Murine leukemia virus glycosylated Gag (gPr80gag) facilitates interferon-sensitive virus release through lipid rafts.

Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65(gag), the precursor for internal structural (Gag) proteins. Whereas gPr80gag is evolutionarily conserved among gammaretroviruses, its mechanism of action has been unclear, although it facilitates virus production at a late assembly or release step. Here, it is shown that gPr80gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular, gPr80gag-facilitated release occurs through lipid rafts, because gPr80gag-negative M-MuLV has a lower cholesterol content, is less sensitive to inhibition of release by the cholesterol-depleting agent MbetaCD, and there is less Pr65gag associated with detergent-resistant membranes in mutant-infected cells. gPr80gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.

Thin Films of a Complex Polymer Compound for the Inhibition of Iron Alloy Corrosion in a H3PO4 Solution.

The etching of iron alloy items in a H3PO4 solution is used in various human activities (gas and oil production, metalworking, transport, utilities, etc.). The etching of iron alloys is associated with significant material losses due to their corrosion. It has been found that an efficient way to prevent the corrosion of iron alloys in a H3PO4 solution involves the formation of thin complex compound films consisting of the corrosion inhibitor molecules of a triazole derivative (TrzD) on their surface. It has been shown that the protection of iron alloys with a mixture of TrzD + KNCS in a H3PO4 solution is accompanied by the formation of a thin film of coordination polymer compounds thicker than 4 nm consisting of TrzD molecules, Fe2+ cations and NCS-. The layer of the complex compound immediately adjacent to the iron alloy surface is chemisorbed on it. The efficiency of this composition as an inhibitor of iron alloy corrosion and hydrogen bulk sorption by iron alloys is determined by its ability to form a coordination polymer compound layer, as experimentally confirmed by electrochemical, AFM and XPS data. The efficiency values of inhibitor compositions 5 mM TrzD + 0.5 mM KNCS and 5 mM TrzD + 0.5 mM KNCS + 200 mM C6H12N4 at a temperature of 20 ± 1 °C are 97% and 98%, respectively. The kinetic parameters of the limiting processes of hydrogen evolution and permeation into an iron alloy in a H3PO4 solution were determined. A significant decrease in both the reaction rate of hydrogen evolution and the rate of hydrogen permeation into the iron alloy by the TrzD and its mixtures in question was noted. The inhibitor compositions 5 mM TrzD + 0.5 mM KNCS and 5 mM TrzD + 0.5 mM KNCS + 200 mM C6H12N4 decreased the total hydrogen concentration in the iron alloy up to 9.3- and 11-fold, respectively. The preservation of the iron alloy plasticity in the corrosive environment containing the inhibitor under study was determined by a decrease in the hydrogen content in the alloy bulk.

The TY3 Gag3 spacer controls intracellular condensation and uncoating.

Cells expressing the yeast retrotransposon Ty3 form concentrated foci of Ty3 proteins and RNA within which virus-like particle (VLP) assembly occurs. Gag3, the major structural protein of the Ty3 retrotransposon, is composed of capsid (CA), spacer (SP), and nucleocapsid (NC) domains analogous to retroviral domains. Unlike the known SP domains of retroviruses, Ty3 SP is highly acidic. The current studies investigated the role of this domain. Although deletion of Ty3 SP dramatically reduced retrotransposition, significant Gag3 processing and cDNA synthesis occurred. Mutations that interfered with cleavage at the SP-NC junction disrupted CA-SP processing, cDNA synthesis, and electron-dense core formation. Mutations that interfered with cleavage of CA-SP allowed cleavage of the SP-NC junction, production of electron-dense cores, and cDNA synthesis but blocked retrotransposition. A mutant in which acidic residues of SP were replaced with alanine failed to form both Gag3 foci and VLPs. We propose a speculative "spring" model for Gag3 during assembly. In the first phase during concentration of Gag3 into foci, intramolecular interactions between negatively charged SP and positively charged NC domains of Gag3 limit multimerization. In the second phase, the NC domain binds RNA, and the bound form is stabilized by intermolecular interactions with the SP domain. These interactions promote CA domain multimerization. In the third phase, a negatively charged SP domain destabilizes the remaining CA-SP shell for cDNA release.

Structural studies of the giant mimivirus.

Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral double-stranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 60 degrees . The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 Angstroms.

Glioma cells display complex cell surface topographies that resist the actions of cytolytic effector lymphocytes.

Gliomas are invasive cancers that resist all forms of attempted therapy. Immunotherapy using Ag-pulsed dendritic cells has improved survival in some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes toward gliomas. Atomic force microscopy of four different glioma types-human U251 and rat T9 and F98 glioma cells, including freshly isolated human glioblastoma multiforme neurosphere cultures (containing "stem cell-like cells")-revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional CTLs, and chimeric Ag-receptor-redirected T cells) better than their nonmicrovilli-expressing counterparts. Killer lymphocytes released perforin, which was detected within the glioma's microvilli/filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Air-dried gliomas revealed nodes within the microvilli/filopodia. The microvilli that penetrated 0.4-µm transwell chamber's pores resisted the actions of CTLs and physical damage. Those nodelike structures may represent a compartmentalization that resists physical damage. These microvilli may play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.

Effect of Quaternary Ammonium Salts and 1,2,4-Triazole Derivatives on Hydrogen Absorption by Mild Steel in Hydrochloric Acid Solution.

The treatment of low-carbon steel items with hydrochloric acid solutions is used in many industrial technologies. This process is accompanied not only by metal corrosion losses, but also by hydrogen absorption by the metal. In this study, the kinetics of hydrogen cathodic reduction on low-carbon steel in 2 M HCl containing corrosion inhibitors, namely, quaternary ammonium salts and a 3-substituted 1,2,4-triazole, have been studied. Adsorption isotherms of corrosion inhibitors on cathodically polarized steel surface have been obtained. XPS data provide valuable information on the composition and structure of protective layers formed on steel in HCl solutions containing inhibitors. The main rate constants of the stages of gaseous hydrogen evolution and incorporation of hydrogen atoms into the metal have been determined. The addition of quaternary ammonium salts or 3-substituted 1,2,4-triazole inhibits the cathodic reduction of hydrogen and its penetration into steel in the HCl solution. 3-substituted 1,2,4-triazole is the most efficient inhibitor of hydrogen absorption. The inhibitory effect of this compound is caused by a decrease in the ratio of the hydrogen concentration in the metal phase to the degree of surface coverage with hydrogen. The maximum decrease in hydrogen concentration in the metal bulk in the presence of the 3-substituted 1,2,4-triazole is 8.2-fold, which determines the preservation of the plastic properties of steel as it corrodes in HCl solutions. The high efficiency of the 3-substituted 1,2,4-triazole as an inhibitor of hydrogen cathodic reduction and absorption results from strong (chemical) adsorption of this compound on the steel surface and the formation of a polymolecular protective layer.

Superhydrophobic Anticorrosive Phosphonate-Siloxane Films Formed on Zinc with Different Surface Morphology.

The composition, structure, and protective and hydrophobic properties of nanoscale films formed layer-by-layer in solutions of sodium dodecylphosphonate (SDDP) and vinyltrimethoxysilane or n-octyltriethoxysilane (OTES) on the zinc surface with different morphologies were studied by SEM, XPS, water contact angle measurements, and electrochemical and corrosion tests. The protective, hydrophobic properties of phosphonate-siloxane films on zinc and their stability in a corrosive media are determined both by the initial surface morphology and composition of the surface oxide layer, and by the nature of inhibitors. It was shown that preliminary laser texturing of the zinc surface is preferable than chemical etching to enhance the anticorrosive properties of the resulting thin films. The most stable films with excellent superhydrophobic and protective properties in atmospheres of high humidity and salt spray are formed on the zinc surface with fractal morphology during layer-by-layer passivation with SDDP and OTES.

Mutual Effect of Components of Protective Films Applied on Copper and Brass from Octadecylamine and 1,2,3-Benzotriazole Vapors.

It has been shown by a set of corrosion, electrochemical and physical methods that a chamber corrosion inhibitor that consists of a mixture of octadecylamine (ODA) and benzotriazole (BTA) efficiently protects copper and brass from atmospheric corrosion and can be used for the temporary protection of metal items. The optimum temperatures of treatment with the ODA + BTA mixed inhibitor is 120 °C for brass and 100 °C for copper. One-hour treatment in ODA + BTA vapors at these temperatures results in the formation of nanosized adsorption films on the surface of these metals. These films stabilize the passive state and provide efficient temporary protection of metal items. The ODA + BTA inhibitor is superior to its components in terms of protective aftereffect. Our analysis of the mutual effect of BTA and ODA indicated that they show an antagonism of protective action on copper, but there is also a synergistic enhancement in the case of brass. Electrochemical impedance spectroscopy studies demonstrate that the inhibitors in question mainly act by using a blocking mechanism on copper and brass. Chamber treatment of the metals studied in vapors of the ODA + BTA mixture resulted in a noticeable hydrophobization of the copper surface and an insignificant effect on the brass surface. Chamber treatment of copper samples with artificially created polymodal roughness made it possible to obtain a superhydrophobic surface.

Fibrillar oligomers nucleate the oligomerization of monomeric amyloid beta but do not seed fibril formation.

Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid beta (Abeta) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 x g, rich in beta-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Abeta FOs do not seed the formation of thioflavin T-positive fibrils from Abeta monomers but instead seed the formation of FOs from Abeta monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril.

Mutual Effects of Components of Protective Films Applied on Steel in Octadecylamine and 1,2,3-Benzotriazole Vapors.

In this work, we used a combination of corrosion, electrochemical, and physical methods to determine the properties of nanoscale films obtained by treatment with octadecylamine (ODA), benzotriazole (BTA) vapors, and their mixtures at elevated temperatures. The mixture of ODA + BTA surpasses its components in protective aftereffect, but an analysis of their mutual effects shows that there is antagonism between them. Electrochemical impedance spectroscopy data indicate that the protection of steel by a mixture of ODA + BTA and its components is characterized by a mixed blocking activation mechanism. The processing of steel in hot vapors of the ODA + BTA mixture leads to hydrophobization of the surface and super-hydrophobization if a polymodal surface is created on the steel before processing in vapors.

The three-dimensional structure of Mimivirus.

Mimivirus, the prototypic member of the new family of Mimiviridae, is the largest virus known to date. Progress has been made recently in determining the three-dimensional structure of the 0.75-microm diameter virion using cryo-electron microscopy and atomic force microscopy. These showed that the virus is composed of an outer layer of dense fibers surrounding an icosahedrally shaped capsid and an internal membrane sac enveloping the genomic material of the virus. Additionally, a unique starfish-like structure at one of the fivefold vertices, required by the virus for infecting its host, has been defined in more detail.

Atomic force microscopy analysis of icosahedral virus RNA.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.

Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity.

Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses.

Mechanisms, kinetics, impurities and defects: consequences in macromolecular crystallization.

The nucleation and growth of protein, nucleic acid and virus crystals from solution are functions of underlying kinetic and thermodynamic parameters that govern the process, and these are all supersaturation-dependent. While the mechanisms of macromolecular crystal growth are essentially the same as for conventional crystals, the underlying parameters are vastly different, in some cases orders of magnitude lower, and this produces very different crystallization processes. Numerous physical features of macromolecular crystals are of serious interest to X-ray diffractionists; the resolution limit and mosaicity, for example, reflect the degree of molecular and lattice order. The defect structure of crystals has an impact on their response to flash-cooling, and terminal crystal size is dependent on impurity absorption and incorporation. The variety and extent of these issues are further unique to crystals of biological macromolecules. All of these features are amenable to study using atomic force microscopy, which provides direct images at the nanoscale level. Some of those images are presented here.

An atomic force microscopy investigation of cyanophage structure.

Marine viruses have only relatively recently come to the attention of molecular biologists, and the extraordinary diversity of potential host organisms suggests a new wealth of genetic and structural forms. A promising technology for characterizing and describing the viruses structurally is atomic force microscopy (AFM). We provide examples here of some of the different architectures and novel structural features that emerge from even a very limited investigation, one focused on cyanophages, viruses that infect cyanobacteria (blue-green algae). These were isolated by phage selection of viruses collected from California coastal waters. We present AFM images of tailed, spherical, filamentous, rod shaped viruses, and others of eccentric form. Among the tailed phages numerous myoviruses were observed, some having long tail fibers, some other none, and some having no visible baseplate. Syphoviruses and a podovirus were also seen. We also describe a unique structural features found on some tailed marine phages that appear to have no terrestrial homolog. These are long, 450 nm, complex helical tail fibers terminating in a unique pattern of 3+1 globular units made up of about 20 small proteins.

Atomic force microscopy in imaging of viruses and virus-infected cells.

Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number (T). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.

Atomic force microscopy investigation of viruses.

Atomic force microscopy (AFM) has proven to be a valuable approach to delineate the architectures and detailed structural features of a wide variety of viruses. These have ranged from small plant satellite viruses of only 17 nm to the giant mimivirus of 750 nm diameter, and they have included diverse morphologies such as those represented by HIV, icosahedral particles, vaccinia, and bacteriophages. Because it is a surface technique, it provides images and information that are distinct from those obtained by electron microscopy, and in some cases, at even higher resolution. By enzymatic and chemical dissection of virions, internal structures can be revealed, as well as DNA and RNA. The method is relatively rapid and can be carried out on both fixed and unfixed samples in either air or fluids, including culture media. It is nondestructive and even non-perturbing. It can be applied to individual isolated virus, as well as to infected cells. AFM is still in its early development and holds great promise for further investigation of biological systems at the nanometer scale.

Nano-fibers produced by viral infection of amoeba visualized by atomic force microscopy.

In the course of an atomic force microscopy investigation of mimivirus, we observed that disrupted virions released masses of fibers that were several hundreds of nanometers in length and that could not be explained as nucleic acid or polysaccharide. The fibers exhibited a strong 7 nm periodicity along their lengths. They existed singly, and also as ribbons, cables, and in multi stranded coils. In the aggregate structures, the periodic bands of the individual fibers aligned laterally to produce ribbons and other superstructures having a corresponding pattern of 7 nm periodic transverse bands. We have not observed such fibers in studies of other virus and cellular systems. The fibers are mechanically flexible and resistant to breakage. Occasionally fibers were associated with toroidal protein complexes, assumed to be processive enzyme complexes, apparently in the act of modifying the fibers.

TY3 GAG3 protein forms ordered particles in Escherichia coli.

The yeast retrovirus-like element Ty3 GAG3 gene encodes a Gag3 polyprotein analogous to retroviral Gag. Gag3 lacks matrix, but contains capsid, spacer, and nucleocapsid domains. Expression of a Ty3 Gag3 or capsid domain optimized for expression in Escherichia coli was sufficient for Ty3 particle assembly. Virus-like ordered particles assembled from Gag3 were similar in size to immature particles from yeast and contained nucleic acid. However, particles assembled from the CA domain were variable in size and displayed much less organization than native particles. These results indicate that assembly can be driven through interactions among capsid subunits in the particle, but that the nucleocapsid domain, likely in association with RNA, confers order upon this process.

Mutation in the glycosylated gag protein of murine leukemia virus results in reduced in vivo infectivity and a novel defect in viral budding or release.

All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80gag) in addition to the polyprotein precursor of the viral capsid proteins (Pr65gag). gPr80gag is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65gag. The role of glyco-Gag in MuLV replication has been unclear, since gPr80gag-negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80gag mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80gag negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80gag in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80gag produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80gag is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.