Transmembrane movement and distribution of cholesterol in the membrane of vesicular stomatitis virus

Abstract

The transmembrane movement and distribution of cholesterol in the vesicular stomatitis virus membrane were studied by following the depletion of cholesterol from virions to interacting phospholipid vesicles and by exchange of radiolabeled cholesterol between virions and phospholipid-cholesterol vesicles. The kinetics of the cholesterol exchange or depletion reactions revealed the presence of two exponential rates: a rapid rate, dependent on the vesicle to virus ratio, and a slower rate, independent of the vesicle to virus ratio. The kinetics of cholesterol movement could be best interpreted by a model of the virion membrane considered as a two pool system in which approximately 30% of the cholesterol resides in the outer monolayer and approximately 70% in the inner monolayer. The half-time for equilibration of the two pools was calculated to be 4--6 h and was assumed to represent the time required for transmembrane movement of cholesterol across the bilayer. The initial rate of transfer of cholesterol from virus into vesicles increased when vesicle phospholipids contained more unsaturated and shorter chain fatty acids. Furthermore, the transfer of cholesterol appeared to occur by a collisional mechanism requiring membrane-membrane contact. Interaction with lipid vesicles did not significantly affect the integrity of the virion membrane as assessed by the relative inaccessibility of internal proteins to lactoperoxidase-catalyzed iodination and by the small loss of [3H]amino acid labeled protein from the virus.

Depletion and exchange of cholesterol from the membrane of vesicular stomatitis virus by interaction with serum lipoproteins or poly(vinylpyrrolidone) complexed with bovine serum albumin

“…Kinetic analyses of cholesterol depletion and exchange from the VSV membrane into lipid vesicles performed in our laboratory have clearly demonstrated that VSV cholesterol is present in two distinct pools, in which we now know less than 30% resides in the inner monolayer and more than 70% in the outer monolayer (Patzer et al, 1978b); an error in the original calculations resulted in presentation in the reverse order of cholesterol bilayer distribution. Most cholesterol depletion/exchange studies done with lipid vesicles, however, suffer from a serious artifact: a considerable number of residual vesicles remain firmly adherent to the surface of the virus (or cells) following interaction with the vesicles (Moore et al, 1978;Patzer et al, 1978b). Such nonspecific adherence of lipid vesicles to VSV may affect the 1 Abbreviations used: VSV, vesicular stomatitis virus; G, glycoprotein; M, matrix protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SPM, sphingomyelin; DPL, dipalmitoyllecithin; BSA, bovine serum albumin; PVP, polyvinylpyrrolidone); BHK, baby hamster kidney; BME, basal medium Eagle; pfu, plaque-forming units; PBS, phosphate-buffered saline; DPH, 1,6diphenyl-l,3,5-hexatriene; FCS, fetal calf serum; THF, tetrahydrofuran; HDL, high-density lipoproteins; LDL, low-density lipoproteins; DOPC, dioleoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine.…”

“…Although exchange reactions of this type have been utilized in many instances for calculating the distribution and transbilayer movement of cholesterol, results in most cases appear to be contradictory (Poznansky & Lange, 1976;Gottlieb, 1976;Lenard & Rothman, 1976;Bloj & Zilversmit, 1977;Lange et al, 1977;Nakagawa et al, 1979;Backer & Dawidowicz, 1979; Sefton & Gaffney, 1979). Kinetic analyses of cholesterol depletion and exchange from the VSV membrane into lipid vesicles performed in our laboratory have clearly demonstrated that VSV cholesterol is present in two distinct pools, in which we now know less than 30% resides in the inner monolayer and more than 70% in the outer monolayer (Patzer et al, 1978b); an error in the original calculations resulted in presentation in the reverse order of cholesterol bilayer distribution. Most cholesterol depletion/exchange studies done with lipid vesicles, however, suffer from a serious artifact: a considerable number of residual vesicles remain firmly adherent to the surface of the virus (or cells) following interaction with the vesicles (Moore et al, 1978;Patzer et al, 1978b).…”

“…kinetic parameters of cholesterol exchange or depletion which are used to derive the t,/2 values for time of cholesterol transposition as well as the value of cholesterol distribution, both of which are calculated from these kinetic parameters (Bloj & Zilversmit, 1976). Nonspecific adherence of vesicles can be reduced, but not eliminated, by removing the glycoprotein spikes of the virus envelope with trypsin (Patzer et al, 1978b;Shaw et al, 1979), but this results in drastic loss in infectivity (Bishop et al, 1975). Although certain experiments can be performed with spikeless virions, others can not, and the problem of residual adherent lipids always poses an unknown factor.…”

Spontaneous phosphatidylcholine transfer by collision between vesicles at high lipid concentration

“…The study by Kremer et al (1977) on phospholipid transfer between model membranes supported collisionally mediated transfer. Also, a study on cholesterol transfer from vesicular stomatitis virus membranes to phospholipid vesicles (Patzer et al, 1978) as well as an investigation of cholesterol transfer from vesicles to brush border membranes (Müsch et al, 1986) suggested this mechanism.…”

Mechanism of cholesterol exchange between phospholipid vesicles

“…Evidence in favor of the collision-dependent mechanism for cholesterol exchange has been presented in several studies (Bruckdorfer & Green, 1967; Lenard & Rothman, 1976; Haran & Shporer, 1977;Poznansky & Lange, 1978;Moore et al, 1978; Patzer et al, 1978;Jonas & Maine, 1979 & Claret, 1979;Gottlieb, 1980), although in some of these the data on which the conclusions were based is somewhat scant. In addition, Bruckdorfer & Graham (1976) argued that the involvement of an aqueous cholesterol intermediate could be dismissed, because Gould et al (1955) had reported that they were unable to observe cholesterol exchange between membranes that were separated from each other by a dialysis membrane.…”