Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil-type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus-soze membrane vesicles. Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.     

Association of sindbis virus capsid protein with phospholipid membranes and the E2 glycoprotein: implications for alphavirus assembly

A late stage in assembly of alphaviruses within infected cells is thought to be directed by interactions between the nucleocapsid and the cytoplasmic domain of the E2 protein, a component of the viral E1/E2 glycoprotein complex that is embedded in the plasma membrane. Recognition between the nucleocapsid protein and the E2 protein was explored in solution using NMR spectroscopy, as well as in binding assays using a model phospholipid membrane system that incorporated a variety of Sindbis virus E2 cytoplasmic domain (cdE2) and capsid protein constructs. In these binding assays, synthetic cdE2 peptides were reconstituted into phospholipid vesicles to simulate the presentation of cdE2 on the inner leaflet of the plasma membrane. Results from these binding assays showed a direct interaction between a peptide containing the C-terminal 16 amino acids of the cdE2 sequence and a Sindbis virus capsid protein construct containing amino acids 19-264. Additional experiments that probed the sequence specificity of this cdE2-capsid interaction are also described. Further binding assays demonstrated an interaction between the 19-264 capsid protein and artificial vesicles containing neutral or negatively charged phospholipids, while capsid protein constructs with N-terminal truncations displayed either little or no affinity for such vesicles. The membrane-binding property of the capsid protein suggests that the membrane may play an active role in alphavirus assembly. The results are consistent with an assembly process involving an initial membrane association, whereby an association with E2 glycoprotein further enhances capsid binding to facilitate membrane envelopment of the nucleocapsid for budding. Collectively, these experiments elucidate certain requirements for the binding of Sindbis virus capsid protein to the cytoplasmic domain of the E2 glycoprotein, a critical event in the alphavirus maturation pathway.     

Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites.

At areas of adhesion between outer membrane (OM) and inner membrane (IM) in gram-negative bacteria, newly synthesized membrane constituents are inserted, and bacteriophage infection occurs. We describe here the isolation of these sites from cell membrane fractions of Salmonella anatum. Sucrose density gradients yielded membrane vesicles of the OM and IM; their mutual cross-contamination was low, as measured by 2-keto-3-deoxyoctonate and beta-NADH-oxidase activities. To mark the areas of lipopolysaccharide synthesis in the envelope (the adhesion sites), we infected S. anatum with phage epsilon 15, which causes a rapid change (conversion) in the cell's O-antigenic composition from serogroup E1 to E2; lipopolysaccharide of type E2 also serves as receptor for phage epsilon 34. We found that the fractions of intermediate density (Int. M) from briefly converted cells bound both phage epsilon 34 and E2-specific antibody. In the electron microscope, epsilon 34 was seen to have absorbed with a high degree of significance to the Int. M fraction of briefly converted cells, but not to the Int. M fraction of unconverted cells. Furthermore, the Int. M fractions of briefly converted cells coagglutinated anti-E2-coated Staphylococcus aureus, whereas the OM and IM fractions showed comparatively little agglutination. In addition, Int. M material exhibited elevated phospholipase A1 and A2 activities comparable to those of the OM fraction; the IM was essentially phospholipase free. Our data indicate that this membrane fractionation allows one to isolate from Int. M regions a variety of activities associated with adhesion sites.     

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.     

pH对菌紫质分子的旋转运动和光电响应的影响

用闪光诱导瞬间二向色性方法测量了不同pH条件下的菌紫质分子在脂质囊泡中的旋转扩散运动.在人工平扳膜(BLM)系统中测量了不同pH条件下菌紫质分子的光电响应.在pH3至8.3的范围内没有明显观察到菌紫质分子在膜中旋转运动上的差别.pH低于3时,菌紫质分子旋转运动受到影响;pH高于11时,观察不到旋转扩散运动.在BLM系统中测量了pH2到pH11范围内菌紫质分子的光电响应信号,随着pH的增加,无论紫膜碎片还是单体菌紫质分子的光电响应逐渐由照光后快速正信号并快速衰减及撤光时的快速负信号并逐渐衰减变成慢的正信号.pH高于9.4时,单体菌紫质分子的光电响应信号由正变负,pH高于11时,观察不到信号.     

Further morphological characterization and structural proteins of infectious bursal disease virus.

An outer layer surrounding the capsid of infectious bursal disease virus was evident from electron micrographs of intact virus particles having diameters of 62 to 63 nm. The capsid was found to be composed of large morphological units or capsomeres, measuring about 12 nm in diameter. The architecture of the capsid appears to be that of T = 3 symmetry, with a probable 32 morphological units by rotational enhancement of image detail. Structural proteins of infectious bursal disease virus consist of seven species, two major and five minor polypeptides. These are P1 to P7, with molecular weights of 133 x 10(3), 124 x 10(3), 98 x 10(3), 51 x 10(3), 33 x 10(3), 26 x 10(3), and 23 x 10(3), respectively.     

脂质泡中的菌紫质旋转运动的介质依赖性

用闪光诱导瞬间二向色性方法测量了蔗糖和甘油对菌紫质（ＢＲ）分子在脂质泡中的旋转扩散运动的影响。结果表明脂质囊泡本身在悬浮液中的运动并不会影响ＢＲ分子在膜中的旋转运动的测量。蔗糖和甘油对ＢＲ旋转运动的影响在相变温度以上和相变温度以下是不同的,相变温度以下的主要作用是相分离,使运动减慢,相变温度以上的作用可能是脂的分散,使运动加快。     

Magnetic anisotropy of egg lecithin membranes.

Magnetic realignment and rotational diffusion of cylindrical egg lecithin vesicles were measured under a phase contrast microscope. The anisotropy of magnetic susceptibility times membrane thickness was calculated from the data for several thin-walled vesicles. The resulting values were assigned to discrete numbers of bilayers. The difference between the susceptibilities parallel and perpendicular to the long axes of the lecithin molecules is deduced to be X parallel - X perpendicular = -(0.28 +/- 0.02) . 10(-8) cgs at 23 degrees C, if a bilayer thickness of 60 A is assumed.     

Increase in lipid microviscosity of unilamellar vesicles upon the creation of transmembrane potential

Summary Diffusion potential of potassium ions was formed in unilamellar vesicles of phosphatidyl choline. The vesicles, which included potassium sulfate buffered with potassium phosphate, were diluted into an analogous salt solution made of sodium sulfate and sodium phosphate. The diffusion potential was created by the addition of the potassium-ionophore, valinomycin. The change in lipid microviscosity, ensuing the formation of membrane potential, was measured by the conventional method of fluorescence depolarization with 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid microviscosity was found to increase with membrane potential in a nonlinear manner, irrespective of the potential direction. Two tentative interpretations are proposed for this observation. The first assumes that the membrane potential imposes an energy barrier on the lipid flow which can be treated in terms of Boltzmann-distribution. The other interpretation assumes a decrease in lipid-free volume due to the pressure induced by the electrical potential. Since increase in lipid viscosity can reduce lateral and rotational motions, as well as increase exposure of functional membrane proteins, physiological effects induced by transmembrane potential could be associated with such dynamic changes.     

The size of the lactose permease derived from rotational diffusion measurements.

The lactose permease of Escherichia coli was labeled with eosinyl-maleimide, reconstituted into vesicles of dimyristoylphosphatidylcholine and subjected to time-dependent phosphorescence anisotropy measurements in order to determine the rotational diffusion coefficient. By comparison with bacteriorhodopsin, the diffusion coefficient is evaluated in terms of an effective radius of the lactose permease in the plane of the membrane. This radius amounts to 20 +/- 2 A which implies that the lactose permease is a monomer. The monomeric state is maintained in the presence of a membrane potential.     

More than one door - Budding of enveloped viruses through cellular membranes

Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.     

Dynamics of DNA ejection from bacteriophage

The ejection of DNA from a bacterial virus (i.e., phage) into its host cell is a biologically important example of the translocation of a macromolecular chain along its length through a membrane. The simplest mechanism for this motion is diffusion, but in the case of phage ejection a significant driving force derives from the high degree of stress to which the DNA is subjected in the viral capsid. The translocation is further sped up by the ratcheting and entropic forces associated with proteins that bind to the viral DNA in the host cell cytoplasm. We formulate a generalized diffusion equation that includes these various pushing and pulling effects and make estimates of the corresponding speedups in the overall translocation process. Stress in the capsid is the dominant factor throughout early ejection, with the pull due to binding particles taking over at later stages. Confinement effects are also investigated, in the case where the phage injects its DNA into a volume comparable to the capsid size. Our results suggest a series of in vitro experiments involving the ejection of DNA into vesicles filled with varying amounts of binding proteins from phage whose state of stress is controlled by ambient salt conditions or by tuning genome length.     

Assembly of gag-beta-galactosidase proteins into retrovirus particles.

We studied the expression of beta-galactosidase (beta-gal) and 15 gag-beta-gal fusion proteins in the presence of Moloney murine leukemia virus wild-type core (gag) proteins. Analysis indicated that proteins retaining the amino-terminal portion of gag through the capsid protein-coding region were incorporated into retrovirus particles. Proteins which deleted portions of the capsid protein were assembled into virions at low efficiency, indicating the importance of capsid protein interactions in retrovirus assembly. Fusion proteins which retained the amino-terminal matrix protein of the gag polyprotein but which lacked the capsid protein were released efficiently from cells in a nonviral form. The nonviral form was characterized by a high sedimentation coefficient and a low density, suggestive of membrane vesicles. While beta-gal was present in the cytoplasm of expressing cells, all fusion constructs were associated with cellular membranes. gag-beta-gal proteins which were capable of release from cells demonstrated a two-component immunofluorescence staining pattern consisting of a circle of fluorescence around the nucleus and a punctate pattern of staining throughout the remainder of the cell. Interestingly, fusions within the matrix protein were trapped intracellularly and yielded distinct perinuclear staining patterns, possibly localizing to the rough endoplasmic reticulum and/or Golgi. This observation suggests that Moloney murine leukemia virus gag proteins travel to the plasma membrane by vesicular transport associated with the cytoplasmic face of intracellular vesicles.     

Acyl Chain Length and Saturation Modulate Interleaflet Coupling in Asymmetric Bilayers: Effects on Dynamics and Structural Order

A long-standing question about membrane structure and function is the degree to which the physical properties of the inner and outer leaflets of a bilayer are coupled to one another. Using our recently developed methods to prepare asymmetric vesicles, coupling was investigated for vesicles containing phosphatidylcholine (PC) in the inner leaflet and sphingomyelin (SM) in the outer leaflet. The coupling of both lateral diffusion and membrane order was monitored as a function of PC and SM acyl chain structure. The presence in the outer leaflet of brain SM, which decreased outer-leaflet lateral diffusion, had little effect upon lateral diffusion in inner leaflets composed of dioleoyl PC (i.e., diffusion was only weakly coupled in the two leaflets) but did greatly reduce lateral diffusion in inner leaflets composed of PC with one saturated and one oleoyl acyl chain (i.e., diffusion was strongly coupled in these cases). In addition, reduced outer-leaflet diffusion upon introduction of outer-leaflet milk SM or a synthetic C24:0 SM, both of which have long interdigitating acyl chains, also greatly reduce diffusion of inner leaflets composed of dioleoyl PC, indicative of strong coupling. Strikingly, several assays showed that the ordering of the outer leaflet induced by the presence of SM was not reflected in increased lipid order in the inner leaflet, i.e., there was no detectable coupling between inner and outer leaflet membrane order. We propose a model for how lateral diffusion can be coupled in opposite leaflets and discuss how this might impact membrane function.     

Southern rice black-streaked dwarf virus hijacks SNARE complex of its insect vector for its effective transmission to rice

Vesicular trafficking is an important dynamic process that facilitates intracellular transport of biological macromolecules and their release into the extracellular environment. However, little is known about whether or how plant viruses utilize intracellular vesicles to their advantage. Here, we report that southern rice black-streaked dwarf virus (SRBSDV) enters intracellular vesicles in epithelial cells of its insect vector by engaging VAMP7 and Vti1a proteins in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The major outer capsid protein P10 of SRBSDV was shown to interact with VAMP7 and Vti1a of the white-backed planthopper and promote the fusion of vesicles into a large vesicle, which finally fused with the plasma membrane to release virions from midgut epithelial cells. Downregulation of the expression of either VAMP7 or Vti1a did not affect viral entry and accumulation in the gut, but significantly reduced viral accumulation in the haemolymph. It also did not affect virus acquisition, but significantly reduced the virus transmission efficiency to rice. Our data reveal a critical mechanism by which a plant reovirus hijacks the vesicle transport system to overcome the midgut escape barrier in vector insects and provide new insights into the role of the SNARE complex in viral transmission and the potential for developing novel strategies of viral disease control.     

Complex formation in vesicle-reconstituted mitochondrial cytochrome P450 systems (CYP11A1 and CYP11B1) as evidenced by rotational diffusion experiments using EPR and ST-EPR.

Rotational diffusion measurements using EPR and saturation transfer EPR were applied to analyze complex formation between the electron-transfer components of the mitochondrial steroid-hydroxylating cytochrome P450 systems (CYP11A1 and CYP11B1) in phosphatidylcholine/phosphatidylethanolamine/cardiolipin vesicles prepared by octyl glucoside dialysis/adsorption. Octyl glucoside reconstitution of P450SCC results in large vesicles, which have an advantage over small vesicles in that vesicle tumbling does not contribute to measured rotational diffusion rates. Immobilization of spin-labeled adrenodoxin by both P450SCC and adrenodoxin reductase indicates equimolar complexation between P450SCC and adrenodoxin as well as between adrenodoxin reductase and adrenodoxin. Combination of rotational diffusion and antibody cross-linking confirmed the complexation of adrenodoxin with P450SCC and for the first time provided direct evidence of a complex between P450SCC and P45011beta in the membrane. In contrast, no evidence was found for the existence of adrenodoxin reductase-P450SCC complexes or a ternary complex of all three proteins. Thus, these experiments confirm the shuttle mechanism of electron transfer to vesicle-reconstituted P450SCC and P45011beta.     

E-type delayed fluorescence depolarization, technique to probe rotational motion in the microsecond range.

E (eosin)-type delayed fluorescence depolarization studies extend the time range for the measurement of rotational diffusion to microseconds and ms, thereby allowing investigation of slow rotational movement of macromolecules like membrane proteins. An apparatus is described for the determination of time-dependent anisotropy in this interesting time range. The method has been tested on eosin-labelled cytochrome P-450 incorporated into phospholipid membrane vesicles.     

Use of phospholipid transfer protein as a probe to study the lipid dynamics and alkaline phosphatase activity in the brush border membrane of human term placenta

Incubation of placental brush border membrane (BBM) along with sonicated vesicles of exogenous lipids (egg yolk PC) in the presence of phospholipid-transfer protein (PL-TP) showed a decrease in the alkaline phosphatase activity due to the change in the membrane micro-environment, such as fluidity. Effect of substrate concentration was tested by Lineweaver-Burk plot, which showed decreased V(max) and K(M). The effect of temperature was probed by the Arrhenius plot, which showed no change in transition temperature, but a decline in the energy of activation both below and above the transition temperature. The protein-catalyzed transfer of phospholipid from the donor unilamellar vesicles resulted in a substantial increase in the BBM phospholipid and a net decrease in cholesterol/phospholipid molar ratio. The change in membrane fluidity was assessed by translational as well as rotational diffusion of membrane extrinsic fluorescent probes, pyrene and diphenyl-hexatriene. An increased lateral mobility was recorded by the increased pyrene excimer formation. A decrease in fluorescent polarization of diphenyl-hexatriene was observed, which led to the decrease in fluorescence anisotropy and order parameter, and therefore, an increase in membrane fluidity (rotational diffusion). Mean anisotropy parameter was also decreased in the presence of PL-TP. Thus, the placental BBM alkaline phosphatase activity showed a distinct lipid dependence which may have important physiological consequences.