【病毒外文文献】1991 Modulation of coronavirus-mediated cell fusion by homeostatic control of cholesterol and fatty acid metabolism

Journal of Medical Virology 35 142 149 1991 Modulation of Coronavirus Mediated Cell Fusion by Homeostatic Control of Cholesterol and Fatty Acid Metabolism Marguerite Cervin and Robert Anderson Department of Microbiology and Infectious Diseases University of Calgary Calgary Alberta Canada Cellular susceptibility to fusion mediated by mu rine coronavirus mouse hepatitis virus MHV strain A59 was separated into lipid dependent and lipid independent mechanisms with the use of subclones and selected mutants of mouse L 2 fibroblasts Fusion resistant L 2 cell mutants had similar cholesterol and fatty acid composition as did their fusion susceptible parent subclone and were presumably deficient in a genetically mu table non lipid host cell factor e g fusion pro tein receptor On the other hand cellular sensi tivity to virus fusion which is known to be influenced by cell cholesterol content Daya et al 19881 was shown further to be modulated by homeostatic alterations in fatty acid metabolism Cholesterol supplementation of mouse L 2 fibro blasts or of peritoneal macrophages from MHV susceptible mice elevated susceptibility to viral fusion Increased fusion susceptibility occurred in cholesterol supplemented L 2 cells in the ab sence of any detectable alterations in host cell fatty acid composition thus demonstrating fu sion enhancement by cholesterol alone L 2 cells cloned by limiting dilution in normal not cho lesterol supplemented medium were found to be heterogeneous in cholesterol content Inter estingly high cholesterol containing subclones had increased levels of C 18 0 C 18 2 C 20 4 and C 22 6 and markedly reduced levels of C 18 l fatty acids when compared to low choles terol containing subclones High cholesterol containing subclones did not show enhanced susceptibility to viral fusion suggesting that homeostatic alteration of fatty acid metabolism compensated for the increased cholesterol levels and countered the normally fusion enhancing effect of cholesterol alone Since these observa tions have potentially important consequences regarding the effects of dietary cholesterol on the severity of virus infection we examined liver titres and pathology of normal and hypercholes terolemic mice infected with MHV Hypercholes terolemia had no significant effect on virus rep lication or on liver pathology in two MHV sensitive strains BalbIc and AIJ or in one MHV 1991 WILEY LISS INC resistant SJLIJ of mice Lipid analyses of the livers from normal and hypercholesterolemic mice showed evidence of two homeostatic mechanisms cholesterol esterification and alter ation of fatty acid composition which likely counteracted the normally exacerbating effect of cholesterol on MHV cytopathology INTRODUCTION Cholesterol plays an important role in determining the physical properties and functions of animal cell membranes In addition to having a modulating effect on membrane fluidity and permeability Demel et al 19721 there is evidence that cholesterol may interact directly with certain membrane proteins Johnson et al 1980 Asano and Asano 19881 and possibly regulate their functional activity McMurchie 1988 Asano and Asano 19881 Cholesterol is an important dietary lipid and has been shown to modulate resistance of mice to infections by some bacteria and viruses including Listeria mono cytogenes Kos et al 1979 Kos et al 19841 and Coxsackie B virus Campbell et al 19821 and also MHV 3 Pereira et al 19871 MHV 3 is strongly hepa totropic in Balb c mice but causes only a mild liver infection in A J mice Pereira et al 19871 hypothe sized that Kupffer cells KC the liver macrophages are involved in resistance to MHV 3 in AIJ mice and that such resistance may be overcome by cholesterol supplementation This and previous studies Ruebner et al 1958 Ruebner and Bramhall 19601 on modula tion of MHV infection by cholesterol or fats employed complex food mixtures in which cholesterol was not the sole variable constituent As a result it has not been possible to relate biological effects to cholesterol per se We have shown previously Daya et al 19881 that supplementation of cultured mouse L 2 fibroblasts with Accepted for publication June 19 1991 Address reprint requests to R Anderson Dept of Microbiology and Infectious Diseases University of Calgary Calgary Alberta Canada Lipid Homeostasis and Viral Fusion cholesterol results in a marked increase in cellular susceptibility to fusion mediated by mouse hepatitis virus MHV Roos et al 19901 have also presented evidence suggesting a role for other lipids particularly saturatediunsaturated fatty acids in modifying cellu lar responsiveness to MHV induced fusion Since these results have potentially important consequences for the spread and severity of virus infections as a function of dietary cholesterol and other lipids we undertook a study of cholesterol metabolism and MHV infection in vitro and in vivo The results identify important ho meostatic control mechanisms which counteract the fusion enhancing effects of excess cholesterol MATERIALS AND METHODS Cells Virus and Culture Conditions Mouse L 2 fibroblasts Rothfels et al 19591 were cultured as monolayers in minimal essential medium MEM supplemented with 10 fetal calf serum Mouse L 2 cell mutants selected for partial resistance against MHV infection were those described by Daya et al 19891 Cholesterol supplemented medium was pre pared as previously described Daya et al 19881 Peritoneal macrophages obtained by peritoneal lavage of starch primed mice were also cultured in normal or cholesterol supplemented medium in the same man ner The A59 strain of MHV Manaker et al 19611 was used throughout Cell Membrane Preparation Cell monolayer cultures 60 mm tissue culture plates were washed with phosphate buffered saline PBS scraped from the plastic plates and spun into pellets 5 min at 1000 x g Cells were resuspended in reticulocyte standard buffer RSB allowed to swell on ice for 10 min and then disrupted by manual glass homogenization Following removal of nucleii by brief centrifugation 1 min at 650 x g total membranes were recovered by centrifugation at 80 000 x g for 1 h Contact Fusion Assays For the assessment of fusion susceptibility contact fusion assays similar to those described previously Mizzen et al 19831 were performed Experiments were performed three times on triplicate cultures Sparsely seeded MHV infected L 2 cells L2 1 subclone were overlaid with a ten fold excess of uninfected test cells Following a two hour incubation at 37 C syncy tial formation was evaluated by light microscopy and expressed as a fusion index Mizzen et al 19831 Mice Diet and MHV Infection Three strains of mice were used for the in vivo studies Balbic mice were purchased from Charles River Quebec Canada while A J and SJLIJ mice were obtained from Jackson Laboratories Bar Harbour Maine USA The control diet consisted of 10 wiw corn oil mixed in with ground Wayne Rodent Blox The cholesterol supplemented diet was adapted from the hypercholesterolemic diet used for inducing atheroscle 143 rosis in rabbits Frank and Fogelman 19891 and con sisted of 2 w w crystalline cholesterol in 10 wiw corn oil in ground Wayne Rodent Blox Mice were fed fresh food and water daily Following 21 days feeding on either the control or cholesterol diet eight mice on each diet were either mock infected with 200 pl PBS or infected with lo6 pfu ml MHV A59 in 200 pl PBS At 3 days post inoculation PI mice were sacrificed the livers extracted and either frozen at 70 C for titration and lipid analysis or preserved in 15 formalin Par affin sections from formalin fixed mouse livers were stained with hematoxylin eosin HiE and the number of lesions present per section was counted Lipid Analyses Cell lipid determinations were performed on tripli cate cultures from three separate experiments Lipids from L 2 cells or from membrane fractions were ex tracted with chloroformimethanol 1 1 Liver lipids were extracted by first homogenizing 0 3 g portions of liver in 3 ml PBS and then mixing 300 pl of homoge nate with 3 ml chloroformimethanol 1 l After 2 h stirring at room temperature extracts were filtered through glass wool Aliquots 300 p1 of the lipid extracts were methanolyzed 5 methanolic HC1 for 2 h at 100 C and acetylated pyridineiacetic anhydride 1 l overnight at room temperature in order to convert total fatty acids and cholesterol to their methyl and acetyl derivatives respectively Fatty acid methyl es ters and cholesterol acetate were then analyzed quan titatively by gas chromatography as previously de scribed Daya et al 19881 To differentiate free cholesterol from cholesterol ester aliquots of the lipid extracts were applied to pasteur pipette columns of Biosil A BioRad and neutral lipid fractions obtained by elution with chloroformimethanol 30 1 Trimeth ylsilylation of the neutral lipids before and after methanolic HC1 hydrolysis yielded the TMS deriva tives of free and total cholesterol respectively which were then determined by gas chromatography RESULTS Enhanced fusion susceptibility results from cho lesterol supplementation in the absence of fatty acid alterations Since membrane fluidity is deter mined primarily by cholesterol content and fatty acid chain lengthiunsaturation we examined the possibility that cholesterol enhanced fusion could be partly brought about by alterations in the host cell fatty acid composition Cultures of L 2 cells were maintained in normal or cholesterol supplemented medium for 24 or 48 hours and subjected to analysis of cholesterol and fatty acid As illustrated in a representative chromato gram Fig 1 and quantitated in Table I cholesterol supplementation caused an increase in the cellular content of total free esterified cholesterol Choles terol ester normally a minor lipid constituent in L 2 cells was particularly elevated in concentration within 24 hours of cholesterol supplementation Increasing the 144 Cervin and Anderson Time h Fig 1 Response of cellular cholesterol metabolism to exogenous supplementation with cholesterol Cultures of L 2 cells were main tained in normal Time 0 or cholesterol supplemented medium for 24 or 48 h Washed cells were lipid extracted and aliquots of the extracts run on TLC which was subsequently visualized by sulfuric acid charring Abbreviations are Ch cholesterol and Ch E cholesterol ester duration of cholesterol supplementation from 24 to 48 hours did not further raise the cellular contents of either free or esterified cholesterol Analysis of the membrane fraction also showed increased cholesterol content in response to cholesterol supplementation Table I It is important to note that although the major cellular response to cholesterol supplementation was an increase in cholesterol ester level there was also considerable increase in free cholesterol which was found predominantly in the membrane fraction Cho lesterol ester was not found in the membrane fraction and was presumably present as a cytoplasmic storage form data not shown Analysis of the cellular fatty acids revealed that essentially no alterations in fatty acid composition had taken place as a result of the considerable infiltration of cholesterol documented above Table I Therefore the enhancement of viral fusion observed in cholester ol supplemented cells Daya et al 1988 and Table I1 cannot be ascribed to fatty acid dependent membrane changes L 2 cell subclones with elevated cholesterol but altered fatty acid composition do not have en hanced susceptibility to viral fusion Analysis of a number of L 2 cell subclones all grown in the same medium without added cholesterol showed a surpris ing variability in cholesterol contents Table 11 Nota bly however and in contrast to L 2 cells supplemented exogenously with cholesterol the high cholesterol containing subclones L2 85 and L2 86 showed marked alterations in their fatty acid composition Table 11 In particular the high cholesterol containing subclones had elevated amounts of C 18 2 C 18 0 C 20 4 and C 22 6 along with drastically reduced levels of C 18 l We exploited this observation to examine the suscepti bility of the various subclones to viral fusion in a contact fusion assay Despite the elevated cholesterol levels found in two of the subclones L2 85 and L2 86 these subclones showed no increased susceptibility to MHV induced fusion over subclones of low cholesterol content L2 1 L2 2 and L2 87 Taken together the results so far suggest that cholesterol enhanced fusion can be counterbalanced by cellular alterations in fatty acid metabolism Fusion resistant L 2 cell mutants have wild type cholesterol and fatty acid compositions In light of the observations above we investigated the fatty acid and cholesterol compositions of L 2 cell mutants selected for their ability to survive acute MHV infec tion and which show a relatively fusion resistant phe notype Daya et al 19891 It was found that all five mutants examined had a cholesterol content and fatty acid composition similar to the parental L 2 cell clone from which they were generated Table 111 Thus an alteration in fatty acid or cholesterol metabolism does not underly the genetic lesion which is responsible for diminished susceptibility to MHV induced fusion in these mutants Nevertheless each of the five L 2 mutant cells showed cholesterol dependent fusion enhancement when supplemented with cholesterol data not shown although the degree of fusion enhancement was not as great as that observed with wild type L 2 cells It would therefore seem that even cells of disparate susceptibil ity to MHV induced fusion possess a window within which cell fusion is subject to cholesterol dependent modulation Cholesterol enhances MHV mediated fusion of infected macrophages In an effort to extend our in vitro results to the in vivo situation we subjected peritoneal macrophages from three strains of mice Balblc AIJ and SJL J to cholesterol supplementation Both control and cholesterol supplemented macro phages were then challenged with MHV and scored for the development of syncytia Macrophages are impor tant in MHV infection for several reasons First hepa totropic viruses may require replication in macro phages prior to invading liver parenchymal cells Mims 1964 Allison 1974 Sabesin and Koft 19741 Second mouse strain susceptibility to MHV has been Lipid Homeostasis and Viral Fusion 145 TABLE I Fusion Enhancement in Cholesterol Supplemented L 2 Cells Occurs in the Absence of Changes in Fatty Acid Composition Fatty acid composition Cholesterol supplementation h Fatty acid 0 24 48 C 16 O C 16 l C 18 O C 18 l C 18 2 2 204 3 226 C 24 O C 24 l Unknowns 11 1 1 6 20 5 39 9 2 3 2 5 1 o 2 4 2 4 16 3 10 4 1 7 20 4 40 4 2 1 2 7 1 3 2 4 2 3 16 3 10 5 1 8 21 6 41 3 1 9 2 5 1 3 2 0 1 9 15 2 Total FA 0 14 f 0 03 0 14 f 0 04 0 16 f 0 04 Total free cholesterol 0 022 5 0 003 0 032 f 0 004 0 032 f 0 004 Total cholesterol ester 0 002 0 001 0 018 f 0 003 0 020 0 005 Cell cholesterol FA 0 17 0 36 0 33 Membrane cholesterol FA 0 29 0 39 0 38 Fusion index Confluent 60 mm tissue culture plates of cells were maintained in normal or cholesterol supplemented medium for 24 or 48 h Lipid extracts were prepared from cells or membrane fractions and their fatty acid composition and cholesterol contents were determined Parallel cultures were assayed for fusion index in a contact fusion assay Results were obtained from three experiments and are expressed as the means standarddeviation except for fatty acid compositions which for clarity are expressed only as the means mg mg protein mg mg protein mg mg protein w w ratio w w Ratio 0 33 0 05 0 61 f 0 10 0 55 0 07 TABLE 11 L 2 Cell Subclones With Elevated Cholesterol Have Compensatory Fatty Acid Alterations Fatty acid composition Fatty acid L2 1 L2 2 L2 85 L2 86 L2 87 C 16 O C 1611 C 18 O C 18 l C 18 2 C 20 4 C 226 C 24 O C 24 l Unknowns Total FA mg mg protein Total free cholesterol mg mg protein Total cholesterol ester mg mg protein Cell cholesterol FA w w ratio Membrane cholesterol FA w w ratio 7 8 1 1 18 7 33 4 6 4 3 6 3 9 2 2 4 3 18 6 0 14 0 025 0 003 0 20 0 29 8 2 0 8 18 0 37 2 4 5 3 2 3 5 2 8 3 7 18 1 0 15 0 022 0 002 0 16 0 28 Fusion index 0 92 0 71 Confluent 60mm tissue culture plates of cells weremaintainedinnormal medium MEM 10 FCS for 48 h Lipid extracts were prepared from cells or membrane fractions and their fatty acid composition and cholesterol contents determined Parallel cultures were assayed for fusion in a contract fusion assay Results are expressed as the means from triplicate cultures from three experiments 7 0 0 5 26 9 8 7 16 8 8 7 7 2 2 4 2 8 19 0 0 15 0 036 0 024 0 41 0 44 0 27 6 8 0 1 25 5 10 4 15 6 9 7 8 5 2 0 2 2 19 2 0 13 0 033 0 019 0 40 0 42 0 43 7 7 0 7 22 6 32 2 5 9 3 7 4 4 2 1 3 4 17 3 0 14 0 025 0 003 0 20 0 29 0 57 146 Cervin and Anderson TABLE 111 Fusion Resistant L 2 Cell Mutants Have Similar Cholesterol Contents and Fatty Acid Compositions as Their Fusion Sensitive Parent Subclone Fatty acid composition L2 1 Fatty acid parent M2 M10 M12 M22 M26 C 16 0 C 16 l c 18 O 2 181 C 18 2 C 20 4 C 22 6 C 24 0 Unknowns Total FA mg mg protein Total free cholesterol mg mg protein Total cholesterol ester mg mg protein Cell cholesterol FA w w ratio Membrane cholesterol FA w w ratio C 24 l Fusion index 7 8 1 1 18 7 33 4 6 4 3 6 3 9 2 2 4 3 18 6 0 14 0 025 0 003 0 20 0 29 0 92 8 0 6 7 7 2 0 9 1 2 1 3 17 7 18 9 16 9 34 6 35 4 32 8 6 6 6 9 7 7 3 8 2 9 4 0 4 1 4 3 3 8 1 9 1 7 2 6 4 4 4 6 3 8 18 0 17 4 19 9 0 15 0 13 0 12 0 029 0 022 0 027 0 004 0 002 0 003 0 22 0 19 0 25 0 33 0 26 0 30 8 3 7 5 0 7 0 5 18 7 17 4 33 3 32 7 7 5 6 7 4 1 4 1 4 3 4 5 2 7 2 5 4 8 4 7 15 6 19 4 0 14 0 12 0 027 0 026 0 003 0 004 0 22 0 25 0 32 0 30 0 15 0 15 0 05 0 20 0 05 Confluent 60mm tissue culture plates of cellswere maintainedinnormal medium MEM 10 FCS for 48 h Lipid extracts were prepared from cells or membrane fractions and their fatty acid composition and cholesterol contents determined Parallel cultures were assayed for fusion in a contact fusion assay Results are expressed as the means from triplicate cultures from three experiments reported to correlate with virus replication in ex planted macrophages IVirelizier and Allison 19761 Third the ability of a virus to replicate in macrophages or any cell involved in the immune response may compromise their role in virus clearance We chose three strains of mice two of which are permissive for MHV A59 Balbic and A J and one which is non permissive SJLIJ due to a single genetic locus Smith et al 19841 Cholesterol supplementation of peritoneal macrophages taken from Balb c and AiJ mice resulted in enhanced fusion following infection with MHV SJLiJ macrophages on the other hand whether sup plemented with cholesterol or not showed no evidence of MHV induced fusion Thus while cholesterol en hances viral fusion in MHV permissive macrophages it is unable to overcome the block to replication of macrophages which are non permissive to MHV In vivo effects of cholesterol supplementation on MHV infection Given our previous evidence that cholesterol enhances cellular susceptibility to MHV induced fusion we examined whether cholesterol sup plementation in vivo has an effect on the course or severity of MHV induced disease Three strains of mice Balbic A J and SJL J were maintained for 21 days on either normal or cholesterol supplemented diet and examined for evidence of hypercholesterolemia and any changes in susceptibility or severity of MHV induced hepatic disease In contrast to the results of Loria et al 19761 and Pereira et al 19871 we found no difference in the rate of growth approx 2g week or in the liver weights of mice maintained in normal or cholesterol supple mented diet Respective liver weights as percentage body weight for normal and cholesterol supplemented animals were 6 1 0 4 and 6 9 0 7 for Balb c 4 2 2 0 2 and 4 8 0 3 for AM and 5 0 0 4 and 5 0 2 0 4 for SJLIJ Livers from cholesterol supple mented mice had a generally paler colour than did those from the control animals Mic