Vesicular Stomatitis Virus Membrane Research Articles

Depletion of β-COP reveals a role for COP-I in compartmentalization of secretory compartments and in biosynthetic transport of caveolin-1

We have utilized small interfering RNA (siRNA)-mediated depletion of the beta-COP subunit of COP-I to explore COP-I function in organellar compartmentalization and protein traffic. Reduction in beta-COP levels causes the colocalization of markers for the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), Golgi, trans-Golgi network (TGN), and recycling endosomes in large, globular compartments. The lack of spatial differentiation of these compartments is not due to a general collapse of all cellular organelles since markers for the early endosomes and lysosomes do not redistribute to the common structures. Anterograde trafficking of the transmembrane cargo vesicular stomatitis virus membrane glycoprotein and of a subset of soluble cargoes is arrested within the common globular compartments. Similarly, recycling traffic of transferrin through the common compartment is perturbed. Furthermore, the trafficking of caveolin-1 (Cav1), a structural protein of caveolae, is arrested within the globular structures. Importantly, Cav1 coprecipitates with the gamma-subunit of COP-I, suggesting that Cav1 is a COP-I cargo. Our findings suggest that COP-I is required for the compartmentalization of the ERGIC, Golgi, TGN, and recycling endosomes and that COP-I plays a novel role in the biosynthetic transport of Cav1.

Presenilin 1 Regulates Epidermal Growth Factor Receptor Turnover and Signaling in the Endosomal-Lysosomal Pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.

Traffic through the Golgi apparatus.

The role of vesicles in cargo transport through the Golgi apparatus has been controversial. Large forms of cargo such as protein aggregates are thought to progress through the Golgi stack by a process of cisternal maturation, balanced by a return flow of Golgi resident proteins in COPI-coated vesicles. However, whether this is the primary role of vesicles, or whether they also serve to transport small cargo molecules in a forward direction has been debated. Two papers (Martínez-Menárguez et al., 2001; Mironov et al., 2001, this issue) use sophisticated light and electron microscopy to provide evidence that the vesicular stomatitis virus membrane glycoprotein (VSV G)* is largely excluded from vesicles in vivo, and does not move between cisternae, whereas resident Golgi enzymes freely enter vesicles as predicted by the cisternal maturation model. Both papers conclude that vesicles are likely to play only a minor role in the anterograde transport of cargo through the Golgi apparatus in mammalian tissue culture cells.

Photoinactivation of virus infectivity by hypocrellin A.

We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV-1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration-dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or beta-carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.

High-efficiency incorporation of functional influenza virus glycoproteins into recombinant vesicular stomatitis viruses.

We derived recombinant vesicular stomatitis virus (VSV) expressing either influenza virus hemagglutinin (HA) or neuraminidase (NA) glycoproteins from extra genes inserted in the viral genome. The HA protein was expressed from a site downstream of the VSV glycoprotein (G) gene, while NA protein was expressed from a site upstream of the VSV G gene. The HA protein was expressed at lower levels than the VSV G protein, while the NA protein was expressed at higher levels, as expected from the gradient of VSV transcription that follows the gene order. The HA and NA proteins were transported to the cell surface and were functional as demonstrated by hemadsorption, hemolysis, and NA assays. Biochemical analysis showed that both HA and NA proteins were incorporated into VSV particles at high levels, although there was a preference for incorporation of the VSV G protein over either of the influenza virus proteins. Immunoelectron microscopy of the recombinants showed that the particles derived from the recombinants were mosaics carrying both the VSV G protein and the influenza virus membrane glycoproteins. These results extend earlier studies showing incorporation of the cellular glycoprotein CD4 and two other viral glycoproteins into VSV particles. Our results indicate that there is significant space in the VSV membrane that can accommodate foreign membrane proteins and that the foreign protein can represent as much as 35% of the total protein in the viral envelope. Incorporation of foreign proteins into VSV virions can, in many cases, occur passively in the absence of specific incorporation signals.

Use of the fluorescent probe Laurdan to investigate structural organization of the vesicular stomatitis virus (VSV) membrane.

We have used 6-dodecanoil-2-dimethylaminonaphtalene (Laurdan) to study the membrane fluidity of Vesicular Stomatitis Virus (VSV) during virus activation at acidic pH 5.8). The fluorescence properties of Laurdan provide a unique possibility to study lipid organization because of the different excitation and emission spectra of this probe in the gel and liquid crystalline phase. Acidification to pH 5.8 (the pH which triggers VSV fusion with target membranes) generates a decrease in VSV membrane fluidity that could be reversed perfectly after neutralization. We conclude that lipid reorganization of the VSV membrane in the endocytic vesicles is needed for virus activation.

Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry.

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.

Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56).

Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56.

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes.

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin

The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.

Structural Domains in Vesicular Stomatitis Virus Membrane as Studied by Differential Scanning Calorimetry

Structural Domains in Vesicular Stomatitis Virus Membrane as Studied by Differential Scanning Calorimetry

Pardaxin, a hydrophobic toxin of the Red Sea flatfish, disassembles the intact membrane of vesicular stomatitis virus.

Reaction of vesicular stomatitis virus with pardaxin, the hydrophobic toxin of the Red Sea flatfish, resulted in a profound morphological change of many virions and dissociation of their membrane and nucleocapsid into components readily separable by density gradient centrifugation. The basic matrix protein and acidic pardaxin segregated largely with the high density nucleocapsid. The dissociated virion membrane formed lipoprotein vesicles which retained glycoprotein spikes and a certain amount of N protein but no appreciable amounts of other nucleocapsid proteins and little if any RNA. Iodination of the tyrosine residue of the glycoprotein tail fragment provided supporting evidence that the COOH terminus of the glycoprotein extends beyond the inner layer of the membrane into the interior of the virion. These data indicate that pardaxin may serve as a probe for studying the organization of viral membranes, and, hopefully, other biological membranes.

Role of matrix protein in assembling the membrane of vesicular stomatitis virus: reconstitution of matrix protein with negatively charged phospholipid vesicles.

The matrix (M) protein of vesicular stomatitis virus (VSV) was reconstituted into phospholipid vesicles by detergent dialysis. Reconstitution of the positively charged M protein occurred only in the presence of negatively charged phospholipids such as phosphatidylserine, phosphatidic acid, or phosphatidylinositol. Preformed vesicles containing negatively charged phospholipids also bound free M protein. Derivatization of the positively charged lysines in M protein with acetic anhydride or succinic anhydride prevented M protein reconstitution but did not affect the biological property of M protein to inhibit in vitro VSV transcription. An additional indication of the electrostatic nature of the M protein binding to the vesicles was that M protein could not be reconstituted in the presence of 0.5 M NaCl. Nonelectrostatic forces also appear to be involved in the association of the M protein with vesicles, since previously reconstituted M protein remained associated with the vesicles upon subsequent exposure to 0.5 M NaCl.

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.

Cholesterol was depleted from the membrane of vesicular stomatitis virus by exposing virion suspensions to serum lipoproteins enriched with phospholipids. Unlike the reaction of virions with phospholipid vesicle, nonspecific adherence of lipoproteins and exogenous lipids to the envelope of the virus was found to be minimal. The extent of cholesterol depletion was dependent upon the type of phospholipid complexed with interacting lipoprotein; sphingomyelin and dipalmitoyllecithin were found to be highly effective depleters of cholesterol compared to egg phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine. Similar depletion of cholesterol from the virion membrane was also observed when vesicular stomatitis virus was exposed to a complex of poly(vinylpyrrolidone) and bovine serum albumin coated with egg phosphatidylcholine or dioleoylphosphatidylcholine. Cholesterol depletion was found to alter the morphology but not the membrane integrity of the virus. Directly correlated with depletion of cholesterol was a substantial loss in the anisotropy of the viral membrane as determined by fluorescence depolarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene. Interaction with poly(vinylpyrrolidone) complexed with albumin, phosphatidylcholine, and cholesterol resulted in exchange of cholesterol from the virion membrane which following biphasic kinetics with a rapid and a slow phase; these data indicate that 75-85% of viral membrane cholesterol is present in the outer monolayer, and 15-25% is located in the inner monolayer. Depletion of cholesterol from the virion membrane resulted in a significant drop in the infectivity of the virus as measured by plating efficiency on L-cell monolayers. Such an effect was not observed when virion cholesterol was exchanged without net reduction in the concentration of viral membrane cholesterol. Part of the loss in infectivity following depletion of cholesterol could be restored by reincorporation of cholesterol in the membrane, thus demonstrating that membrane cholesterol partly contributes to the infectivity of vesicular stomatitis virus.

Alteration of the membrane lipid composition and infectivity of vesicular stomatitis virus by growth in a Chinese hamster ovary cell sterol mutant and in lipid-supplemented baby hamster kidney clone 21 cells.

The cholesterol and phospholipid composition of the membrane of vesicular stomatitis (VS) virus was altered by growth in a sterol auxotroph Chinese hamster ovary (CHO MI) host cell and by infection of CHO MI and baby hamster kidney (BHK)-21 cells supplemented with fatty acids and dimethylethanolamine. VS virus released from infected CHO MI sterol auxotroph cells grown in delipidated serum had a 50% lower ratio of cholesterol to phospholipid and an 80% drop in infectivity measured by plaque formation on L-929 cells compared with VS virus released from infected CHO MI cells grown in fetal calf serum. When VS virus was harvested from infected BHK-21 cells fed the choline analogue dimethylethanolamine, 29% of the membrane phospholipids were phosphatidyldimethylethanolamine (PDME); 87% of the PDME was located in the external monolayer of the virus membrane as determined by phospholipase C hydrolysis. Exogenous fatty acids added to the medium of cells infected with VS virus comprised up to 30% of the fatty acyl chains of the viral glycerophospholipids. The presence of PDME or unusual fatty acyl chains in the viral membrane had no effect on viral infectivity. These data indicate that the lipid composition of the VS virus membrane is determined primarily by the lipids available in the host cell and that only cholesterol content affects the biological activity of the virus membrane.

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

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.

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the

Lipid organization of the membrane of vesicular stomatitis virus

Lipid organization of the membrane of vesicular stomatitis virus

Assembly of viral membranes: nature of the association of vesicular stomatitis virus proteins to membranes

To explore the interaction of vesicular stomatitis virus (VSV) proteins with cellular membranes, we have isolated membranes from infected cells that have been radioactively pulse-labeled. We have found conditions of isolation that result in membrane preparation which contain primarily the VSV membrane protein (M) and glycoprotein (G). Both of these proteins are very firmly attached to membranes: conditions known to release peripherally associated membrane proteins from membranes (S. Razin, Biochim, Biophys. Acta 265:241-246, 1972; S. J. Singer, Annu. Rev. Biochem. 43:805-826, 1974; S. J. Singer and G. L. Nicholson, Science 175:720-731, 1972) are ineffective in detaching either the G or the M protein. The results of trypsin digestion of these membrane fractions suggest that the M protein resides primarily on one side, the cytoplasmic side of cellular membranes, whereas the glycoprotein has been transported to the lumen of the membrane vesicle. However, we present evidence that the glycoprotein is transmembranal and that approximately 3,000 daltons of one end of the molecule is on the cytoplasmic side of the membrane. We have also found that undenatured VSV M protein contains a trypsin-resistant core with a molecular weight of 22,000. This region of the M protein is trypsin-resistant regardless of its association with membranes.

In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes.

Translation in vitro of the mRNA coding for the vesicular stomatitis virus membrane glycoprotein G in a membrane-free ribosomal extract from HeLa cells allowed the synthesis of only the unglycosylated protein G1 (molecular weight, 63,000). Addition of stripped crude microsomal membranes from HeLa cells resulted in the conversion of G1 to the glycosylated protein G2 (molecular weight, 67,000). The G2 protein synthesized by the reconstructed microsomal membrane/ribosome system was found to be segregated inside the microsomal membrane vesicles and was thus protected from the proteolytic action of trypsin and chymotrypsin. Stripped membranes were required at an early stage of protein synthesis for the synthesized protein to be inserted into the membrane vesicles and to be glycosilated. The segregated protein G2, however, was not completely protected from proteolytic digestion, showing that a portion of the polypeptide chain of about 3000 daltons was present on the cytoplasmic side of the membrane vesicle. Our data thus suggest that, unlike the secretory proteins, the membrane glycoproteins are not completely discharged across the microsomal membranes.