scholarly journals Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

2021 ◽  
Author(s):  
Katherine N Liu ◽  
Steven G. Boxer
Keyword(s):  
Membrane Fusion ◽  
Influenza A ◽  
Pore Formation ◽  
Fluorescent Labeling ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Enveloped Viruses ◽  
Virus Assay ◽  
Virus Content ◽  
Target Membrane

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.

The Role of the Transmembrane and of the Intraviral Domain of Glycoproteins in Membrane Fusion of Enveloped Viruses

2000 ◽  
Vol 20 (6) ◽  
pp. 571-595 ◽  
Author(s):  
Britta Schroth-Diez ◽  
Kai Ludwig ◽  
Bolormaa Baljinnyam ◽  
Christine Kozerski ◽  
Qiang Huang ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Fusion Process ◽  
Fusion Pore ◽  
Minimum Length ◽  
Viral Fusion ◽  
Fusion Activity ◽  
Enveloped Viruses

Fusion of enveloped viruses with their target membrane is mediated by viral integral glycoproteins. A conformational change of their ectodomain triggers membrane fusion. Several studies suggest that an extended, triple-stranded rod-shaped α-helical coiled coil resembles a common structural and functional motif of the ectodomain of fusion proteins. From that, it is believed that essential features of the fusion process are conserved among the various enveloped viruses. However, this has not been established so far for the highly conserved transmembrane and intraviral sequences of fusion proteins. The article will focus on the role of both sequences in the fusion process. Recent studies from various enveloped viruses strongly imply that a transmembrane domain with a minimum length is required for later steps of membrane fusion, i.e., the formation and enlargement of the aqueous fusion pore. Although no specific sequence of the TM is necessary for pore formation, distinct properties and motifs of the domain may be obligatory to ascertain full fusion activity. However, with some exceptions, the intraviral domain seems to be not required for fusion activity of viral fusion proteins.

scholarly journals Precise Triggering and Chemical Control of Single-Virus Fusion within Endosomes

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Sourav Haldar ◽  
Kenta Okamoto ◽  
Rebecca A. Dunning ◽  
Peter M. Kasson
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Influenza A ◽  
Living Cells ◽  
Membrane Curvature ◽  
Endosomal Trafficking ◽  
Viral Fusion ◽  
Enveloped Viruses ◽  
Endosomal Membrane ◽  
Virus Fusion

ABSTRACT Many enveloped viruses infect cells within endocytic compartments. The pH drop that accompanies endosomal maturation, often in conjunction with proteolysis, triggers viral proteins to insert into the endosomal membrane and drive fusion. Fusion dynamics have been studied by tracking viruses within living cells, which limits the precision with which fusion can be synchronized and controlled, and reconstituting viral fusion to synthetic membranes, which introduces nonphysiological membrane curvature and composition. To overcome these limitations, we report chemically controllable triggering of single-virus fusion within endosomes. We isolated influenza (A/Aichi/68; H3N2) virus:endosome conjugates from cells, immobilized them in a microfluidic flow cell, and rapidly and controllably triggered fusion. Observed lipid-mixing kinetics were surprisingly similar to those of influenza virus fusion with model membranes of opposite curvature: 80% of single-virus events had indistinguishable kinetics. This result suggests that endosomal membrane curvature is not a key permissive feature for viral entry, at least lipid mixing. The assay preserved endosomal membrane asymmetry and protein composition, providing a platform to test how cellular restriction factors and altered endosomal trafficking affect viral membrane fusion. IMPORTANCE Many enveloped viruses infect cells via fusion to endosomes, but controlling this process within living cells has been challenging. We studied the fusion of influenza virus virions to endosomes in a chemically controllable manner. Extracting virus:endosome conjugates from cells and exogenously triggering fusion permits precise study of virus:endosome fusion kinetics. Surprisingly, endosomal curvature does not grossly alter fusion kinetics, although membrane deformability does. This supports a model for influenza virus entry where cells restrict or permit membrane fusion by changing deformability, for instance, using interferon-induced proteins.

Mechanics of membrane fusion/pore formation

2015 ◽  
Vol 185 ◽  
pp. 109-128 ◽  
Author(s):  
Marc Fuhrmans ◽  
Giovanni Marelli ◽  
Yuliya G. Smirnova ◽  
Marcus Müller
Keyword(s):  
Membrane Fusion ◽  
Pore Formation ◽  
Fusion Pore

scholarly journals Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion.

1994 ◽  
Vol 127 (6) ◽  
pp. 1885-1894 ◽  
Author(s):  
J Zimmerberg ◽  
R Blumenthal ◽  
D P Sarkar ◽  
M Curran ◽  
S J Morris
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Pore Formation ◽  
Fusion Pore ◽  
Restricted Movement ◽  
Influenza Hemagglutinin ◽  
Simultaneous Measurements ◽  
Cell Membrane Capacitance ◽  
Total Fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".

Modulating the influenza A virus – target membrane fusion interface with synthetic DNA-lipid receptors

2021 ◽  
Author(s):  
Elizabeth Webster ◽  
Katherine Liu ◽  
Robert Rawle ◽  
Steven Boxer
Keyword(s):  
Cell Membrane ◽  
Influenza A Virus ◽  
Influenza A ◽  
Viral Fusion ◽  
Fusion Peptides ◽  
Viral Receptor ◽  
Synthetic Dna ◽  
Rate Limiting Step ◽  
Target Membrane ◽  
Fusion Cell

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell surface glycans are highly heterogenous in length and glycosylation density, which leads to variation in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion, or the low-pH triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

Modulating the influenza A virus – target membrane fusion interface with synthetic DNA-lipid receptors

2021 ◽  
Author(s):  
Elizabeth Webster ◽  
Katherine Liu ◽  
Robert Rawle ◽  
Steven Boxer
Keyword(s):  
Cell Membrane ◽  
Influenza A Virus ◽  
Influenza A ◽  
Viral Fusion ◽  
Fusion Peptides ◽  
Viral Receptor ◽  
Synthetic Dna ◽  
Rate Limiting Step ◽  
Target Membrane ◽  
Fusion Cell

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell surface glycans are highly heterogenous in length and glycosylation density, which leads to variation in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion, or the low-pH triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

scholarly journals pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Thomas Gerlach ◽  
Luca Hensen ◽  
Tatyana Matrosovich ◽  
Janina Bergmann ◽  
Michael Winkler ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Fusion ◽  
Influenza A ◽  
Target Cells ◽  
Viral Fusion ◽  
Influenza A Viruses ◽  
Ph Optimum ◽  
High Ph ◽  
Innate Defense ◽  
Antiviral State

ABSTRACT The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.

scholarly journals Reevaluating Herpes Simplex Virus Hemifusion

2010 ◽  
Vol 84 (22) ◽  
pp. 11814-11821 ◽  
Author(s):  
Julia O. Jackson ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Viral Fusion Proteins ◽  
Simplex Virus

ABSTRACT Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.

scholarly journals Herpesvirus Nuclear Egress across the Outer Nuclear Membrane

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2356
Author(s):  
Richard J. Roller ◽  
David C. Johnson
Keyword(s):  
Membrane Fusion ◽  
Nuclear Membrane ◽  
Membrane Curvature ◽  
Fusion Pore ◽  
Second Step ◽  
Second Phase ◽  
Viral Fusion ◽  
Outer Nuclear Membrane ◽  
Viral Fusion Protein ◽  
Nuclear Egress

Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.

Sign in / Sign up

Export Citation Format

Share Document