Fusion of influenza virus in an intracellular acidic compartment measured by fluorescence dequenching

Stegmann, Toon; Morselt, Henriëtte W. M.; Scholma, Janny; Wilschut, Jan

doi:10.1016/0005-2736(87)90100-3

Cited by 76 publications

(62 citation statements)

References 21 publications

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“…The observation (Nussbaum & Loyter, 1987) that fusion proceeded without a lag phase, which would have been required for endocytosis and endosome acidification, is puzzling. Stegmann et al (1987a) found that fusion with adherent cell lines proceeded after a lag phase of 10 to 15 min.…”

Section: Discussionmentioning

confidence: 99%

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

Düzgüneş

Lima²,

Stamatatos³

et al. 1992

Journal of General Virology

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The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma ($49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4-9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5-4, different from that of the virus preincubated at low pH without target membranes.

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Section: Discussionmentioning

confidence: 99%

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

Düzgüneş

Lima²,

Stamatatos³

et al. 1992

Journal of General Virology

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“…Pretreatment of cells with the lysosomotropic agents NH&l or monensin (Mellman et al, 1986;Stegmann et al, 1987b) or the endocytosis inhibitor sodium azide (Blumenthal et al, 1987) did not affect fluorescence dequenching (not shown).…”

Section: Time (Min)mentioning

confidence: 90%

“…This procedure has been described to reduce cell endocytic activity (Blumenthalet al, 1987). IncubationofPC-12cells(5 X 106 cells) with either 30 to 100 mM NH4Cl or with 6-12 pM monensin (1 5 min, 37 OC, totalvolume 1.9 mL, and continuous stirring) was carried out to increase the pH in intracellular acidic compartments (Mellman et al, 1986;Stegmann et al, 1987b). Following the incubations, labeled influenza virus (2 pg of viral protein) was added to the cells, and fusion was monitored (pH 7.4, 37 "C) as described above.…”

Section: Virusmentioning

confidence: 99%

A common mechanism for influenza virus fusion activity and inactivation

Ramalho‐Santos

Nir²,

Düzgüneş³

et al. 1993

Biochemistry

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The fusion of influenza virus (A/PR/8/34 strain) with PC-12 cells was monitored by a fluorescence assay, and the results were analyzed with a mass-action model which could explain and predict the kinetics of fusion. The model accounted explicitly for the reduction in the fusion rate constant upon exposure of the virus to low pH, either for the virus alone in suspension or for the virus bound to the cells. When the pH was lowered without previous viral attachment to cells, an optimal fusion activity was detected at pH 5.2. When the virus was prebound to the cells, however, reduction of pH below 5.2 resulted in enhanced fusion activity at the initial stages. These results were explained by the fact that the rate constants of both fusion and inactivation increased severalfold at pH 4.5 or 4, compared to those at pH 5.2. At pH 5.2, lowering the temperature from 37 to 20 or 4 "C resulted in a decrease in the fusion rate constant by more than 30-or 1000-fold, respectively. Inactivation of the virus when preincubated in the absence of target membranes at pH 5 was found to be rapid and extensive at 37 OC, but was also detected at 0 OC. Our results indicate a strong correlation between fusion and inactivation rate constants, suggesting that the rate-limiting step in viral hemagglutinin (HA)-mediated fusion, that is, rearrangement of viral glycoproteins at the contact points with the target membrane, is similar to that involved in fusion inactivation.

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“…The virus can use nonclathrin endocytosis for productive entry and infection (9), and fusion can be observed from smooth-surfaced vesicles as well as coated vesicles (7). Studies from several different groups have shown that the virus fuses out of a low-pH compartment, with fusion occurring at pH 5.0-5.5 in vitro (10,11). Once fusion has occurred from the endosomal compartment, the uncoated virus is released into the cytoplasm and the genomic ribonucleoproteins enter the nucleus (12,13).…”

mentioning

confidence: 99%

Influenza virus entry and infection require host cell N-linked glycoprotein

Chu

Whittaker

2004

Proc. Natl. Acad. Sci. U.S.A.

183

144

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A widely held view of influenza virus infection is that the viral receptor consists of cell surface carbohydrate sialic acid, which can be present as glycoprotein or glycolipid. Here, we examined influenza virus entry and infection in Lec1 cells, a mutant CHO cell line deficient in terminal N-linked glycosylation caused by a mutation in the N-acetylglucosaminyltransferase I (GnT1) gene. We show that influenza virus cannot infect Lec1 cells, despite having full capacity to undergo virus binding and fusion. Lec1 cells also show no virus replication defect, and infection was restored in Lec1 cells expressing wild-type GnT1. Viruses were apparently arrested at the level of internalization from the plasma membrane and were not endocytosed. Lec1 cells were refractory to infection by several strains of influenza virus, including H1 and H3 strains of influenza A, as well as influenza B virus. Finally, cleavage of N-glycans from wild-type CHO cells markedly reduced infection by influenza virus. We suggest that influenza virus specifically requires N-linked glycoprotein for entry into cells, and that sialic acid, although acting as an efficient attachment factor, is not sufficient as an influenza virus receptor in vivo.

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Fusion of influenza virus in an intracellular acidic compartment measured by fluorescence dequenching

Cited by 76 publications

References 21 publications

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

A common mechanism for influenza virus fusion activity and inactivation

Influenza virus entry and infection require host cell N-linked glycoprotein

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