The amino acid sequences of coat proteins (CPs) of potexviruses such as potato virus X (PVX) and alternanthera mosaic virus (AltMV) share about 40% sequence identity. However, the N-terminal CP domains of these virions differ both in length (the N-terminal CP domain of PVX is longer by 28 residues, ∆N = 28), and in amino acid sequence. In this work, we determined the effect of the N-terminal CP domain on the structure and physicochemical properties of the entire PVX and AltMV virions. It was shown that the melting point of PVX samples is 10-12°C higher than that of AltMV preparations; the circular dichroism spectra of these viruses also differ significantly. Spatial alignment of the existing high-resolution potexvirus CP structures showed that the RMSD value between Cα-atoms was the largest for the N-terminal domains of the two compared models. From computer simulations the ∆N-terminal CP domain of PVX is completely disordered. According to synchrotron small-angle X-ray scattering (SAXS) data, the structure of CP of PVX and AltMV virions differs, in particular, CP PVX has a larger size of crystallinity regions and, therefore, is more ordered. Using SAXS, virion diameters and helix parameters in solution are calculated. The influence of the conformation and localization of the N-terminal domain of PVX CP relative to the surface of the virion on its structure was revealed. Presumably, the increased thermal stability of PVX virions compared to AltMV is provided by the elongated N-terminal domains (ΔN = 28), which ensures additional contact between the adjacent CP subunits in the PVX virion.

Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.IMPORTANCE The matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to the Avulavirus subfamily of the Paramyxoviridae family of Mononegavirales that include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here, we investigate the solution structure and membrane binding properties of this protein at both acidic and neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals for the possible use of the virus as an adequate template for future drug or vaccine delivery.

The structure of tobacco mosaic virus (TMV) virions and stacked disk aggregates of TMV coat protein (CP) in solution was analyzed by synchrotron-based small-angle X-ray scattering (SAXS) and negative contrast transmission electron microscopy (TEM). TMV CP aggregates had a unique stability but did not have helical symmetry. According to the TEM data, they were stacked disks associated into transversely striated rod-shaped structures 300 to 800 Å long. According to modeling based on the crystallographic model of the 4-layer TMV CP aggregate (PDB: 1EI7), the stacked disks represented hollow cylinders. The calculated SAXS pattern for the disks was compared to the experimental one over the entire measured range. The best correlation with the SAXS data was found for the model with the repeating central pair of discs; the SAXS curves for the stacked disks were virtually identical irrespectively of the protein isolation method. The positions of maxima on the scatter curves could be used as characteristic features of the studied samples; some of the peaks were assigned to the existing elements of the quaternary structure (periodicity of aggregate structure, virion helix pitch). Low-resolution structural data for the repolymerized TMV CP aggregates in solution under conditions similar to natural were produced for the first time. Analysis of such nano-size objects is essential for their application in biomedicine and biotechnology.

Small-angle X-ray scattering is an important structural tool for studying biological membranes; however, interpretation of scattering data remains a challenging problem. In most cases, analysis makes it possible to determine some structural parameters and the electron density profile of lipid bilayers, but no methods providing more detailed information (e.g., about the structural organization of vesicles) have been proposed yet. An approach making it possible to determine the main integral characteristics of liposomes using small-angle scattering is presented in this study. Within this approach a quasi-atomic model of liposome is built from individual lipid molecules, which form a sphere or a hollow ellipsoid. The method has been implemented in a computer program, verified on experimental small-angle X-ray scattering data, and proposed to analyze the structure of lipid vesicles and their interactions with proteins.

The efficiency of tritium labeling of salts of pantothenic (HO–R–COOH), hopantenic (HO–R–CH2–COOH), and 4-D-phosphopantothenic [(HO)2P(=O)–O–R–COOH] acids [R = CH2C(CH3)2CHOH–CONH(CH2)2] with thermal activation of tritium at target temperatures of 77 and 295 K was studied. The phosphate group inhibits the isotope exchange. The tritium-labeled compounds were used for studying the adsorption of pantothenic acid derivatives from aqueous solutions and in the presence of 0.9% NaCl at 297 ± 3 K onto nanodiamonds prepared by detonation synthesis (NDs). Preparation of stable ND suspensions in advance enhances the ability of NDs to adsorb the compounds studied. The parameters of the equation describing the sorption isotherms at different ionic strengths of the solution were calculated. The strength of the adsorbate retention in contact with water, 0.9% NaCl solution, 0.01 M HCl solution, and 40 g L−1 bovine serum albumin (BSA) solution was determined. The data obtained allow two mechanisms of the adsorbate retention on the ND surface to be considered: reversible adsorption due to ionic interactions and irreversible binding due to hydrophobic interactions. The strongly bound molecules undergo slow desorption in the presence of BSA. The revealed trends confirm high potential of NDs as a drug delivery platform.

This paper reports on a complex structural analysis of the potato virus A coat protein using a set of complementary physico-chemical methods. We have demonstrated previously that this protein does not exist as individual subunits in solution and undergoes association into oligomers with subsequent transition to β‑conformation. The purpose of the present work was to study the possible mechanisms of this transformation and to search for methods that dissociate protein oligomers. To analyze the low resolution protein structure in solution, small-angle X-ray scattering was used. Stable particles representing clusters of 30 coat protein subunits were present even in an aqueous salt solution with a high ionic strength and pH (pH 10.5; 0.5 M NaCl). The particles did not dissociate in the presence of 10 mM dextran sulfates (15 and 100 kDa). Dissociation in the presence of 5.2 mM sodium dodecyl sulfate results in the formation of the subunit–detergent complexes consisting of 10–12 small particles joined together like “beads on a string”. Similar effects of sodium dodecyl sulfate were shown for serum albumins (bovine and human). Denaturation of the potato virus A coat protein molecules occurs in the presence of detergent concentrations that are seven times lower than that in albumins (5.2 and 35 mM), which confirms low stability of the potato virus A coat protein. Using spectral methods, preservation of the secondary structure and loss of the tertiary structure of the protein in its complex with sodium dodecyl sulfate have been demonstrated. Possible mechanism for protein particle formation through the interaction between unordered terminal domains and their transformation into β‑structures has been suggested.

This paper reports on a complex structural analysis of the potato virus A coat protein using a set of complementary physico-chemical methods. We have demonstrated previously that this protein does not exist as individual subunits in solution and undergoes association into oligomers with subsequent transition to β-conformation. The purpose of the present work was to study the possible mechanisms of this transformation and to search for methods that dissociate protein oligomers. To analyze the low resolution protein structure in solution, small-angle X-ray scattering was used. Stable particles representing clusters of 30 coat protein subunits were present even in an aqueous salt solution with a high ionic strength and pH (pH 10.5; 0.5 M NaCl). The particles did not dissociate in the presence of 10 mM dextran sulfates (15 and 100 kDa). Dissociation in the presence of 5.2 mM sodium dodecyl sulfate results in the formation of the subunit-detergent complexes consisting of 10-12 small particles joined together like "beads on a string". Similar effects of sodium dodecyl sulfate were shown for serum albumins (bovine and human). Denaturation of the potato virus A coat protein molecules occurs in the presence of detergent concentrations that are seven times lower than that in albumins (5.2 and 35 mM), which confirms low stability of the potato virus A coat protein. Using spectral methods, preservation of the secondary structure and loss of the tertiary structure of the protein in its complex with sodium dodecyl sulfate have been demonstrated. Possible mechanism for protein particle formation through the interaction between unordered terminal domains and their transformation into β-structures has been suggested.

A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological рН value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development.

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.

ABSTRACT Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ∼6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ∼5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. IMPORTANCE Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coat of protein inside the viral surface reacts to the changes in acid that come soon after internalization. We found that acid makes the molecules of the protein coat push each other while they are still stuck to the virus, so that they would like to rip the membrane apart. This ripping force is known to promote membrane fusion, the process by which infection actually occurs.

The kinetics of interaction of influenza A virus M1 matrix protein with hydrophilic polymer-supported lipid bilayers formed by 1,2-diphytanoyl-sn-glycero-3-phosphocholine and 1,2-diphytanoyl-sn-glycerophospho-l-serine at pH 7.0 was studied by surface plasmon resonance spectroscopy. The M1 protein was shown to bind to the lipid bilayer almost irreversibly to form a monolayer; this is in line with the key function of the M1 protein, that is, formation of the protein envelope of the virion. An increase in the percentage of negatively charged lipids from 0 to 30% leads to a considerable increase in the protein monolayer density and a several-fold increase in the binding constant (K a) of the M1 protein with the lipid bilayer up to (3.60±0.11)·108 L mol–1. The negatively charged lipids in the bilayer appear to promote the manifestation of anisotropic properties of the M1 protein, which enable the protein to form reversible non-monolayer structures on the surface. The M1—M1 interaction was found to be reversible and to be characterized by the binding constant K a = (6.3±0.1)·107 L mol–1.