Sulfated Polysaccharide,Curdlan Sulfate,Efficiently Prevents Entry/Fusion and Restricts Antibody-Dependent Enhancement of Dengue Virus Infection In Vitro: A Possible Candidate for Clinical Application

Curdlan sulfate (CRDS), a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV). CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E) protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion). The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.     

Roles of neuraminidase in the initial stage of influenza virus infection

We propose a concept that neuraminidase (NA) promotes virus entry into target cells during the initial stage of viral infection, in addition to the generally accepted concept that influenza virus NA promotes the release of progeny virus from a host cell at the final stage of viral replication. When NA activity was inhibited with specific inhibitors such as zanamivir and oseltamivir carboxylate, infection efficiency of the virus to MDCK and A549 cells was reduced to approximately 1/4 and 1/8, respectively. NA inhibitors did not significantly affect virus binding and envelope fusion activities, when assessed using an erythrocyte and virus system. Since the initial stage of viral infection involves binding of the virus to the target cell, virus entry into an endosome and envelope fusion with the endosomal membrane, our results indicated that NA inhibitors interfered with the virus entry step. Thus, NA is thought to promote virus entry, and thereby enhances infection efficiency.     

New insights into the role of endosomal proteins for African swine fever virus infection

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.     

Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.     

Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release

Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl beta-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4 degrees C but shifted to lipid rafts at 37 degrees C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.     

Polypyrimidine tract-binding protein influences negative strand RNA synthesis of dengue virus

Flavivirus non-structural protein 4A (NS4A) induces membrane rearrangements to form viral replication complex and functions as interferon antagonist. However, other non-structural roles of NS4A protein in relation to virus life-cycle are poorly defined. This study elucidated if dengue virus (DENV) NS4A protein interacts with host proteins and contributes to viral pathogenesis by screening human liver cDNA yeast-two-hybrid library. Our study identified polypyrimidine tract-binding protein (PTB) as a novel interacting partner of DENV NS4A protein. We reported for the first time that PTB influenced DENV production. Gene-silencing studies showed that PTB did not have an effect on DENV entry and DENV RNA translation. Further functional studies revealed that PTB influenced DENV production by modulating negative strand RNA synthesis. This is the first study that enlightens the interaction of DENV NS4A protein with PTB, in addition to demonstrating the novel role of PTB in relation to mosquito-borne flavivirus life-cycle.     

麻疹病毒受体与病毒侵入

麻疹病毒是一种具囊膜的负链RNA病毒,两种主要的囊膜蛋白血凝素蛋白(H)和膜融合蛋白(F)表达在膜表面负责病毒侵入过程中与宿主受体的结合和膜融合过程.病毒囊膜蛋白与受体的相互作用是病毒侵入宿主的关键步骤,决定了病毒感染能力、种属和组织嗜性.因此,囊膜病毒与受体的结合位点往往成为重要的抗病毒药物的靶点.目前已发现的3种麻疹病毒受体包括CD46、SLAM和Nectin-4.以下综述了麻疹病毒受体的特征及在病毒侵入中的作用、麻疹病毒H蛋白与受体的相互作用机制,为抗病毒药物设计及麻疹病毒作为肿瘤治疗性载体的应用提供理论依据.     

Differential requirements in endocytic trafficking for penetration of dengue virus

The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection.     

Hepatitis C virus entry depends on clathrin-mediated endocytosis

Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.     

Mechanism of dengue virus broad cross-neutralization by a monoclonal antibody

The dengue virus (DENV) complex is composed of four distinct but serologically related flaviviruses, which together cause the present-day most important emerging viral disease. Although DENV infection induces lifelong immunity against viruses of the same serotype, the antibodies raised appear to contribute to severe disease in cases of heterotypic infections. Understanding the mechanisms of DENV neutralization by antibodies is, therefore, crucial for the design of vaccines that simultaneously protect against all four viruses. Here, we report a comparative, high-resolution crystallographic analysis of an "A-strand" murine monoclonal antibody, Mab 4E11, in complex with its target domain of the envelope protein from the four DENVs. Mab 4E11 is capable of neutralizing all four serotypes, and our study reveals the determinants of this cross-reactivity. The structures also highlight the mechanism by which A-strand Mabs disrupt the architecture of the mature virion, inducing premature fusion loop exposure and concomitant particle inactivation.     

Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.     

A model of the binding, entry, uncoating, and RNA synthesis of Semliki Forest virus in baby hamster kidney (BHK-21) cells

A quantitative understanding of viral trafficking would be useful in treating viral-mediated diseases, designing protocols for viral gene therapy, and optimizing heterologous protein production. In this article, a model for the trafficking of Semliki Forest virus and its RNA synthesis in baby hamster kidney (BHK-21) cells is presented. This model includes the various steps leading to infection such as attachment, endocytosis, and viral fusion in the endosome. The model estimates a mean fusion time of 4 to 6 min for the wild-type virus, and 38 min for Fus-1, an SFV mutant which requires a lower pH for fusion. These mean fusion times are consistent with the time-scale of endosomal acidification, suggesting viruses fuse almost instantaneously with the endosomal membrane as soon as the pH of the endosome drops below the pH threshold of the virus. Infection is most likely controlled at the level of viral uncoating, as shown by the close agreement between the efficiency of uncoating and the experimentally determined fraction of viruses that is infectious. The viral RNA synthesized per cell is best described by assuming that it depends on the number of uncoated viruses prior to the onset of replication according to a saturation-type expression. A Poisson distribution is used to determine the distribution of uncoated viruses among the cells. Because attachment is the rate-limiting step in the uncoating of the virus, increasing the attachment rate can lead to enhanced RNA synthesis and, hence, new virion production. Such an increase in the attachment rate may be obtained by lowering the medium pH or the addition of a polycation. (c) 1995 John Wiley & Sons, Inc.     

Nef does not affect the efficiency of human immunodeficiency virus type 1 fusion with target cells

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by an unknown mechanism. Recent studies have suggested that Nef may act by regulating the efficiency of virus entry into cells. Here we provide evidence to the contrary. Using a quantitative assay of HIV-1 virus-cell fusion, we observed equivalent rates and extents of fusion of wild-type and Nef-defective HIV-1 particles with MT-4 cells and CD4-expressing HeLa cells. In studies using soluble CD4 (sCD4) to inhibit infection, wild-type and Nef-defective HIV-1 escaped the sCD4 block with similar kinetics. We conclude that Nef acts at a postentry step in infection, probably by facilitating intracellular transport of the HIV-1 ribonucleoprotein complex.     

The helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry

The envelope (E) of dengue virus (DENV) is a determinant of tropism and virulence. At the C terminus of E protein, there is a stem region containing two amphipathic α-helical domains (EH1 and EH2) and a stretch of conserved sequences in between. The crystal structure of E protein at the postfusion state suggested the involvement of the stem during the fusion; however, the critical domains or residues involved remain unknown. Site-directed mutagenesis was carried out to replace each of the stem residues at the hydrophobic face with an alanine or proline in a DENV serotype 4 (DENV4) precursor membrane (prM)/E expression construct. Most of the 15 proline mutations at either EH1 or EH2 severely affected the assembly of virus-like particles (VLPs). Radioimmunoprecipitation and membrane flotation assays revealed that EH1 mutations primarily affect prM-E heterodimerization and EH2 mutations affect the membrane binding of the stem. Introducing four proline mutations at either EH1 or EH2 into a DENV2 replicon packaging system greatly affects assembly and entry. Moreover, introducing these mutations into a DENV2 infectious clone confirmed the impairment in assembly and infectivity. Sequencing analysis of adaptive mutations in passage 5 viruses revealed a change to a leucine or wild-type residue at the original site, suggesting the importance of maintaining the helical structure. Collectively, these findings suggest that the EH1 and EH2 domains are involved in both assembly and entry steps of the DENV replication cycle; this feature, together with the high degree of sequence conservation, suggests that the stem region is a potential target of antiviral strategies.     

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.     

Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.     

Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells

Infection of mammalian cells with Semliki Forest virus requires the endocytosis of the virus, its delivery to prelysosomal endosomes, and fusion of the viral envelope with the endosome membrane. Previous studies have indicated that the low endosomal pH triggers a conformational change in the viral spike glycoproteins rendering them fusogenic. In this paper, we demonstrate an additional factor(s) which regulates virus fusion in endosomes. We found that Semliki Forest virus is unable to penetrate or infect baby hamster kidney (BHK-21) cells grown in medium containing reduced Na+ concentrations. Virus endocytosis and degradation are nearly normal, the virus is transported to endosomes where a characteristic low pH-induced loss of trypsin-sensitivity of the E1 spike glycoprotein occurs. Nevertheless, the viral envelope fails to fuse with the endosomal membrane and the viral RNA is not released into the cytosol. As judged by the uptake of the voltage-sensitive probe [3H]triphenylmethyl phosphonium we observed a close correlation between conditions which inhibit virus infection and which cause depolarization of the cells. We propose that in intact cells, the fusion of Semliki Forest virus with the endosome membrane depends not only on acidic endosomal pH, but also on the maintenance of the potential.     

Interactions among measles virus hemagglutinin, fusion protein and cell receptor signaling lymphocyte activation molecule (SLAM) indicating a new fusion-trimer model

For measles viruses, fusion on the cell membrane is an important initial step in the entry into the infected cells. The recent research indicated that hemagglutinin firstly leads the conformational changes in the fusion protein then co-mediates the membrane fusion. In the work, we use the co-immunoprecipitation and pull-down techniques to identify the interactions among fusion protein, hemagglutinin and signaling lymphocyte activation molecule (SLAM), which reveal that the three proteins can form a functional complex to mediate the SLAM-dependent fusion. Moreover, under the confocal microscope, fusion protein and hemagglutinin protein can show the cocapping mediated by the SLAM. So fusion protein not only is involved in the fusion but also might directly interact with the SLAM to be a new fusion-trimer model, which might account for the infection mechanism of measles virus.     

Sphingolipids: Modulators of HIV-1 Infection and Pathogenesis

HIV-1 infects host cells by sequential interactions of its fusion protein (gp120-gp41) with receptors CD4, CXCR4 and/or CCR5 followed by fusion of viral and host membranes. Studies indicate that additional factors such as receptor density and composition of viral and cellular lipids can dramatically modulate the fusion reaction. Lipid rafts, which primarily consist of sphingolipids and cholesterol, have been implicated for infectious route of HIV-1 entry. Plasma membrane Glycosphingolipids (GSLs) have been proposed to support HIV-1 infection in multiple ways: (a) as alternate receptor(s) for CD4-independent entry in neuronal and other cell types, (b) viral transmission, and (c) gp120-gp41-mediated membrane fusion. However, the exact mechanism(s) by which GSLs support fusion is still elusive. This article will focus on the contribution of target membrane sphingolipids and their metabolites in modulating viral entry. We will discuss the current working hypotheses underlying the mechanisms by which these lipids promote and/or block HIV-1 entry. Recent approaches in the design and development of novel glycosyl derivatives, as anti-HIV agents will be summarized.