Molecular mechanisms of virus spread and virion components as tools of virulence. A review

Despite of differences in replication strategy among virus families, some basic principles have remained similar. Analogous mechanisms govern virus entry into cells and the use of enzymes which direct the replication of the virus genome. The function of many cell surface receptors (such as glycosoaminoglycans, glycoproteins, proteins) which interact with viral capsid proteins or envelope glycoproteins has recently been elucidated. The list of cellular receptors (Table I) is still far from being final. The capsid components, similarly as the envelope glycoproteins, may form specific pocket like sites, which interact with the cell surface receptors. Neutralizing antibodies usually react with antigenic domains adjacent to the receptor binding site(s) and hamper the close contact inevitable for virion attachment. In the case of more complex viruses, such as herpes simplex virus, different viral glycoproteins interact with several cellular receptors. At progressed phase of adsorption the virions are engulfed into endocytic vesicles and the virion fusion domain(s) become(s) activated. The outer capsid components of reoviruses which participate in adsorption and fusion may get activated already in the lumen of digestive tract, i.e. before their engulfment by resorptive epithelium cells. Activation of the hydrophobic fusion domain(s) is a further important step allowing to pass through the lipid bilayer when penetrating the cell membrane in order to reach the cytosol. Activation of the virion fusion domain is accomplished by a conformation change, which occurs at acid pH (influenza virus hemagglutinin, sigma 1 protein of the reovirus particle) and/or after protease treatment. The herpes simplex virus fusion factors (gD and gH) undergo conformation changes by a pH-independent mechanism triggered due to interaction with the cell surface receptor(s) and mediated by mutual interactions with the viral envelope glycoproteins. The virion capsid or envelope components participating in the entry and membrane fusion are not the only tools of virulence. The correct function of virus coded proteins, which participate in replication of the viral genome, and/or in the supply of necessary nucleotides, may be very essential. In the case of enteroviruses, which RNA interacts with ribosomes directly, the correct configuration of the non-coding viral RNA sequence is crucial for initiation of translation occurring in the absence of the classical "cap" structure.     

Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry

During the entry process many icosahedral viruses must adopt a lower-order symmetry or incur a symmetry mismatch to release their genome through a single site. A membrane model system in which poliovirus was bound to receptor-decorated liposomes was used to pioneer techniques that studied the break in the symmetry of the initial attachment complex by cryo-electron microscopy. Novel methods involving a fiducial marker for the membrane contact point were developed to objectively determine the symmetry of this complex and provide a starting model to initiate a bootstrap orientation refinement. Here we analyze how errors in the subjective assignment of this position affect the determination of symmetry, and the accuracy of calculating Euler angles for each raw image. In this study we have optimized the method and applied it to study the membrane-attachment complex of Semliki Forest virus (SFV), a model system for enveloped virus fusion. The resulting reconstruction of the SFV-membrane complex with a fiducial provides the first experimental evidence that this pre-fusion cell entry intermediate approaches the membrane along the viral 5-fold axis. The analysis reported here, and its subsequent application to enveloped virus fusion, indicate that this is a robust tool for solving the structures of mixed-symmetry complexes.     

疱疹病毒膜融合的分子机制

囊膜病毒与宿主细胞的膜融合是病毒入侵宿主细胞的重要过程,这一过程涉及到病毒囊膜表面糖蛋白与宿主细胞表面受体之间的相互作用和构象变化．疱疹病毒有多个糖蛋白及不同类型的细胞作用受体,相应的受体-糖蛋白复合体构成方式也有多种,其引致的膜融合机制被认为是目前病毒融合机制研究中最复杂的,近年来被广泛研究并取得突破性进展．从病毒糖蛋白与相应受体的结构与功能、受体-糖蛋白复合体的形成与入侵途径,以及膜融合模式几个方面,全面综述疱疹病毒膜融合的分子机制,并展望了未来研究趋势．     

Novel Entry Pathway of Bovine Herpesvirus 1 and 5

Herpesviruses enter cells by a yet poorly understood mechanism. We visualized the crucial steps of the entry pathway of bovine herpesvirus 1 (BHV-1) and BHV-5 by transmission and scanning electron microscopy, employing cryotechniques that include time monitoring, ultrarapid freezing, and freeze substitution of cultured cells inoculated with virus. A key step in the entry pathway of both BHV-1 and BHV-5 is a unique fusion of the outer phospholipid layer of the viral envelope with the inner layer of the plasma membrane and vice versa resulting in “crossing” of the fused membranes and in partial insertion of the viral envelope into the plasma membrane. The fusion area is proposed to function as an axis for driving the virus particle into an invagination that is concomitantly formed close to the fusion site. The virus particle enters the cytoplasm through the opened tip of the invagination, and the viral envelope defuses from the plasma membrane. There is strong evidence that the intact virus particle is then transported to the nuclear region.     

The earliest steps in hepatitis B virus infection

The early steps in hepatitis B virus (HBV) infection, a human hepadnavirus, initiates from cell attachment followed by entry and delivery of the genetic information to the nucleus. Despite the fact that these steps determine the virus-related pathogenesis, their molecular basis is poorly understood. Cumulative data suggest that this process can be divided to cell attachment, endocytosis, membrane fusion and post-fusion consecutive steps. These steps are likely to be regulated by the viral envelope proteins and by the cellular membrane, receptors and extracellular matrix. In the absence of animal model for HBV, the duck hepadnavirus DHBV turned out to be a fruitful animal model. Therefore data concerning the early, post-attachment steps in hepadnaviral entry are largely based on studies performed with DHBV in primary duck liver hepatocytes. These studies are now starting to illuminate the mechanisms of hepadnavirus route of cell entry and to provide some new insights on the molecular basis of the strict species specificity of hepadnavirus infection.     

The earliest steps in hepatitis B virus infection

The early steps in hepatitis B virus (HBV) infection, a human hepadnavirus, initiates from cell attachment followed by entry and delivery of the genetic information to the nucleus. Despite the fact that these steps determine the virus-related pathogenesis, their molecular basis is poorly understood. Cumulative data suggest that this process can be divided to cell attachment, endocytosis, membrane fusion and post-fusion consecutive steps. These steps are likely to be regulated by the viral envelope proteins and by the cellular membrane, receptors and extracellular matrix. In the absence of animal model for HBV, the duck hepadnavirus DHBV turned out to be a fruitful animal model. Therefore data concerning the early, post-attachment steps in hepadnaviral entry are largely based on studies performed with DHBV in primary duck liver hepatocytes. These studies are now starting to illuminate the mechanisms of hepadnavirus route of cell entry and to provide some new insights on the molecular basis of the strict species specificity of hepadnavirus infection.     

X-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein

Although many viral receptors have been identified, the ways in which they interact with their cognate viruses are not understood at the molecular level. We have determined the X-ray structure of a complex between calcium-containing modules of the very low-density lipoprotein receptor and the minor group human rhinovirus HRV2. The receptor binds close to the icosahedral five-fold vertex, with only one module per virus protomer. The binding face of this module is defined by acidic calcium-chelating residues and, in particular, by an exposed tryptophan that is highly conserved. The attachment site on the virus involves only residues from VP1, particularly a lysine strictly conserved in all minor group HRVs. The disposition of the attached ligand-binding repeats around the five-fold axis, together with the proximity of the N- and C-terminal ends of adjacent modules, suggests that more than one repeat in a single receptor molecule might attach simultaneously.     

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.     

Receptor binding and pH stability — How influenza A virus hemagglutinin affects host-specific virus infection

Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus.     

How Viruses Use the Endoplasmic Reticulum for Entry,Replication, and Assembly

To cause infection, a virus enters a host cell, replicates, and assembles, with the resulting new viral progeny typically released into the extracellular environment to initiate a new infection round. Virus entry, replication, and assembly are dynamic and coordinated processes that require precise interactions with host components, often within and surrounding a defined subcellular compartment. Accumulating evidence pinpoints the endoplasmic reticulum (ER) as a crucial organelle supporting viral entry, replication, and assembly. This review focuses on the molecular mechanism by which different viruses co-opt the ER to accomplish these crucial infection steps. Certain bacterial toxins also hijack the ER for entry. An interdisciplinary approach, using rigorous biochemical and cell biological assays coupled with advanced microscopy strategies, will push to the next level our understanding of the virus-ER interaction during infection.To trigger infection, a virus binds to receptors on a host cell’s plasma membrane. This interaction induces virus internalization, and initiates a complex journey of the viral particle into the host’s interior that leads to either nonproductive or productive infection (Mercer et al. 2010). In nonproductive infection, the virus may be targeted to and trapped in organelles unsupportive of viral membrane fusion or penetration, events which normally enable the viral nucleic acid access to the host cytosol or nucleus. Alternatively, the virus could be transported to a degradative intracellular compartment in which it is destroyed. In contrast, for productive infection, a viral particle must avoid these nonproductive routes and traffic along a pathway that allows it to reach the appropriate replication and assembly site. Successful infection is usually completed when the newly assembled particle is released into the extracellular milieu, in which it can promote another infection round. Thus, the ability to co-opt a host cell entry pathway leading to efficient replication and assembly ultimately dictates the fate of an incoming virus.For proper entry, replication, and assembly, viruses often rely on the complex membranous network surrounding and residing within the host cell, such as the plasma, endolysosomal, and endoplasmic reticulum (ER) membranes. Selecting the suitable membrane system requires several considerations. To support entry, the membranous system must possess triggers capable of inducing the necessary conformational changes that facilitate viral membrane fusion or penetration (Inoue et al. 2011). Examples of cellular triggers include receptors, low pH, proteases, chaperones, and reductases. Additionally, because viral replication and assembly often occur in the context of virus-induced membranous structures derived from host membranes, the membranous network of choice should accommodate these remodeling reactions (Miller and Krijnse-Locker 2008). Moreover, as a virus commonly manipulates the host immune system to sustain infection, a membrane’s ability to provide the virus with such an opportunity would offer tremendous advantages during the infection course (Takeuchi and Akira 2009).A wealth of data implicates the endoplasmic reticulum (ER), one of the most elaborate membranous networks in a cell (Shibata et al. 2009), as the organelle many viruses exploit during infection. This review focuses on how viruses co-opt the ER to enter, replicate, and assemble in the target cell. We will also draw parallels from the mechanisms by which bacterial toxins use the ER for entry. Together, these insights should unveil clues regarding why many viruses select the ER during infection.     

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

ABSTRACT: Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (-) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (-) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.     

Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.     

Dynamic tradeoffs in the raft-mediated entry of human immunodeficiency virus type 1 into cells

To initiate an infection human immunodeficiency virus type 1 (HIV-1) particles must first bind to receptors on the surface of their host cells, a process that eventually leads to fusion of viral and cellular membranes and release of the viral genome into the cytoplasm. Understanding the molecular mechanisms of these processes may enable the development of new anti-HIV strategies. Disagreement currently prevails on the role in virus entry of microdomains within the cellular plasma membrane known as lipid rafts. Experiments have suggested that lipid rafts, in their interactions with cellular receptors and viral particles, either promote or have minimal effect on viral entry. Here we develop a dynamic model for HIV-1 entry that enables us to identify and quantitatively assess tradeoffs that can arise from the clustering of receptors in rafts. Specifically, receptor clustering can be detrimental to the initiation of viral infection by reducing the probability that a virus particle finds its primary receptor, CD4. However, receptor clustering can also enable a virus particle, once bound, to rapidly form multivalent interactions with receptors and co-receptors that are required for virus-cell membrane fusion. We show how the resolution of such tradeoffs hinges on the level and spatial distribution of receptors and co-receptors on the cell surface, and we discuss implications of these effects for the design of therapeutics that inhibit HIV-1 entry.     

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality.Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells,followed by fusion of virus-cell membrane also mediated by GP.Using an human immunodeficiency virus (HIV)-based pseudotyping system,the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions.We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry.An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry.It was found that R64 and K95 are involved in receptor binding.In contrast,some residues such as I170 are important for viral entry,but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data,suggesting that these residues are involved in post-binding steps of viral entry.Furthermore,our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.     

The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins–a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion–the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.     

Characterization of the ion channels formed by poliovirus in planar lipid membranes.

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.     

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.     

Stoichiometry of envelope glycoprotein trimers in the entry of human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs) function as a trimer, mediating virus entry by promoting the fusion of the viral and target cell membranes. HIV-1 Env trimers induce membrane fusion through a pH-independent pathway driven by the interaction between an Env trimer and its cellular receptors, CD4 and CCR5/CXCR4. We studied viruses with mixed heterotrimers of wild-type and dominant-negative Envs to determine the number (T) of Env trimers required for HIV-1 entry. To our surprise, we found that a single Env trimer is capable of supporting HIV-1 entry; i.e., T = 1. A similar approach was applied to investigate the entry stoichiometry of envelope glycoproteins from amphotropic murine leukemia virus (A-MLV), avian sarcoma/leukosis virus type A (ASLV-A), and influenza A virus. When pseudotyped on HIV-1 virions, the A-MLV and ASLV-A Envs also exhibit a T = 1 entry stoichiometry. In contrast, eight to nine influenza A virus hemagglutinin trimers function cooperatively to achieve membrane fusion and virus entry, using a pH-dependent pathway. The different entry requirements for cooperativity among Env trimers for retroviruses and influenza A virus may influence viral strategies for replication and evasion of the immune system.     

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Authors Summary

Herpes simplex virus (HSV) is a widespread human pathogen. Four viral glycoproteins (gD, gB, gH/gL) are required for HSV entry into host cells. gD binding to a cell surface receptor triggers conformational changes in the other viral glycoproteins leading to membrane fusion and viral entry. Two structurally unrelated cellular protein receptors, nectin-1 and HVEM, can mediate HSV entry upon binding to gD. The structure presented here reveals the molecular basis for the stable interaction between HSV-1 gD and the receptor nectin-1. Comparison with the previously determined structures of the gD/HVEM complex and unliganded gD shows that, despite the fact that the two receptors interact with different binding sites, they both cause a similar conformational change in gD. Therefore, our data point to a conserved mechanism for receptor mediated activation of the HSV entry process. In addition, the gD/Nectin-1 structure reveals that the gD-binding site overlaps with a surface involved in nectin-1 homo-dimerization. This observation explains how gD interferes with the cell adhesion function of nectin-1. Finally, the gD/Nectin-1 complex displays similarities with other viral ligands bound to immunoglobulin-like receptors suggesting a convergent mechanism for receptors selection and usage.