The in situ structural characterization of the influenza A virus matrix M1 protein within a virion.

The first attempt has been made to suggest a model of influenza A virus matrix M1 protein spatial structure and molecule orientation within a virion on the basis of tritium planigraphy data and theoretical prediction results. Limited in situ proteolysis of the intact virions with bromelain and surface plasmon resonance spectroscopy study of the M1 protein interaction with lipid coated surfaces were used for independent confirmation of the proposed model.

One more probable structural transition in potato virus X virions and a revised model of the virus coat protein structure.

We found that a 2-h incubation of potato virus X (PVX) virions in 10 mM Tris-HCl buffer pH 7.5 at -20 degrees C results in a strong but reversible drop in virion stability. Under these conditions, the PVX virions are completely disrupted by low (starting from 50 mM) concentrations of LiCl and CaCl(2) but not of NaCl. Incubation of PVX samples with 0.05-2 M LiCl at +4 degrees C did not result in virion disassembly and the virions were not disrupted upon incubation at -20 degrees C in 10 mM Tris-HCl buffer pH 7.5 without LiCl. We suggest that a 2-h incubation of the PVX virions at -20 degrees C in 10 mM Tris-HCl pH 7.5 results in a structural transition in the virus particles. A revised model of the three-dimensional organization of coat protein subunits in the PVX virions is proposed. This two-domain model explains better the high plasticity of the PVX CP structure.

Atomic tritium as an instrument for study of protein behavior at the air-water interface.

Atomic tritium was successfully applied as an instrument for study of protein behavior at the air-water interface. Samples of lysozyme solution in 20 mM phosphate buffer (pH 7.0) with concentration of 2 mg/ml incubated at the room temperature for 1 h were exposed to bombardment with tritium atoms generated on hot tungsten wire in special vacuum device. This procedure resulted in substitution of hydrogen atoms by radioactive tritium in the thin surface layer of studied preparations. Analysis of experimental data on intramolecular radioactivity distribution in lysozyme and computer simulation of tritium bombardment allowed us to suggest two equally probable opposite orientations of lysozyme molecule in the adsorption layer at the air-water interface.

Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles.

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm(-1) region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm(-1)), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.