Calcium, cells and virus—Alterations caused by paramyxoviruses

Components involved in virally mediated membrane fusion and permeability changes

“…Alternatively, for the experiment described in Table 1, viral envelope components were solubilized in Triton X-100 (Sigma Chemical Co., Poole, Dorset, U.K.) by the method of Volsky & Loyter (1978a (Pasternak & Micklem, 1973, 1974a. Uptake was determined by spinning through oil as described by Impraim et al (1979). Binding of 1251-labelled virus was determined by incubating virus (100 haemagglutination units) with Lettree cells (8 x 106) in a final volume of 0.7ml at 40C or 350C for 15min, washing three times in ice-cold Hepes-buffered saline and measuring 125I in the pellet.…”

“…The amino acid or peptide (2,umol) was dissolved in 0.2 ml of phosphate-buffered saline (Dulbecco & Vogt, 1954) adjusted to pH 8 with NaOH, and 0.02 ml of fluorescamine (Sigma Chemical Co.) in ethanol (5 mg/ml) was added rapidly with shaking. After 15 min at room temperature, each sample (0.05 ml) was added to 0.5 ml of a suspension (1.2 x 107cells/ ml) of Lettree cells that had previously been treated with or without virus (200 haemagglutination units/ ml) for 10min at 370 C. Incubation was continued at 370C, and samples (0.1 ml) were removed at intervals and centrifuged through oil (Impraim et al, 1979) (Hosaka & Shimizu, 1972) to provide a milieu conducive to membrane fusion; secondly, to reconstitute the 'pores' (see below) present in intact virus; thirdly, to reassemble particles of a sufficient size. Although Hosaka & Shimizu (1972) and Volsky & Loyter (1978b) claim to have successfully reassembled preparations showing haemolysis or cell-cell fusion, and hence by implication (Knutton & Pastemak, 1979;Impraim et al, 1980) permeability changes also, the retention of trace amounts of detergent was not rigorously…”

“…Permeability changes elicited by '1-day' virus Virus harvested after growth in eggs for only 1 day is non-haemolytic and does not mediate giantcell formation, but agglutinates cells and is infective (Impraim et al, 1979). Treatments that render such virus haemolytic, and capable of promoting giantcell formation, such as freeze-thawing or incubation at 370C , cause it to elicit permeability changes also (Table 2; Impraim et al, 1979).…”

“…Another of our postulates is that virus-cell fusion, in contrast with subsequent permeability changes leading to haemolysis and giant-cell formation, is not affected by Ca2+ (Knutton & Pasternak, 1979;Impraim et al, 1979Impraim et al, , 1980. At present few convenient methods for assay of F glycoprotein or virus-cell fusion exist.…”

Protein Synthesis in Semliki Forest Virus-infected Cells is Not Controlled by Permeability Changes

“…It might be argued that [3H]GppCH2p associated with cells is not inside cells but bound at the cell surface. This is also unlikely since in the presence of 2 mu-Ca 2 +, which is known to block membrane permeability changes (Pasternak & Micklem, 1974;Impraim et al, 1979Impraim et al, , 1980, the uptake of GppCH2p ( Fig. 1) and its action on Sendai virustreated cells are inhibited (data not shown).…”