The envelope of intracellular African swine fever virus is composed of a single lipid bilayer

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.     

Phospholipase C activity-induced fusion of pure lipid model membranes. A freeze fracture study

The structural effects of in situ production of diacylglycerol by phospholipase C in pure lipid model membranes have been examined by freeze fracture electron microscopy. Phospholipase C-activity induces massive aggregation and fusion of large unilamellar lipid vesicles and leads to the formation of a 'sealed' lipid aggregate; the outer membrane of this aggregate appears to be continuous. In some areas lipid arranges into a honeycomb structure; this structure is probably a precursor of a discontinuous inverted (type II) cubic phase. Similarly, enzyme treatment of multilamellar vesicles leads to extensive membrane fusion and vesiculation. Thus morphological evidence is obtained showing the ability of phospholipase C to induce bilayer destabilization and fusion. It is speculated that phospholipase C-induced membrane fusion involves a type II fusion intermediate induced by diacylglycerol produced locally.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

Study of adsorption of Influenza virus matrix protein M1 on lipid membranes by the technique of fluorescent probes

Matrix protein M1 of Influenza virus, which forms its inner scaffold, is the most abundant amongst viral proteins. Functions of M1 protein are highly diverse, as it has to ensure both the entry of the viral genetic material into the cytoplasm of the infected cell and the assembly of new viral particles for multiplication of infection. In all these processes matrix protein interacts with lipid membranes–either viral external lipid envelope or plasma membrane of a virus-infected cell. However, molecular mechanisms of such interactions are still unclear. In this work, we used the method of fluorescent probes on the example of 1-anilinonaphthalene- 8-sulfonate to determine components of the lipid bilayer required for binding of the M1 protein to the membrane, as well as possible orientations of the protein relative to the lipid membrane. We found that for the adsorption of matrix protein M1 lipid bilayer had to contain phosphatidylserines, while neither phosphatidylethanolamine nor cholesterol promoted protein binding to the membrane. Furthermore, our data suggest that M1 protein binds negatively charged lipid bilayer by positively charged amino acids exhibiting outward anionic sites.     

An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes.

The peptide fragment of the carboxy-terminal region of the human immunodeficiency virus (HIV) transmembrane protein (gp41) has been implicated in T-cell death. This positively charged, amphipathic helix (amino acids 828 to 848) of the envelope protein is located within virions or cytoplasm. We studied the interaction of the isolated, synthetic amphipathic helix of gp41 with planar phospholipid bilayer membranes and with Sf9 cells using voltage clamp, potentiodynamic, and single-cell recording techniques. We found that the peptide binds strongly to planar membranes, especially to the negatively charged phosphatidylserine bilayer. In the presence of micromolar concentrations of peptide sufficient to make its surface densities comparable with those of envelope glycoprotein molecules in HIV virions, an increase in bilayer conductance and a decrease in bilayer stability were observed, showing pore formation in the planar lipid bilayers. These pores were permeable to both monovalent and divalent cations, as well as to chloride. The exposure of the inner leaflet of cell membranes to even 25 nM peptide increased membrane conductance. We suggest that the carboxy-terminal fragment of the HIV type 1 envelope protein may interact with the cell membrane of infected T cells to create lipidic pores which increase membrane permeability, leading to sodium and calcium flux into cells, osmotic swelling, and T-cell necrosis or apoptosis.     

A trans-Golgi network resident protein, golgin-97, accumulates in viral factories and incorporates into virions during poxvirus infection

Poxviruses are the only DNA viruses known to replicate and assemble in the cytoplasm of infected cells. Poxvirus morphogenesis is a complicated process in which four distinct infectious forms of the virus are produced: intracellular mature virus, intracellular enveloped virus, cell-associated enveloped virus, and extracellular enveloped virus. The source of primary membrane wrapping the intracellular mature virus, the first infectious form, is still unknown. Although the membrane was suggested to originate from the endoplasmic reticulum-Golgi intermediate compartment, none of the marker proteins from this or any other cell compartments has been found in the intracellular mature virus. Thus, it was hypothesized that the membrane is either extensively modified by the virus or synthesized de novo. In the work described here, we demonstrate that a host cell protein residing in the trans-Golgi network membrane, golgin-97, is transported to the sites of virus replication and assembly and becomes incorporated into the virions during poxvirus infection. Inside the virion, golgin-97 is associated with the insoluble core protein fraction. Being able to adopt a long rod-like structure, the protein apparently extends through the virion envelope and protrudes from its surface. Here we discuss the potential role and functions of golgin-97 in poxvirus replication and propose two working models.     

ULTRASTRUCTURAL ORGANIZATION OF CHLOROPLAST THYLAKOIDS OF THE GREEN ALGA "OOCYSTIS MARSSONII"

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85–90 Å diameter particles. Adjacent membranes in stacked regions evidently share 170 Å diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 Å particles or only the upper portion of such particles, creating in the latter case images of 125–135 Å diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.     

Isolation, Chemical Composition, and Ultrastructural Features of the Cell Membrane of the Mycoplasma-Like Organism Spiroplasma citri

Thin sections of Spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with "Stubborn" disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm(3). Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 mug/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily.     

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Intramembrane changes occurring during maturation of herpes simplex virus type 1: freeze-fracture study.

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.     

DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures.

A molecular level theory is presented for the thermodynamic stability of two (similar) types of structural complexes formed by (either single strand or supercoiled) DNA and cationic liposomes, both involving a monolayer-coated DNA as the central structural unit. In the "spaghetti" complex the central unit is surrounded by another, oppositely curved, monolayer, thus forming a bilayer mantle. The "honeycomb" complex is a bundle of hexagonally packed DNA-monolayer units. The formation free energy of these complexes, starting from a planar cationic/neutral lipid bilayer and bare DNA, is expressed as a sum of electrostatic, bending, mixing, and (for the honeycomb) chain frustration contributions. The electrostatic free energy is calculated using the Poisson-Boltzmann equation. The bending energy of the mixed lipid layers is treated in the quadratic curvature approximation with composition-dependent bending rigidity and spontaneous curvature. Ideal lipid mixing is assumed within each lipid monolayer. We found that the most stable monolayer-coated DNA units are formed when the charged/neutral lipid composition corresponds (nearly) to charge neutralization; the optimal monolayer radius corresponds to close DNA-monolayer contact. These conclusions are also valid for the honeycomb complex, as the chain frustration energy is found to be negligible. Typically, the stabilization energies for these structures are on the order of 1 k(B)T/A of DNA length, reflecting mainly the balance between the electrostatic and bending energies. The spaghetti complexes are less stable due to the additional bending energy of the external monolayer. A thermodynamic analysis is presented for calculating the equilibrium lipid compositions when the complexes coexist with excess bilayer.     

Membrane remodeling by the double-barrel scaffolding protein of poxvirus

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors.     

X-Ray Diffraction Pattern from a Bilayer with Protein Outside

The X-ray diffraction pattern from a lipid bilayer has been reported previously; a series of fairly regularly spaced bands was both predicted and observed. In this note it is predicted that adding protein molecules at one or both surfaces of the bilayer will give rise to a cross-interference effect. For smaller amounts of protein, a more or less obvious ripple will be introduced into the bilayer pattern. The amount of protein, its thickness, and the distance from the bilayer to the protein layer all can be readily estimated from an observed ripple. Deciding whether the protein is all on one side or else distributed on both sides of the bilayer may be more difficult; by carefully recording and measuring the intensity near the center of the pattern one may be able to distinguish between the two possibilities. For larger amounts of protein, there will be more profound changes in the diffraction pattern. The theory developed here is applied in the following paper to a lipid dispersion incubated with cytochrome c and will be applied in a subsequent paper to a bacterial envelope. In an appendix it is shown that the patterns reported previously for several natural membranes do not confirm prediction for a normal, continuous lipid bilayer with all the protein outside. Thus it is doubtful that a structure of this kind is valid for these membranes.     

HIV-1, lipid rafts,and antibodies to liposomes: implications for anti-viral-neutralizing antibodies (Review)

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1.     

HIV-1, lipid rafts, and antibodies to liposomes: implications for anti-viral-neutralizing antibodies

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1.     

Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ~45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [(3)H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.     

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABA_A receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50–200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABA_A receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABA_A receptor activity and their effects on a lipid bilayer model system containing cholesterol.     

Vaccinia virus induces strong immunoregulatory cytokine production in healthy human epidermal keratinocytes: a novel strategy for immune evasion

Iatrogenic cutaneous infection with vaccinia virus (VV) and naturally occurring systemic infection with variola virus both lead to the characteristic skin "pox" lesions. Despite significant medical experience with both viruses, surprisingly little is understood about the interactions between these poxviruses and healthy resident skin cells. In recent years, it has become clear that skin plays an essential role in modulating both innate and adaptive immune responses, in part by producing and responding to a variety of cytokines and chemokines upon stimulation. Antagonists of many of these compounds are encoded in poxvirus genomes. Infection of skin cells with poxvirus may lead to a unique pattern of cytokine and chemokine production that might alter the cutaneous immune surveillance function. In this study, we infected primary cultures of human skin cells with VV and monitored antigen expression, virus replication, and cytokine production from the infected cells. While T cells, Langerhans cells, and dermal dendritic cells were infected abortively, keratinocytes, dermal fibroblasts, and dermal microvascular endothelial cells (HMVEC-d) all supported the complete virus life cycle. In contrast to the robust viral replication in fibroblasts and HMVEC-d, only limited viral replication was observed in keratinocytes. Importantly, VV infection of keratinocytes led to up-regulation of immunoregulatory and Th2 cytokines, including transforming growth factor beta, interleukin-10 (IL-10), and IL-13. We propose that the rapid induction of keratinocyte Th2 and immunoregulatory cytokines represents a poxvirus strategy to evade immune surveillance, and the limited viral multiplication in keratinocytes may be a protective mechanism to help the immune system "win the race."     

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.