scholarly journals Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Dependent Viral Penetration into the Cytosol and Not for Viral Uncoating

2004 ◽  
Vol 78 (19) ◽  
pp. 10433-10441 ◽  
Author(s):  
Richard J. O. Barnard ◽  
Shakti Narayan ◽  
Geethanjali Dornadula ◽  
Michael D. Miller ◽  
John A. T. Young
Keyword(s):  
Viral Entry ◽  
Strong Support ◽  
Low Ph ◽  
Viral Envelope ◽  
Specific Cell ◽  
Dependent Manner ◽  
Surface Receptors ◽  
Ph Dependent ◽  
Avian Sarcoma ◽  
Hiv 1

ABSTRACT A novel entry mechanism has been proposed for the avian sarcoma and leukosis virus (ASLV), whereby interaction with specific cell surface receptors activates or primes the viral envelope glycoprotein (Env), rendering it sensitive to subsequent low-pH-dependent fusion triggering in acidic intracellular organelles. However, ASLV fusion seems to proceed to a lipid mixing stage at neutral pH, leading to the suggestion that low pH might instead be required for a later stage of viral entry such as uncoating (L. J. Earp, S. E. Delos, R. C. Netter, P. Bates, and J. M. White. J. Virol. 77:3058-3066, 2003). To address this possibility, hybrid virus particles were generated with the core of human immunodeficiency virus type 1 (HIV-1), a known pH-independent virus, and with subgroups A or B ASLV Env proteins. Infection of cells by these pseudotyped virions was blocked by lysosomotropic agents, as judged by inhibition of HIV-1 DNA synthesis. Furthermore, by using HIV-1 cores that contain a Vpr-β-lactamase fusion protein (Vpr-BlaM) to monitor viral penetration into the cytosol, we demonstrated that virions bearing ASLV Env, but not HIV-1 Env, enter the cytosol in a low-pH-dependent manner. This effect was independent of the presence of the cytoplasmic tail of ASLV Env. These studies provide strong support for the model, indicating that low pH is required for ASLV Env-dependent viral penetration into the cytosol and not for viral uncoating.

scholarly journals Detection of Cell-Cell Fusion Mediated by Ebola Virus Glycoproteins

2006 ◽  
Vol 80 (6) ◽  
pp. 2815-2822 ◽  
Author(s):  
Séverine Bär ◽  
Ayato Takada ◽  
Yoshihiro Kawaoka ◽  
Marc Alizon
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Ebola Virus ◽  
Low Ph ◽  
Viral Envelope ◽  
Target Cells ◽  
Direct Role ◽  
Influenza Virus Hemagglutinin ◽  
Ph Dependent

ABSTRACT Ebola viruses (EboV) are enveloped RNA viruses infecting cells by a pH-dependent process mediated by viral glycoproteins (GP) involving endocytosis of virions and their routing into acidic endosomes. As with well-characterized pH-dependent viral entry proteins, in particular influenza virus hemagglutinin, it is thought that EboV GP require activation by low pH in order to mediate fusion of the viral envelope with the membrane of endosomes. However, it has not yet been possible to confirm the direct role of EboV GP in membrane fusion and the requirement for low-pH activation. It was in particular not possible to induce formation of syncytia by exposing cells expressing EboV GP to acidic medium. Here, we have used an assay based on the induction of a β-galactosidase (lacZ) reporter gene in target cells to detect cytoplasmic exchanges, indicating membrane fusion, with cells expressing EboV GP (Zaire species). Acidic activation of GP-expressing cells was required for efficient fusion with target cells. The direct role of EboV GP in this process is indicated by its inhibition by anti-GP antibodies and by the lack of activity of mutant GP normally expressed at the cell surface but defective for virus entry. Fusion was not observed when target cells underwent acidic treatment, for example, when they were placed in coculture with GP-expressing cells before the activation step. This unexpected feature, possibly related to the nature of the EboV receptor, could explain the impossibility of inducing formation of syncytia among GP-expressing cells.

scholarly journals The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

2003 ◽  
Vol 77 (5) ◽  
pp. 3058-3066 ◽  
Author(s):  
Laurie J. Earp ◽  
Sue E. Delos ◽  
Robert C. Netter ◽  
Paul Bates ◽  
Judith M. White
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Low Ph ◽  
Target Cells ◽  
Dependent Manner ◽  
Neutral Ph ◽  
Subtype A ◽  
Avian Sarcoma ◽  
Virus Subtype ◽  
Cell Cell

ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

scholarly journals The Baculovirus GP64 Protein Mediates Highly Stable Infectivity of a Human Respiratory Syncytial Virus Lacking Its Homologous Transmembrane Glycoproteins

2004 ◽  
Vol 78 (1) ◽  
pp. 124-135 ◽  
Author(s):  
A. G. P. Oomens ◽  
Gail W. Wertz
Keyword(s):  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Low Ph ◽  
Human Respiratory Syncytial Virus ◽  
Dependent Manner ◽  
Practical Applications ◽  
Ph Dependent ◽  
Syncytial Virus

ABSTRACT Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP64/F, a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33°C than at 37°C. The open reading frames (ORFs) encoding GP64 or GP64/F, along with two marker ORFs encoding green fluorescent protein (GFP) and β-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP64/F protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RSΔsh,g,f/GP64 or RSΔsh,g,f/GP64/F, respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RSΔsh [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP64/F proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RSΔsh,g,f/GP64 and RSΔsh,g,f/GP64/F replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.

scholarly journals Cholesterol Dependence of Varicella-Zoster Virion Entry into Target Cells

2007 ◽  
Vol 81 (14) ◽  
pp. 7548-7558 ◽  
Author(s):  
S. Hambleton ◽  
S. P. Steinberg ◽  
M. D. Gershon ◽  
A. A. Gershon
Keyword(s):  
Lipid Rafts ◽  
Viral Entry ◽  
Membrane Lipid ◽  
Viral Envelope ◽  
Target Cells ◽  
Cholesterol Depletion ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Focus Formation ◽  
Varicella Zoster

ABSTRACT The entry of inhaled virions into airway cells is presumably the initiating step of varicella-zoster infection. In order to characterize viral entry, we studied the relative roles played by lipid rafts and clathrin-mediated transport. Virus and target cells were pretreated with agents designed to perturb selected aspects of endocytosis and membrane composition, and the effects of these perturbations on infectious focus formation were monitored. Infectivity was exquisitely sensitive to methyl-β-cyclodextrin (MβCD) and nystatin, which disrupt lipid rafts by removing cholesterol. These agents inhibited infection by enveloped, but not cell-associated, varicella-zoster virus (VZV) in a dose-dependent manner and exerted these effects on both target cell and viral membranes. Inhibition by MβCD, which could be reversed by cholesterol replenishment, rapidly declined as a function of time after exposure of target cells to VZV, suggesting that an early step in viral infection requires cholesterol. No effect of cholesterol depletion, however, was seen on viral binding; moreover, there was no reduction in the surface expression or internalization of mannose 6-phosphate receptors, which are required for VZV entry. Viral entry was energy dependent and showed concentration-dependent inhibition by chlorpromazine, which, among other actions, blocks clathrin-mediated endocytosis. These data suggest that both membrane lipid composition and clathrin-mediated transport are critical for VZV entry. Lipid rafts are likely to contribute directly to viral envelope integrity and, in the host membrane, may influence endocytosis, evoke downstream signaling, and/or facilitate membrane fusion.

scholarly journals The Avian Coronavirus Infectious Bronchitis Virus Undergoes Direct Low-pH-Dependent Fusion Activation during Entry into Host Cells

2006 ◽  
Vol 80 (7) ◽  
pp. 3180-3188 ◽  
Author(s):  
Victor C. Chu ◽  
Lisa J. McElroy ◽  
Vicky Chu ◽  
Beverley E. Bauman ◽  
Gary R. Whittaker
Keyword(s):  
Cell Fusion ◽  
Conformational Changes ◽  
Infectious Bronchitis Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Surface Proteins ◽  
Host Cells ◽  
Dependent Manner ◽  
Infectious Bronchitis ◽  
Ph Dependent

ABSTRACT Coronaviruses are the causative agents of respiratory disease in humans and animals, including severe acute respiratory syndrome. Fusion of coronaviruses is generally thought to occur at neutral pH, although there is also evidence for a role of acidic endosomes during entry of a variety of coronaviruses. Therefore, the molecular basis of coronavirus fusion during entry into host cells remains incompletely defined. Here, we examined coronavirus-cell fusion and entry employing the avian coronavirus infectious bronchitis virus (IBV). Virus entry into cells was inhibited by acidotropic bases and by other inhibitors of pH-dependent endocytosis. We carried out fluorescence-dequenching fusion assays of R18-labeled virions and show that for IBV, coronavirus-cell fusion occurs in a low-pH-dependent manner, with a half-maximal rate of fusion occurring at pH 5.5. Fusion was reduced, but still occurred, at lower temperatures (20°C). We observed no effect of inhibitors of endosomal proteases on the fusion event. These data are the first direct measure of virus-cell fusion for any coronavirus and demonstrate that the coronavirus IBV employs a direct, low-pH-dependent virus-cell fusion activation reaction. We further show that IBV was not inactivated, and fusion was unaffected, by prior exposure to pH 5.0 buffer. Virions also showed evidence of reversible conformational changes in their surface proteins, indicating that aspects of the fusion reaction may be reversible in nature.

scholarly journals HIV-1 Envelope Glycoprotein at the Interface of Host Restriction and Virus Evasion

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 311 ◽  
Author(s):  
Saina Beitari ◽  
Yimeng Wang ◽  
Shan-Lu Liu ◽  
Chen Liang
Keyword(s):  
Viral Infection ◽  
Adaptive Immunity ◽  
Viral Entry ◽  
Envelope Protein ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Innate Immune ◽  
Host Restriction ◽  
Hiv 1 ◽  
Viral Envelope Proteins

Without viral envelope proteins, viruses cannot enter cells to start infection. As the major viral proteins present on the surface of virions, viral envelope proteins are a prominent target of the host immune system in preventing and ultimately eliminating viral infection. In addition to the well-appreciated adaptive immunity that produces envelope protein-specific antibodies and T cell responses, recent studies have begun to unveil a rich layer of host innate immune mechanisms restricting viral entry. This review focuses on the exciting progress that has been made in this new direction of research, by discussing various known examples of host restriction of viral entry, and diverse viral countering strategies, in particular, the emerging role of viral envelope proteins in evading host innate immune suppression. We will also highlight the effective cooperation between innate and adaptive immunity to achieve the synergistic control of viral infection by targeting viral envelope protein and checking viral escape. Given that many of the related findings were made with HIV-1, we will use HIV-1 as the model virus to illustrate the basic principles and molecular mechanisms on host restriction targeting HIV-1 envelope protein.

scholarly journals Histone Deacetylase 6 Regulates Human Immunodeficiency Virus Type 1 Infection

2005 ◽  
Vol 16 (11) ◽  
pp. 5445-5454 ◽  
Author(s):  
Agustín Valenzuela-Fernández ◽  
Susana Álvarez ◽  
Mónica Gordon-Alonso ◽  
Marta Barrero ◽  
Ángeles Ursa ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Membrane Dynamics ◽  
Fusion Pore ◽  
Dependent Manner ◽  
Histone Deacetylase 6 ◽  
Surface Receptors ◽  
Immunodeficiency Virus ◽  
Syncytia Formation ◽  
Hiv 1

Efficient human immunodeficiency virus (HIV)-1 infection depends on multiple interactions between the viral gp41/gp120 envelope (Env) proteins and cell surface receptors. However, cytoskeleton-associated proteins that modify membrane dynamics may also regulate the formation of the HIV-mediated fusion pore and hence viral infection. Because the effects of HDAC6-tubulin deacetylase on cortical α-tubulin regulate cell migration and immune synapse organization, we explored the possible role of HDAC6 in HIV-1-envelope-mediated cell fusion and infection. The binding of the gp120 protein to CD4+-permissive cells increased the level of acetylated α-tubulin in a CD4-dependent manner. Furthermore, overexpression of active HDAC6 inhibited the acetylation of α-tubulin, and remarkably, prevented HIV-1 envelope-dependent cell fusion and infection without affecting the expression and codistribution of HIV-1 receptors. In contrast, knockdown of HDAC6 expression or inhibition of its tubulin deacetylase activity strongly enhanced HIV-1 infection and syncytia formation. These results demonstrate that HDAC6 plays a significant role in regulating HIV-1 infection and Env-mediated syncytia formation.

scholarly journals Imaging Viral Infection by Fluorescence Microscopy: Focus on HIV-1 Early Stage

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 213
Author(s):  
Soumajit Mukherjee ◽  
Emmanuel Boutant ◽  
Eleonore Réal ◽  
Yves Mély ◽  
Halina Anton
Keyword(s):  
Host Cell ◽  
Viral Entry ◽  
Integration Site ◽  
Viral Envelope ◽  
Integration Step ◽  
Imaging Studies ◽  
Cytoplasmic Transport ◽  
Capsid Shell ◽  
Wide Range ◽  
Hiv 1

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.

scholarly journals gp120 Alters Its Conformation to Enhance Evasiveness and Infectivity

2021 ◽  
Author(s):  
Joseph A. Ayariga ◽  
Logan Gildea ◽  
Ayodeji Ipimoroti ◽  
Qiana L. Matthews
Keyword(s):  
Structural Dynamics ◽  
Viral Entry ◽  
Antiviral Agents ◽  
Host Immunity ◽  
Viral Envelope ◽  
Cell Surface Receptor ◽  
Type I ◽  
Structural Constraints ◽  
Conformational States ◽  
Hiv 1

Infection by human immunodeficiency virus type I (HIV-1) requires virus particle binding to host cell-surface receptor CD4 via the viral envelope glycoprotein gp120. HIV-1 therapy and prevention efforts involve development of mimetic or recombinant gp120 vaccines or deployment of antiviral agents that target specific epitopes of gp120. The unliganded conformational state of gp120 is closed, whereas the CD4-bound state is open. However, in between, there exist dynamic conformational states, indicating intrinsically flexible region(s) of structural dynamics, imposing a structural challenge for developing drug or antibody targets. Known conformational states of gp120 were determined by X-ray crystallographic and cryo-electron microscopy, and neither method captures the population of gp120 species arising from conformational plasticity, motions, and transitions. gp120 plasticity brings up several important questions. How will differences in conformation affect receptor binding, antibody recognition, and neutralization? Which regions are crucial for gp120 structural plasticity? How could structural dynamics influence HIV-1 evasiveness against host immunity and drugs or vaccines, and facilitate the viral entry into its host? This review explores the structural constraints presented by conformational states of the glycoprotein to antibodies or drugs and how these conformational states provide structural avenues for the virus to escape neutralizing agents and evade host immunity.

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