Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells.

Helenius, Ari; Kielian, Margaret; Wellsteed, Jennifer; Mellman, Ira; Rudnick, Gary

doi:10.1016/s0021-9258(18)89078-0

Cited by 49 publications

(10 citation statements)

References 32 publications

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“…The heterodimeric interactions are interrupted and the El subunits are organized into new oligomers, whereas the E2 subunits appear to remain monomeric. Concomitant with the change in tertiary structure of the spike proteins, the El subunit undergoes conformational alterations as detected by the exposure of the anti-El" epitope and by the previously described increased resistance of El towards trypsin digestion (Helenius et al, 1985) .…”

Section: Discussionmentioning

confidence: 99%

“…The assays for [3sS]methionine-labeled SFV binding to, and endocytosis into, BHK cells were performed essentially as described (Marsh and Helenius, 1980 ;Helenius et al ., 1985) . Typically, 80% confluent BHK cells in 60-mm dishes were washed and preincubated with cold MEM containing 0.2% BSA, 10 mM Hepes, and 2 mM glutamine for 20 min on ice .…”

Section: Binding and Internalization Assaysmentioning

confidence: 99%

“…rpsin digesdons . The appearance of the trypsin-resistant form of El during entry of SFV was assayed as described earlier (Helenius et al ., 1985) . At various times after starting the 37°C incubation virus cell samples were solubilized and treated with a final concentration of 200 pg/ml trypsin (Sigma Chemical Co.) for 10 min at 37°C.…”

Section: Analysis Ofsolubilized Cell and Wrus Samplesmentioning

confidence: 99%

“…By analogy with the low pH-induced changes of HA it is believed that the acidic pH alters the El structure into a form that is competent to perform a membrane fusion reaction. The fact that El changes into a trypsin-resistant form during entry into acidic endosomes and exposes new immunological epitopes as a result of low pH treatment might reflect this conformational change (Edwards et al, 1983;Helenius et al, 1985 ;Kielian and Helenius, 1985;Kielian et al, , 1990.…”

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confidence: 99%

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Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.

Wahlberg

Garoff

1992

The Journal of Cell Biology

162

160

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Abstract. The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells . Existing evidence suggests that the El protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the LPHAVIRUSES, such as Semliki Forest virus (SFV)l and Sindbis virus, are enveloped animal viruses A IL which mature by budding at the plasma membrane (PM) ofinfected cells and enter new cells by an acid-induced membrane fusion process inside the endosomal compartment Schlesinger and Schlesinger, 1986). Because of their simple structure and efficient replication these viruses represent useful systems for studying the molecular mechanisms of membrane budding and fusion. SFV directs the synthesis of three structural proteins, the capsid protein and two transmembrane glycoproteins, p62 and El (Garoff et al., 1982). The capsid protein associates in the cell cytoplasm with the viral RNA genome into nucleocapsids (NCs). The two membrane proteins oligomerize soon after synthesis in the ER into p62E1 heterodimers and are then transported to the cell surface in order to take part in the budding process (Ziemiecki et al., 1980;Wahlberg et al., 1989). In the released virus particles the heterodimers are clustered into groups of three, each representing a spikelike projection on the virus surface (Vogel et al., 1986;Fuller, 1987). Furthermore, the original p62E1 heterodimer is processed by a limited proteolytic attack 66 residues from the NH2 terminus of p62 to form mature E2E1 complexes . This cleavage occurs at a very late stage during cell surface transport of the heterodimer and is probably mediated by a host protease (de Curtis and Simons, 1988) .1. Abbreviations used in this paper: HA, hemagglutinin; NC, nucleocapsid; pfu, plaque-forming units ; PM, plasma membrane ; SFV, Semliki Forest virus .

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Section: Discussionmentioning

confidence: 99%

Section: Binding and Internalization Assaysmentioning

confidence: 99%

Section: Analysis Ofsolubilized Cell and Wrus Samplesmentioning

confidence: 99%

mentioning

confidence: 99%

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Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.

Wahlberg

Garoff

1992

The Journal of Cell Biology

162

160

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“…The kinetics of [35S]methionine-labeled virus uptake into CHO cells, degradation in lysosomes to TCA-soluble counts (Marsh and Helenius, 1980;Schmid et al, 1988), and intracellular conversion oftbe viral E1 glycoprotein to trypsin resistance (Helenius et al, 1985;Kielian et al, 1986) have been previously described. For all assays, radiolabeled virus was prebound to cells on 35-ram plates at 4°C with continuous shaking in RPMI binding media containing 0.2% BSA and buffered with 10 mM Hepes at either pH 6.8 for wt SFV or pH 6.5 forJhsd.…”

Section: Assays For Endocytosis Degradation and Intraceuar Conversion Of Viral E1 Proteinmentioning

confidence: 99%

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

Schmid

Fuchs

Kielian

et al. 1989

The Journal of Cell Biology

Self Cite

144

107

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Abstract. During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes" constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoreticaUy distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH ~6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH <5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH <6.2 in early endosomes with a t~/2 of 5 min. Although a fraction of the virus reached a pH of <5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t~/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41°C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH -,<5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H + pump to fluctuations in the endosomal membrane potential. WE have previously shown, using cell fractionation by free flow electrophoresis (FFE) ~ that internalized Semliki Forest virus (SFV) passes sequentially through two physically distinct subpopulations of endosomes en route to lysosomes in Chinese hamster ovary (CHO) cells (Schmid et al., 1988). Internalized SFV first appears (tja = 3-5 min) in an anodally deflected endosomal compartment of relatively low electrophoretic mobility, defined as "early" endosomes. After an •2 min lag, SFV next appears (t,i2 = Dr. S. Schmid's current address is Research

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Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

Kielian¹,

Marsh²,

Helenius³

1986

The EMBO Journal

110

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The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK‐21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus‐1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus‐1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.

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Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells.

Cited by 49 publications

References 32 publications

Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.

Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

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