Viral Phospholipids Research Articles

Functional Delineation of a Protein-Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8.

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.

Labyrinthopeptin A1 inhibits dengue and Zika virus infection by interfering with the viral phospholipid membrane

To date, there are no broad-spectrum antivirals available to treat infections with flaviviruses such as dengue (DENV) and Zika virus (ZIKV). In this study, we determine the broad antiviral activity of the lantibiotic Labyrinthopeptin A1. We show that Laby A1 inhibits all DENV serotypes and various ZIKV strains with IC50 around 1 μM. The structurally related Laby A2 also displayed a consistent, but about tenfold lower, antiviral activity. Furthermore, Laby A1 inhibits many viruses from divergent families such as HIV, YFV, RSV and Punta Torovirus. Of interest, Laby A1 does not show activity against non-enveloped viruses. Its antiviral activity is independent of the cell line or the used evaluation method, and can also be observed in MDDC, a physiologically relevant primary cell type. Furthermore, Laby A1 demonstrates low cellular toxicity and has a more favorable SI compared to duramycin, a well-described lantibiotic with broad-spectrum antiviral activity. Time-of-drug addition experiments demonstrate that Laby A1 inhibits infection and entry processes of ZIKV and DENV. We reveal that Laby A1 performs its broad antiviral activity by interacting with a viral factor rather than a cellular factor, and that it has virucidal properties. Finally, using SPR interaction studies we demonstrate that Laby A1 interacts with several phospholipids (i.e. PE and PS) present in the viral envelope. Together with other recent Labyrinthopeptin antiviral publications, this work validates the activity of Laby A1 as broad antiviral entry inhibitor with a unique mechanism of action and demonstrates its potential value as antiviral agent against emerging flaviviruses.

Reactivity of allyl methyl sulphide, the in-vitro metabolite of garlic, with some amino acids and with phospholipid involved in viral infections

Garlic, as well as several natural food ingredients such as basil, ginger, turmeric, cinnamon, clove, pepper etc., has long been traditionally used as routine anti-viral and anti-bacterial remedy. Allyl methyl sulfide (AMS) is reportedly a persistent main active metabolite component of allicin after garlic ingestion accounting for at least 90% of the allicin consumed. Several studies have reported the presence of AMS in organs such as lung, kidney etc. and body fluids such as mucous, and blood-plasma. Glycoproteins of enveloped viruses are actively involved in viral pathogenesis. N-acetylneuraminic acid (sialic acid) and N-Acetylglucosamine, are some of the vital amino acids involved in several viral infections using glycoproteins via glycosylation. Simulations studies based on First-principles density functional theory show that these amino acids attach with AMS, and the reactions are thermodynamically spontaneous (ΔG and ΔS negative are at 310.15 K as well as lower and higher temperatures). Further, phospholipid phosphatidylethanolamine (a component of some viral envelops) also attaches readily with AMS and the reaction is spontaneous. AMS molecules attachment with viral phospholipids and amino-acids involved in viral infection would denature the virus and prevent its attachment to the host cell. Communicated by Ramaswamy H. Sarma

Stoichiometry of Influenza Hemagglutinin Neutralization by Broadly Acting Antibodies using Single Virus-Particle Visualization

One of the first steps of influenza infection is fusion of the viral phospholipid envelope to a cellular membrane, opening a pore for the viral genome to enter a cell. The viral surface protein hemagglutinin (HA) mediates the fusion between these two lipid membranes and is a principal antigenic target for our immune system. Recently, antibodies capable of neutralizing both group 1 and group 2 influenza A viruses have been described. Here, we have visualized the interplay between a virus and antibodies during neutralization using our recently developed single-particle fusion assay. To this end, antibodies (mAb) or antibody fragments (Fab) and infectious viral particles were fluorescently labeled and incubated together. Viruses were then surface-immobilized to sialic acid decorated proteins incorporated into a planar, supported phospholipid bilayer and fusion induced by addition of an acidic buffer. Individual hemifusion events were observed as a dequenching spike, resulting from the rapid release of lipophilic dye from the viral membrane into the target bilayer. using this novel setup, we directly link the action of individual HA-binding mAb or Fab to viral fusion events and confirm that both act directly to inhibit HA-mediated membrane fusion in a dose-dependent fashion. Furthermore, we use the fluorescence intensity per virion to calculate the number of virus-bound mAb or Fab - information obfuscated in traditional cell-cell and bulk fusion assays. Our data indicate that total hemifusion inhibition occurs in a mAb/Fab coverage regime where hundreds of HA trimers remain fusion competent. Physical modeling further indicates that within this binding regime, these unbound HA can occur in spatial geometries allowing for the formation of fusogenic clusters. Overall, our results have explicit implications for the model of bilayer fusion arising from the coordinated efforts of multiple hemagglutinin molecules.

Quantitative dissociation of archaeal virus SH1 reveals distinct capsid proteins and a lipid core

Viruses infecting archaeal cells are less well understood than those infecting eukaryotic and bacterial cells. Here we study the distribution of the structural proteins between the capsid and the membrane of icosahedral SH1 virus, an archaeal virus infecting extreme halophilic Haloarcula hispanica cells. General features such as morphology, linear dsDNA genome and presence of lipids suggest that it may belong to the recently proposed PRD1-adenovirus lineage of viruses. To investigate this we have initiated structural studies of the virion. Quantitative dissociation of SH1 by 3 M urea or by lowering the salt concentration identified a number of soluble capsid-associated proteins (VP2, VP3, VP4, VP6, VP7 and VP9). These released proteins left behind a particle, or lipid core, containing two major proteins VP10 and VP12 and viral phospholipids. VP1 was released from the lipid core in low ionic strength conditions but not with 3 M urea. Approximately half of the protein VP5 stayed with the lipid core and the other half was released. Analysis of the soluble capsid-associated proteins by their sedimentation and hydrodynamic properties suggests that the most abundant proteins, putative capsomers VP4 and VP7, form an intricate pattern of protein complexes. We also observed large differences in the sizes of the complexes determined by the two different methods suggesting an elongated overall structure for most of the capsid-associated proteins or protein complexes. This work verifies that there is an internal membrane vesicle residing inside the complex icosahedral capsid that is akin to the overall structure of PRD1-like viruses.

Interaction of reconstituted Sendai viral envelopes with sperm cells: reconstituted Sendai virus envelope-induced fusion-mediated introduction of foreign material into bull sperm cells.

Reconstituted Sendai virus envelopes (RSVE), i.e. membrane vesicles bearing the viral envelope glycoproteins and phospholipids, are able to fuse with bull sperm cells. This was inferred from the increase in the degree of fluorescence dequenching (DQ) obtained following incubation of fluorescently labeled (R18 labeled) RSVE with bull sperm cells and from electron microscopy studies of RSVE-sperm interaction. Only a low degree of DQ was observed, under the same conditions, with non-fusogenic fluorescently labeled RSVE. This, and electron microscopy results, show that binding and membrane fusion events occur between RSVE and sperm cells. In addition, DQ was observed following incubation of RSVE that had been pre-loaded with the self-quenched fluorochrome Calcein, with bull sperm cells, indicating fusion-mediated injection of the dye from the RSVE space into the sperm cells.

Calorimetric detection of influenza virus induced membrane fusion

Membrane fusion induced by the hemagglutinin glycoprotein of influenza virus has been extensively characterized, but the mechanism whereby the protein achieves the merger of the viral and target membrane lipids remains enigmatic. Various lipid intermediate structures have been proposed, and the energies required for their formation predicted. Here, we have analyzed the enthalpies of fusion of influenza with liposomes by titration calorimetry. If a small sample of virus in a weak neutral pH buffer was added to an excess of liposomes at low pH, a two-component reaction was seen, composed of an exothermic reaction and a slower endothermic reaction. The exothermic reaction was the result of acid-base reactions between the neutral pH virus sample and low pH buffer and low-pH-induced changes in the virus. The endothermic reaction was not observed in the absence of liposomes and much reduced if acid-inactivated virus, which had lost its fusion but not its binding activity, was added to liposomes. The endothermic reaction was more temperature dependent than the exothermic reaction; its pH dependence corresponded with that of fusion and its enthalpy was higher if fusion was more extensive. These data indicate that most of the endothermic reaction was due to membrane fusion. The experimentally determined enthalpy of fusion, 0.6-0.7 kcal per mol of viral phospholipids, is much higher than expected on the basis of current theories about the formation of lipid intermediates during membrane fusion.

Structural Studies of the Enveloped dsRNA Bacteriophage θof Pseudomonas syringae by Raman Spectroscopy: I. The Virion and Its Membrane Envelope

We report and interpret the first Raman spectrum of a double-stranded RNA virus containing a membrane envelope. Spectra of the native bacteriophage θ6 and of its isolated host-attachment (spike) protein and phospholipid-free core assembly were collected from aqueous solutions over a wide range of temperature. Comparison of the vibrational spectra by digital difference methods permits the following structural conclusions regarding molecular constituents of the fully assembled virion. (1) The double-stranded RNA, phospholipid and protein components of the phage exhibit Raman amplitudes in accordance with their biochemically determined compositions in the native virion (10, 20 and 70%, respectively). (2) α-Helix and irregular conformations are the dominant secondary structures in proteins of both the viral membrane and nucleocapsid. This represents a departure from previously examined icosahedral phage and plant viruses, which are dominated by β-sheet structures. (3) The phospholipids of the viral membrane are liquid crystalline throughout the determined range of virus thermostability (0 to 40°C). (4) The P3 spike protein of θ6, which is anchored to, but not sequestered within the viral membrane, is largely α-helical (∼35%) and highly thermolabile. Denaturation of P3 at temperatures above 30°C leads to appreciable loss (∼ 20%) of α-helix in favor of β-strand structure, and alters significantly the environments of many aromatic side-chains. (5) The secondary structures of integral membrane proteins of θ6 are overwhelmingly α-helical (∼70 to 80%) and also thermolabile. In contrast to P3, which exhibits aspartate and glutamate carboxyls in the ionized form (CO-2), the integral membrane proteins exhibit only protonated carboxyl groups (COOH).Treatment of θ6 with butylated hydroxytoluene (BHT), which has been shown to remove the P3 spike protein, does not significantly perturb phospholipids and associated integral proteins of the viral membrane or structural proteins and packaged double-stranded RNA of the nucleocapsid. However, P3 subunits, which are recovered after BHT treatment, exhibit radically altered secondary and tertiary structures, including the loss of most subunit α-helices. Among the P3 side-chains affected by BHT treatment, we note a general trend toward greater hydrophilicity and greater solvent exposure of the aromatic residues Trp and Tyr. On the other hand, the cysteine sulfhydryl groups of the BHT-isolated P3 monomer are not solvent exposed and function as strong hydrogen-bond donors in the protein core. The BHT-mediated structure transition of P3 is discussed in relation to putative quaternary rearrangements involving the membrane anchor protein, P6, and homomultimer complexes of the P3 monomer.The present studies show that RNA, nucleocapsid and viral membrane components of an enveloped double-stranded RNA virus can be distinguished and structurally characterized by Raman spectroscopy. Importantly, we have demonstrated the ability to obtain Raman spectra from the intact viral membrane, establishing the feasibility of Raman spectroscopy for structural analysis of protein molecules embedded in a viral phospholipid bilayer. The results obtained also serve as the basis for detailed spectroscopic analyses of the θ6 nucleocapsid, the θ6 polymerase complex (nucleocapsid core), and the packaged θ6 genome, as described in the accompanying paper (Bamford et al., 1993).

Enrichment of the bacteriophage PR4 membrane in phosphatidylglycerol is not essential for phage assembly and infectivity.

The membrane phospholipids of bacteriophage PR4 grown on wild-type Escherichia coli are markedly enriched in phosphatidylglycerol (PG) relative to host phospholipids. To investigate the role of PG in phage assembly and infectivity, we propagated PR4 on an E. coli mutant defective in PG synthesis. The PG content of PR4 grown on the mutant host accounted for 0.4% of the total viral phospholipids, representing a 90-fold decrease in PG relative to the PG content of phage grown on a wild-type host. Phosphatidylethanolamine and phosphatidic acid, the two major phospholipid species present in these phage preparations, accounted for 88.4 and 9.4% of the total viral phospholipids, respectively. This drastic alteration of the phage phospholipid composition had little or no adverse effect on either the stability or infectivity of the phage. We conclude that the enrichment of the PR4 virion in PG does not reflect an absolute structural requirement of the phage and is not essential for phage infectivity.

Location of DNA-binding proteins and disulfide-linked proteins in vaccinia virus structural elements.

Treatment with sodium dodecyl sulfate (SDS) converted the vaccinia virus strain IHD-J into particles of two types: (i) ghosts which possessed a thin-membrane vesicle derived from basement part of the virus membrane with attached lateral bodies and a membranous structure derived from the core wall and (ii) aggregates of a DNA-nucleoprotein eluted from the core. These particles lacked lipids, and all the viral phospholipids were detected in the SDS-soluble fraction. The viral membrane was composed of an SDS-soluble coat layer and the basement membrane, and the basement membrane was maintained by a mechanism other than the lipid bilayer. By comparisons of protein species in morphologically distinct subviral particles prepared by several solubilizing methods, protein compositions of viral structural elements were suggested as follows: 25,000-molecular-weight viral protein-17,000-molecular-weight viral protein ( VP25K - VP17K ), viral basement membrane; VP13 . 8K , major component of the lateral body; VP70K , VP69K , VP66K , and VP64K , minor components of the lateral body; VP61K , outer layer of core wall; VP57K - VP22K , inner layer of core wall; and VP27K - VP13K , nucleoprotein. These structural elements found in the SDS-insoluble particles dissolved in the same SDS solution under reducing conditions, indicating that the disulfide linkages seem to have a principal role in maintaining their morphological integrity. VP57K , VP27K , VP13 . 8K , and VP13K were revealed to possess affinity for DNA. Denatured calf thymus DNA and viral DNA in double- or single-stranded form associated equally well with these proteins, but RNA did not bind. Therefore, it was strongly suggested that disulfide-linked VP27K - VP13K represented the nucleoproteins of vaccinia virus. A structural model of vaccinia virus is proposed and discussed.

A new method for reconstitution of highly fusogenic sendai virus envelopes

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.

The lipid-containing bacteriophage PR4: Effects of altered lipid composition on the virion

Phage PR4 was grown on a variety of Escherichia coli mutants defective in fatty acid and phospholipid metabolism. The composition of the phage lipids was modified by changing the composition of the host membrane phospholipids. The compositions of both the polar and the acyl moieties of the phospholipids were altered. The proportion of saturated fatty acids in the phage phospholipids was increased in increments from 44% of the total fatty acids to 55%, 61% and 69% of the total fatty acids using a host mutant with a temperature-sensitive defect in unsaturated fatty acid biosynthesis. The increase in saturated fatty acids led to a pronounced loss of infectivity when the phage were incubated at temperatures between 2 degrees C and 30 degrees C (temperatures below those at which the phage were grown). The greater the level of saturated fatty acids in the phage phospholipids, the higher the temperature below which the phage were inactivated. Our results strongly suggest that the phage membrane undergoes a lipid phase transition, which can disrupt and inactivate the virion. The phospholipid composition of PR4 was also altered by using host mutants defective in phosphatidylethanolamine and/or cardiolipin synthesis. Phage PR4 grown on wild-type host strains contains 56% phosphatidylethanolamine, 37% phosphatidylglycerol, 4.6% cardiolipin and no detectable phosphatidylserine. However, in response to changes in the host, PR4 preparations were obtained with phospholipid compositions varying from 28% to 60% in phosphatidylethanolamine, from 22% to 39% in phosphatidylglycerol, from 1% to 15% in cardiolipin and containing as much as 35% phosphatidylserine. These changes in phospholipid composition did not affect the infectivity of the phage. Moreover, the increased level of phosphatidylglycerol in the phage relative to the host was not altered by these manipulations. It is concluded that the net charge of the phage membrane phospholipids is not involved in the selection or function of the viral phospholipids. We also present evidence suggesting that the phage and host membranes do not fuse during the course of infection.

Localization of membrane proteins by the use of a photoreactive fatty acid incorporated in vivo into vesicular stomatitis virus.

Vesicular stomatitis virus grown in the presence of omega-[9-3H]diazirinophenoxy nonanoate resulted in the biosynthetic incorporation of this photoreactive fatty acid into viral phospholipids as well as into the membrane anchoring domain of the viral G glycoprotein. Photolysis of the isolated virus at 360 nm resulted in extensive labeling of the G protein but none of the other proteins by the viral phospholipids. In addition, a new product was obtained and was identified as a G-G dimer by its molecular weight and its reaction with anti-G antibody. These results demonstrate the use of photoreactive fatty acid to identify integral membrane proteins and to make photoaffinity probes of fatty acylated membrane proteins.

Acyl Bis(monoacylglycero)phosphate, assumed to be a marker for lysosomes, is a major phospholipid of vaccinia virions

A hitherto unknown major phospholipid of purified vaccinia virions harvested from lysed cells was identified as acyl bis(monoacylglycero)phosphate by chromatographic comparison with synthetic bisphosphatidic acid and various analogs. It accounts for 20–30% of the total viral phospholipids. Digestion with pancreatic phospholipase A 2 liberates fatty acids and results in bis(monoacylglycero)phosphate, which is thought to represent a phospholipid marker for cellular lysosomes. Possible implications for the intracellular virus dissemination are discussed.

Reconstituted G protein-lipid vesicles from vesicular stomatitis virus and their inhibition of VSV infection.

The single glycoprotein (G) of vesiclar stomatitis virus (VSV) was isolated in nearly quantitative yield by extraction of the purified virions with 0.05 M octyl-beta-D- glucoside (OG) in 0.01 M sodium phosphate, pH 8.0. The extract contained essentially all of the viral phospholipids and glycolipids, and was free of other essentially all of the viral phospholipids and glycolipids, and was free of other viral proteins. Dialysis to remove OG resulted in the formation of G protein-viral lipid vesicles having a lipid-G protein ratio similar to that of the intact virions. The vesicles were 250-1,000 A in diameter, with a "fuzzy" external layer also similar to that of intact virions. The vesicles were predominantly unilamellar and sealed, with both phosphatidyl ethanolamine and gangliosides symmetrically distributed in the bilayer. G protein was asymmetrically oriented, with about 80 percent accessible to exogenous protease. Addition of soybean phospholipid to the viral extract before dialysis resulted in vesicles that incorporated viral proteins and lipids quantitatively, but that were markedly decreased in buoyant density. The G neutralized protein-lipid vesicles were effective in eliciting specific anti-G antibodies that neutralized viral infectivity. Competitive radioimmunoassay showed that both reconstituted vesicles and a soluble form of G protein (Gs) were indistinguishable from purified VSV in their antibody binding properties. Addition of G protein-lipid vesicles of BHK-21 cells before, or simultaneously with, infection by VSV inhibited viral infectivity, as measured by two independent techniques (viral RNA production in the presence of actinomycin D and a neutral red assay of cell viability). The total inhibitory activity of G protein in the vesicular form was, however, less than 5 percent of that found for intact virus particles that have been inactivated by ultraviolet light irradiation. Gs was inactive as an inhibitor as determined by the RNA production assay.

Origin of the phospholipids of a lipid-containing virus that replicates in Escherichia coli: bacteriophage PR4.

Bacteriophage PR4 contains lipid and can reproduce in strains of Escherichia coli that carry an appropriate drug-resistance plasmid. Cultivated in either of two E. coli strains, PR4 acquires a lipid region that contains a relatively high level of phosphatidylglycerol and significant amounts of three phospholipids, including phosphatidylserine, which are present in only very low levels in the host cell membranes. To do this, however, PR4 does not significantly alter the relative levels of synthesis of the various E. coli phospholipids after infection. Production of PR4 virions from E. coli cultures labeled with 32PO4 either before or after infection showed that about two-thirds of the viral phospholipid is synthesized after infection. The use of E. coli as the host organism for PR4 should allow a detailed understanding of the assembly process of this lipid-containing virus due to the wealth of biochemical and genetic techniques available.

Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus.

Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.

Temporal origin of viral phospholipids of the enveloped bacteriophage phi 6.

The enveloped bacteriophage phi 6 contains a higher relative level of the negatively charged phospholipid phosphatidylglycerol than is found in the membranes of the host bacterium. During infection of Pseudomonas phaseolicola with phi 6, the level of phosphatidylglycerol synthesis increases significantly. The lipid used to form the viral envelope consists almost entirely of cellular phospholipids synthesized before infection and phosphatidylglycerol synthesized after infection. Based on these and previously published results, a speculative model for this viral envelope formation process is presented.

Structure and synthesis of a lipid-containing bacteriophage XIX. Reconstitution of bacteriophage PM2 In Vitro

The lipid-containing bacteriophage PM2 was reconstituted stepwise from its separated subunits: the superhelical DNA, the two nucleocapsid proteins, the external coat protein, the spike protein, and the two lipids, phosphatidylglycerol and phosphatidylethanolamine. In the first step the nucleocapsid was reconstituted from the DNA and the two nucleocapsid proteins. Since the yield of nucleocapsid was very low, and since the reconstituted nucleocapsid was identical to that obtained by partial degradation of the virion with urea, we used nucleocapsid obtained from the virion for the further studies on reconstitution. Combination of the nucleocapsid from virions with various mixtures of viral lipids resulted in formation of a lipid-containing nucleocapsid that was not able to bind the remaining coat and spike proteins. The ratio of phosphatidylethanolamine to phosphatidylglycerol in the reconstituted lipid-containing nucleocapsid was identical with the ratio of phospholipids added to the incubation mixture. Combination of the nucleocapsid from virions with the coat and spike proteins in the presence of the viral lipids resulted in the formation of infectious virus. In this case the ratio of phosphatidylethanolamine to phosphatidylglycerol in the reconstituted virus was independent of the ratio of these phospholipids in the incubation mixture and identical with that of the purified bacteriophage. The basis of this effect was demonstrated in two-phase partition experiments. The nucleocapsid proteins do not selectively bind viral phospholipids, whereas the coat protein specifically binds phosphatidylglycerol. The affinity of the coat protein to phosphatidylglycerol, or vice versa , is partially responsible for the assembly in vitro of active virus particles containing the correct ratio of phospholipids.

Phospholipids in newcastle disease virus infected cells

Infection of chicken cells with Newcastle Disease Virus modifies phosphatidylserine and phosphatidylcholine systhesis in the host cell. The virion contains cellullar phospholipids synthesized both before and after infection. Relative concentration of various labeled phospholipids in the virus differ from those in the corresponding cells and their surface membranes. Late in infection, fragments of membranes with a distribution of labeled phospholipids similar but not identical to that of the virus can be found in the supernatant of infected cells. The significance of these findings is discussed in relation to origin of viral phospholipids and the intervention of the host cell membrane in the assembly of the viral envelope.