The biochemistry of virus-induced cell fusion. Changes in membrane integrity

Abstract

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.

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).…”

“…1), even though methionine incorporation in infected cells is inhibited more strongly by GppCH2p at this time (Table 1). The lack of any difference in the uptake of [3H]GppCH2p cannot be accounted for by greater leakage from the infected cells during the washing procedure since cells treated with haemolytic Sendai virus which are leaky to nucleotides and other phosphorylated compounds (Pasternak & Micklem, 1973, 1974Impraim et al, 1980), nevertheless retain more [3H]GppCH2p than untreated cells (Fig. 1).…”

“…The proposals for non-specific increases in membrane permeability following virus infection of mammalian cells were based partly on the observation that certain paramyxoviruses cause just such a change (Pasternak & Micklem, 1973, 1974Poste & Pasternak, 1978;Impraim et al, 1980) when fusing (Knutton, 1978) with susceptible cells. By exposing BHK cells to haemolytic Sendai virus (which, although haemolytic is not cytolytic : Pasternak & Micklem, 1973;Knutton et al, 1976;Poste & Pasternak, 1978;Wyke et al, 1980), we have confirmed that 'permeabilized' cells indeed take up more [3H]GppCH2p (Fig.…”

Mechanisms of Membrane Fusion

“…Metabolic alteration of phospholipids, such as PI turnover (Hokin and Hokin 1953;Michell 1975) is involved in triggering some of the early (ca 2 + dependent) events connected with exocytosis, in addition to which the formation of diaclglycerol has been proposed to aid the fusion process itself (Whalley and Whitaker 1988); in the case of virus-cell fusion, such turnover has been ruled out (pasternak and Micklem 1974) Just as in a catalysed chemical reaction the products are of lower energy than the catalytic intermediate (i.e. the activated state), so the bilayer structure is of lower energy than any non-bilayer structure.…”

Myxoviruses do not induce non-specific alterations in membrane permeability early on in infection

“…t h a t t h e y were a cause and not a consequence of subsequent cytopathic changes. We have therefore measured the p e r m e a b i l i t y characteristics of cells by methods previously shown to reveal non-specific leakiness of the t y p e p o s t u l a t e d to occur by C~A S C O and colleagues: n a m e l y the ability to accumulate low molecular weight nutrients, to m a i n t a i n intracellular K + and to retain a N a + gradient across the plasma m e m b r a n e (23,36,37,38,42). W e chose for this initial s t u d y cells infected with myxovimses, since in these systems cytopathie changes leading to cell lysis, which obviously complicate the i n t e r p r e t a t i o n of permeability changes, are generally delayed well beyond the time at which viral protein synthesis commences.…”