located near the binding sites for viral entry factors CD81 and scavenger receptor B1 (20,29). There is also some evidence for the existence of neutralizing determinants in E1 (45). Patient neutralizing antibodies have been identified that target virus interactions with its coreceptors and block glycoprotein-mediated membrane fusion (31).A major hurdle in the development of an effective HCV vaccine is the lack of an immunization strategy to elicit broadly protective antibodies and sustained cell-mediated immunity (32, 66). Studies with chimpanzees have shown the importance of total anti-E1/E2 antibody titers in conferring protection (15). Moreover, neutralizing immunity and functional CD4 ϩ and CD8 ϩ Tcell responses induced early in HCV infection correlate with clearance or viral control in patients (7,37,51).In contrast, the measles virus (MV) vaccine has an outstanding record of efficacy and safety. The MV Moraten vaccine strain is credited with the temporary elimination of indigenous measles transmission on the American continent (14), and the World Health Organization is implementing a global measles eradication program (72,73). After completion of the two-dose vaccination schedule, nearly 100% of recipients develop lasting neutralizing immunity that may be lifelong (2). In addition, the availability of established production methods makes MV an appealing platform for delivering foreign antigens (r...