scholarly journals EWI-2 Inhibits Cell–Cell Fusion at the HIV-1 Virological Presynapse

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1082
Author(s):  
Emily E. Whitaker ◽  
Nicholas J. Matheson ◽  
Sarah Perlee ◽  
Phillip B. Munson ◽  
Menelaos Symeonides ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Virological Synapse ◽  
Virus Particles ◽  
Fusion Inhibitors ◽  
Producer Cell ◽  
Cellular Components ◽  
Cell To Cell Transfer ◽  
Hiv 1 ◽  
Cell Cell

Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell–cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell–cell fusion. Using quantitative fluorescence microscopy, shRNA knockdowns, and cell–cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also find that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell–cell fusion and provides novel insight into how syncytia are prevented from fusing indefinitely.

scholarly journals EWI-2 Inhibits Cell-Cell Fusion at the Virological Presynapse

2018 ◽  
Author(s):  
Emily E. Whitaker ◽  
Menelaos Symeonides ◽  
Phillip B. Munson ◽  
Markus Thali
Keyword(s):  
Cell Fusion ◽  
Virological Synapse ◽  
Viral Receptor ◽  
Virus Particles ◽  
History Of ◽  
Producer Cell ◽  
Cellular Components ◽  
Cell To Cell Transfer ◽  
Hiv 1 ◽  
Cell Cell

AbstractCell-to-cell transfer of virus particles through the virological synapse (VS) is a highly efficient mode of HIV-1 transmission. Formation of the VS, a transient multiform adhesion structure, is mediated through an interaction between the HIV-1 envelope glycoprotein (Env) and the viral receptor CD4 on the surface of infected cell and target cell, respectively. Given that Env, unlike many other viral fusogens, can mediate the merger of membranes at neutral pH, the close encounter of infected and uninfected cells would seem prone to result in cell-cell fusion and thus the formation of syncytia. However, while it is being recognized now that small, T cell-based syncytia are indeed a defining feature of the natural history of HIV-1, the majority of VSs nevertheless resolve without fusion, thus securing continued virus spread. Gag, the main viral structural component, is partially responsible for restraining Env and preventing it from becoming fusogenic before being incorporated into particles. In addition, a few cellular factors, including tetraspanins and ezrin, have also been shown to inhibit Env’s activity while this fusogen is still part of the producer cell.Here, we identify EWI-2, a protein that was previously shown to associate with the tetraspanins CD9 and CD81 and also with ezrin, as a host factor that contributes to the inhibition of Env­mediated cell-cell fusion. Using fluorescence microscopy, flow cytometry, and TZM-bl fusion assays, we show that EWI-2, comparable to tetraspanins, while overall being downregulated upon HIV-1 infection, accumulates at the producer cell side of the VS (i.e. the presynapse), where it contributes to the fusion-preventing activities of the other viral and cellular components.

scholarly journals Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 326
Author(s):  
Danwei Yu ◽  
Yang Su ◽  
Xiaohui Ding ◽  
Yuanmei Zhu ◽  
Bo Qin ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Secondary Mutation ◽  
Fusion Inhibitors ◽  
Hiv 1

Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

scholarly journals Derivation and Characterization of a Simian Immunodeficiency Virus SIVmac239 Variant with Tropism for CXCR4

2009 ◽  
Vol 83 (19) ◽  
pp. 9911-9922 ◽  
Author(s):  
Gregory Q. Del Prete ◽  
Beth Haggarty ◽  
George J. Leslie ◽  
Andrea P. O. Jordan ◽  
Josephine Romano ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Cell Fusion ◽  
High Efficiency ◽  
Rhesus Macaques ◽  
Immunodeficiency Virus ◽  
Specific Antagonist ◽  
Hiv 1 ◽  
Cell Cell

ABSTRACT Like human immunodeficiency virus type 1 (HIV-1), most simian immunodeficiency virus (SIV) strains use CCR5 to establish infection. However, while HIV-1 can acquire the ability to use CXCR4, SIVs that utilize CXCR4 have rarely been reported. To explore possible barriers against SIV coreceptor switching, we derived an R5X4 variant, termed 239-ST1, from the R5 clone SIVmac239 by serially passaging virus in CD4+ CXCR4+ CCR5− SupT1 cells. A 239-ST1 env clone, designated 239-ST1.2-32, used CXCR4 and CCR5 in cell-cell fusion and reporter virus infection assays and conferred the ability for rapid, cytopathic infection of SupT1 cells to SIVmac239. Viral replication was inhibitable by the CXCR4-specific antagonist AMD3100, and replication was abrogated in a novel CXCR4− SupT1 line. Surprisingly, parental SIVmac239 exhibited low-level replication in SupT1 cells that was not observed in CXCR4− SupT1 cells. Only two mutations in the 239-ST1.2-32 Env, K47E in the C1 domain and L328W in the V3 loop, were required for CXCR4 use in cell-cell fusion assays, although two other V3 changes, N316K and I324M, improved CXCR4 use in infection assays. An Env cytoplasmic tail truncation, acquired during propagation of 239-ST1 in SupT1 cells, was not required. Compared with SIVmac239, 239-ST1.2-32 was more sensitive to neutralization by five of seven serum and plasma samples from SIVmac239-infected rhesus macaques and was approximately 50-fold more sensitive to soluble CD4. Thus, SIVmac239 can acquire the ability to use CXCR4 with high efficiency, but the changes required for this phenotype may be distinct from those for HIV-1 CXCR4 use. This finding, along with the increased neutralization sensitivity of this CXCR4-using SIV, suggests a mechanism that could select strongly against this phenotype in vivo.

scholarly journals Human immunodeficiency virus 1 (HIV-1) envelope-dependent cell–cell fusion modulation by HIV-positive sera is related to disease progression

2005 ◽  
Vol 86 (7) ◽  
pp. 1961-1966 ◽  
Author(s):  
L. Huerta ◽  
G. Gómez-Icazbalceta ◽  
L. Soto-Ramírez ◽  
M. Viveros-Rogel ◽  
R. Rodríguez ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Cell Damage ◽  
Hiv Positive ◽  
Immunodeficiency Virus ◽  
Load Removal ◽  
Dependent Cell ◽  
Hiv 1 ◽  
Cell Cell

Fusion of CD4+ cells by HIV-1 envelope proteins (Env) is a mechanism of virus spread and cell damage. Production of antibodies able to influence cell–cell fusion in vivo may affect the course of the infection. The effect of sera from 49 HIV-1-positive patients was tested on an in vitro fusion assay using Env-expressing and normal Jurkat T cells labelled with DiI and DiO dyes, and flow cytometry for quantification of cell–cell fusion. Sera varied in their activity on fusion: 69·4 % inhibited, 24·5 % had no effect and 6·1 % enhanced cell fusion. Fusion activity correlated positively with the CD4+ T-cell count and inversely with the viral load. Removal of IgG or IgM from sera reduced or eliminated inhibition and enhancing activities, respectively. Antibodies with inhibitory activity predominate in early and intermediate stages of infection, whereas loss of inhibition or enhancement of fusion correlates with progression to AIDS.

Studies of membrane fusion. I. Paramyxovirus-induced cell fusion, a scanning electron-microscope study

1977 ◽  
Vol 28 (1) ◽  
pp. 179-188
Author(s):  
S. Knutton ◽  
D. Jackson ◽  
M. Ford
Keyword(s):  
Cell Surface ◽  
Cell Fusion ◽  
Hela Cells ◽  
Cell Swelling ◽  
Cell Contact ◽  
Cell Agglutination ◽  
Scanning Electron Microscope Study ◽  
Virus Particles ◽  
Scanning Electron ◽  
Cell Cell

Fusion of erythrocytes and HeLa cells with Sendai and Newcastle disease viruses has been studied by scanning electron microscopy. Most virus particles are spherical but vary in diameter from approximately 200 to approximately 600 nm. At 4 degrees C virus particles bind randomly to the cell surface and at high cell densities cross-linking of adjacent cells by virus particles results in cell agglutination. Cell-cell fusion takes place when the agglutinated cell suspension is warmed to 37 degrees C. Fusion is initiated at sites of cell-cell contact and is accompanied in all cases by cell swelling. In the case of suspension HeLa cells, virally mediated cell swelling involves an ‘unfolding’ of cell surface microvilli and results in the formation of smooth-surfaced single or fused cells. With erythrocytes, swelling results in haemolysis. There is a dramatic reduction in the numbers of virus particles bound to cells following fusion.

scholarly journals Cell–cell fusion and internalization of the CNS-based, HIV-1 co-receptor, APJ

Virology ◽  
2003 ◽  
Vol 307 (1) ◽  
pp. 22-36 ◽  
Author(s):  
Naiming Zhou ◽  
Xuejun Fan ◽  
Muhammad Mukhtar ◽  
Jianhua Fang ◽  
Charvi A Patel ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Hiv 1 ◽  
Cell Cell

scholarly journals Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Danwei Yu ◽  
Jing Xue ◽  
Huamian Wei ◽  
Zhe Cong ◽  
Ting Chen ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Therapeutic Efficacy ◽  
Rhesus Macaques ◽  
Heptad Repeat ◽  
Fusion Inhibitor ◽  
Resistance Mutations ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance. IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

scholarly journals Cell–cell and virus–cell fusion assay–based analyses of alanine insertion mutants in the distal α9 portion of the JRFL gp41 subunit from HIV-1

2019 ◽  
Vol 294 (14) ◽  
pp. 5677-5687 ◽  
Author(s):  
Mizuki Yamamoto ◽  
Qingling Du ◽  
Jiping Song ◽  
Hongyun Wang ◽  
Aya Watanabe ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Hiv 1 ◽  
Insertion Mutants ◽  
Cell Cell

scholarly journals Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

2019 ◽  
Vol 218 (9) ◽  
pp. 3117-3133 ◽  
Author(s):  
Frank van Drogen ◽  
Ranjan Mishra ◽  
Fabian Rudolf ◽  
Michal J. Walczak ◽  
Sung Sik Lee ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Cell Fusion ◽  
Polarized Growth ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Pheromone Signaling ◽  
Stress Signals ◽  
Cell Cell

Cells continuously adapt cellular processes by integrating external and internal signals. In yeast, multiple stress signals regulate pheromone signaling to prevent mating under unfavorable conditions. However, the underlying crosstalk mechanisms remain poorly understood. Here, we show that mechanical stress activates Pkc1, which prevents lysis of pheromone-treated cells by inhibiting polarized growth. In vitro Pkc1 phosphorylates conserved residues within the RING-H2 domains of the scaffold proteins Far1 and Ste5, which are also phosphorylated in vivo. Interestingly, Pkc1 triggers dispersal of Ste5 from mating projections upon mechanically induced stress and during cell–cell fusion, leading to inhibition of the MAPK Fus3. Indeed, RING phosphorylation interferes with Ste5 membrane association by preventing binding to the receptor-linked Gβγ protein. Cells expressing nonphosphorylatable Ste5 undergo increased lysis upon mechanical stress and exhibit defects in cell–cell fusion during mating, which is exacerbated by simultaneous expression of nonphosphorylatable Far1. These results uncover a mechanical stress–triggered crosstalk mechanism modulating pheromone signaling, polarized growth, and cell–cell fusion during mating.

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