Efficient Infection Mediated by Viral Receptors Incorporated into Retroviral Particles

Many host cell surface proteins, including viral receptors, are incorporated into enveloped viruses. To address the functional significance of these host proteins, murine leukemia viruses containing the cellular receptors for Rous sarcoma virus (Tva) or ecotropic murine leukemia virus (MCAT-1) were produced. These receptor-pseudotyped viruses efficiently infect cells expressing the cognate viral envelope glycoproteins, with titers of up to 10⁵ infectious units per milliliter for the Tva pseudotypes. Receptor and viral glycoprotein specificity and functional requirements are maintained, suggesting that receptor pseudotype infection recapitulates events of normal viral entry. The ability of the Tva and MCAT-1 pseudotypes to infect cells efficiently suggests that, in contrast to human immunodeficiency virus type 1 entry, neither of these retroviral receptors requires a coreceptor for membrane fusion. In addition, the ability of receptor pseudotypes to target infected cells suggests that they may be useful therapeutic reagents for directing infection of viral vectors. Receptor-pseudotyped viruses may be useful for identifying new viral receptors or for defining functional requirements of known receptors. Moreover, this work suggests that the production of receptor pseudotypes in vivo could provide a mechanism for expanded viral tropism with potential effects on the pathogenesis and evolution of the virus.     

Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.     

Stochastic entry of enveloped viruses: fusion versus endocytosis

Infection by membrane-enveloped viruses requires the binding of receptors on the target cell membrane to glycoproteins, or "spikes," on the viral membrane. The initial entry mechanism is usually classified as fusogenic or endocytotic. However, binding of viral spikes to cell surface receptors not only initiates the viral adhesion and the wrapping process necessary for internalization, but can simultaneously initiate direct fusion with the cell membrane. Both fusion and internalization have been observed to be viable pathways for many viruses. We develop a stochastic model for viral entry that incorporates a competition between receptor-mediated fusion and endocytosis. The relative probabilities of fusion and endocytosis of a virus particle initially nonspecifically adsorbed on the host cell membrane are computed as functions of receptor concentration, binding strength, and number of spikes. We find different parameter regimes where the entry pathway probabilities can be analytically expressed. Experimental tests of our mechanistic hypotheses are proposed and discussed.     

Glycosylation in viral hepatitis

BackgroundThe interaction between hepatitis viruses and host cells is regulated by glycans exposed on the surfaces of human and viruses cells. As the biosynthesis and degradation of human glycoproteins take place at the highest level in the liver, the changes in glycosylation of serum proteins may potentially be useful in the diagnosis of liver pathology. On the other hand, specific alterations in viruses envelope glycans could cause large changes in the entry process of hepatitis viruses into a host cells.Scope of reviewUnique alterations in glycosylation of specific proteins can be detected in HBV and HCV infected patients especially with confirmed fibrosis/cirrhosis. On the other hand, viral envelope proteins that bind to host cells are glycosylated. These glycosylated proteins play a key role in recognition, binding and penetration of the host cells. In this review we summarized the knowledge about significance of glycosylation for viral and host factors.Major conclusionsGlycosylation changes in single serum glycoproteins are noticed in the sera of patients with viral hepatitis. However, a more specific biomarker for the diagnosis of chronic hepatitis than that of a single glycosylated molecule is systemic investigation of complete set of glycan structures (N-glycome). Glycans play important roles in the viral biology cycle especially as a connecting element with host receptors.General significanceThe interaction between virus glycoproteins and cellular receptors, which are also glycoproteins, determines the possibility of virus penetration into host cells. Therefore these glycans can be the targets for the developing of novel treatment strategies of viral hepatitis.     

Gangliosides and N-glycoproteins function as Newcastle disease virus receptors

The interaction of enveloped viruses with cell surface receptors is the first step in the viral cycle and an important determinant of viral host range. Although it is established that the paramyxovirus Newcastle Disease Virus binds to sialic acid-containing glycoconjugates the exact nature of the receptors has not yet been determined. Accordingly, here we attempted to characterize the cellular receptors for Newcastle disease virus. Treatment of cells with tunicamycin, an inhibitor of protein N-glycosylation, blocked fusion and infectivity, while the inhibitor of O-glycosylation benzyl-N-acetyl-alpha-D-galactosamide had no effect. Additionally, the inhibitor of glycolipid biosynthesis 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol blocked viral fusion and infectivity. These results suggest that N-linked glycoproteins and glycolipids would be involved in viral entry but not O-linked glycoproteins. The ganglioside content of COS-7 cells was analyzed showing that GD1a was the major ganglioside component; the presence of GM1, GM2 and GM3 was also established. In a thin-layer chromatographic binding assay, we analyzed the binding of the virus to different gangliosides, detecting the interaction with monosialogangliosides such as GM3, GM2 and GM1; disialogangliosides such as GD1a and GD1b, and trisialogangliosides such as GT1b. Unlike with other viruses, our results seem to point to the absence of a specific pattern of gangliosides that interact with Newcastle disease virus. In conclusion, our results suggest that Newcastle disease virus requires different sialic acid-containing compounds, gangliosides and glycoproteins for entry into the target cell. We propose that gangliosides would act as primary receptors while N-linked glycoproteins would function as the second receptor critical for viral entry.     

The Mechanism of Henipavirus Fusion： Examining the Relationships between the Attachment and Fusion Glycoproteins

The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.     

Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts

Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection.     

Virus entry paradigms

Viruses, despite being relatively simple in structure and composition, have evolved to exploit complex cellular processes for their replication in the host cell. After binding to their specific receptor on the cell surface, viruses (or viral genomes) have to enter cells to initiate a productive infection. Though the entry processes of many enveloped viruses is well understood, that of most non-enveloped viruses still remains unresolved. Recent studies have shown that compared to direct fusion at the plasma membrane, endocytosis is more often the preferred means of entry into the target cell. Receptor-mediated endocytic pathways such as the dynamin-dependent clathrin and caveolar pathways are well characterized as viral entry portals. However, many viruses are able to utilize multiple uptake pathways. Fluid phase uptake, though relatively non-specific in terms of its cargo, potentially aids viral infection by its ability to intersect with the endocytic pathway. In fact, many viruses despite using specialized pathways for entry are still able to generate productive infection via fluid phase uptake. Macropinocytosis, a major fluid uptake pathway found in epithelial cells and fibroblasts, is stimulated by growth factor receptors. Many viruses can induce these signaling cascades in cells leading to macropinocytosis. Though endocytic trafficking is utilized by both enveloped and non-enveloped viruses, key differences lie in the way membranes are traversed to deposit the viral genome at its site of replication. This review will discuss recent developments in the rapidly evolving field of viral entry.     

Binding-site interactions between Epstein-Barr virus fusion proteins gp42 and gH/gL reveal a peptide that inhibits both epithelial and B-cell membrane fusion

Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.     

Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography

Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion. We used cryo-electron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH resulted in virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are ∼3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Subtomogram averaging showed that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5-nm-long stalks and is partially disordered, compared with its native conformation. Upon lowering the pH, fusion took place. Fusion is a stochastic process that, once initiated, must be rapid, as only final (postfusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied by patches of dense material but without capsids, implying their disassembly. In addition, some of the products presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process.     

Prediction of antiviral peptides derived from viral fusion proteins potentially active against herpes simplex and influenza A viruses

There are very few antiviral drugs available to fight viral infections and the appearance of viral strains resistant to these antivirals is not a rare event. Hence, the design of new antiviral drugs is important. We describe the prediction of peptides with antiviral activity (AVP) derived from the viral glycoproteins involved in the entrance of herpes simplex (HSV) and influenza A viruses into their host cells. It is known, that during this event viral glycoproteins suffer several conformational changes due to protein-protein interactions, which lead to membrane fusion between the viral envelope and the cellular membrane. Our hypothesis is that AVPs can be derived from these viral glycoproteins, specifically from regions highly conserved in amino acid sequences, which at the same time have the physicochemical properties of being highly exposed (antigenic), hydrophilic, flexible, and charged, since these properties are important for protein-protein interactions. For that, we separately analyzed the HSV glycoprotein H and B, and influenza A viruses hemagglutinin (HA), using several bioinformatics tools. A set of multiple alignments was carried out, to find the most conserved regions in the amino acid sequences. Then, the physicochemical properties indicated above were analyzed. We predicted several peptides 12-20 amino acid length which by docking analysis were able to interact with the fusion viral glycoproteins and thus may prevent conformational changes in them, blocking the viral infection. Our strategy to design AVPs seems to be very promising since the peptides were synthetized and their antiviral activities have produced very encouraging results.     

Maturation of HIV envelope glycoprotein precursors by cellular endoproteases

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.     

Acquisition of viral receptor by NK cells through immunological synapse

Occasional EBV infection of human NK cells may lead to malignant diseases such as naso-pharyngeal NK lymphoma although NK cells do not express CD21, the primary receptor for EBV. Here we show that during early EBV infection in patients, NK cells attacked EBV-infected autologous B cells. In vitro, NK cells activated by conjugation to CD21(+) B-EBV cell targets transiently acquired a weak CD21(+) phenotype by synaptic transfer of few receptor molecules onto their own membrane. In the presence of viral particles, these ectopic receptors allowed EBV binding to the novel NK cell host. Hence, trans-synaptic acquisition of viral receptor from target cells might constitute an unsuspected mode of infection for otherwise unreachable lymphoid hosts.     

Modulation of expression of insulin and IGF-I receptor by Epstein-Barr virus and its gene products LMP and EBNA-2 in lymphocyte cell lines

The receptors for insulin and insulin-like growth factor I (IGF-I) are two closely related integral membrane glycoproteins involved in signalling of cell growth and metabolism. We have used the unique paradigm of pairs of Burkitt lymphoma cell lines (BLO2, BL30, BL41) with or without Epstein-Barr Virus (EBV) infection and cells transfected with EBV-related genes to examine effects of EBV on expression of these receptors at the gene and protein functional level. In BL30 and BL41 cells, EBV infection increased surface insulin binding and total receptor number by 2-and 18-fold, respectively. By contrast, EBV infection decreased total IGF-I receptors by 29 to 87% in all three cell lines. In general, there was a correlation between total receptor concentration and the level of insulin or IGF-I receptor mRNAs, although in one cell line insulin binding increased while receptor mRNA levels decreased slightly, suggesting posttranslational effects. BL41 cells transfected with a vector expressing the EBV latent membrane protein (LMP) exhibited a 2.6- to 3.2-fold increase in insulin receptor expression, whereas cells transfected with EBNA-2 (one of the EBV nuclear antigens) alone exhibited no effect. However, EBNA-2 appears to be required for the EBV effect on insulin receptor expression since cells infected with a mutant virus, P3JHRI, which lacks the EBNA-2 gene failed to show an increase in insulin receptor number. These data indicate that EBV infection of lymphocytes increases expression of insulin receptors while simultaneously decreasing expression of IGF-I receptors. The magnitude and sometimes even the direction of change, depends on host cell factors. A maximal increase in insulin receptors appears to require the coordinate action of several of the EBV proteins including LMP and EBNA-2. © 1993 Wiley-Liss, Inc.     

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review)

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.     

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review)

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.     

人类疱疹病毒7型糖蛋白gB、gH、gL、gO介导细胞融合

人类疱疹病毒 7 型(HHV-7)的感染依赖于包膜糖蛋白在病毒生命周期的多个阶段发挥功能. 这些蛋白质可以介导病毒吸附,病毒包膜和宿主细胞膜融合以及病毒在细胞间的接触传播. 将表达 HHV-7 糖蛋白的 293T 细胞与 HHV-7 易感的SupT1 细胞共培养,检测虫荧光素酶报告基因的表达,以鉴定介导膜融合的 HHV-7 糖蛋白. 研究发现,HHV-7 糖蛋白 gB、gH、gL、gO 能介导 293T 细胞与 SupT1 细胞的融合,且融合可被抗 CD4 单抗所抑制. 结果表明,糖蛋白 gB、gH、gL、gO对于 HHV-7 引发的膜融合是必需的,其中某个蛋白质或所形成的蛋白质复合物可能是 CD4 的配体.     

Mechanisms of Receptor/Coreceptor-Mediated Entry of Enveloped Viruses

Enveloped viruses enter host cells either through endocytosis, or by direct fusion of the viral envelope and the membrane of the host cell. However, some viruses, such as HIV-1, HSV-1, and Epstein-Barr can enter a cell through either mechanism, with the choice of pathway often a function of the ambient physical chemical conditions, such as temperature and pH. We develop a stochastic model that describes the entry process at the level of binding of viral glycoprotein spikes to cell membrane receptors and coreceptors. In our model, receptors attach the cell membrane to the viral membrane, while subsequent binding of coreceptors enables fusion. The model quantifies the competition between fusion and endocytotic entry pathways. Relative probabilities for each pathway are computed numerically, as well as analytically in the high viral spike density limit. We delineate parameter regimes in which fusion or endocytosis is dominant. These parameters are related to measurable and potentially controllable quantities such as membrane bending rigidity and receptor, coreceptor, and viral spike densities. Experimental implications of our mechanistic hypotheses are proposed and discussed.     

Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.