[Flu virion as a substrate for proteolytic enzymes].

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the basic protein components of the virion environment: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently deleted HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA2 185-211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a basic site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM beta-mercaptoethanol than in the presence of 50 mM. However, the complete removal of exodomains of HA, HA, and low-activity enzyme by the HA high- and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein Ml1. The action of trans-epoxysuccinyl-L-leucylamido)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl2 on the hydrolysis of proteins HA and M1 by bromelain was investigated.

[An approach the quantitative determination of the area of glycoprotein spikes at the surface of enveloped viruses].

The density of distribution of glycoproteins on virion surface seriously influences the virus infectivity and pathogenicity. In the present work a method of quantitative determination of the area occupied by the surface glycoprotein spikes is proposed for influenza virus (strain A/PR/8/34) based on data of tritium bombardment and dynamic light scattering (DLS). The method of DLS was used for measuring the diameter of the intact virions and the subviral particles (influenza virions lacking glycoprotein spikes after bromelain digestion). The intact virions and the subviral particles were bombarded by the hot tritium atom flux followed by the analysis of the specific radioactivity of the matrix M1 protein. It was shown that the tritium label was incorporated into the amino acid residues of a thin exposed protein layer and partially penetrated through the lipid bilayer of the viral envelope. As a result, the matrix M1 protein which is located under the lipid bilayer became labeled. The tritium label distribution among different amino acid residues was the same for the M1 protein isolated from the subviral particles and the one isolated from the intact virions. This testifies that the M1 protein spatial structure remains unchanged during proteolysis of the glycoprotein spikes. The difference between the specific radioactivity of the M1 protein isolated from the intact virions and that of the M1 protein isolated from the subviral particles allowed us to calculate the portion of the viral surface which is free of the glycoprotein spikes. If approximate the influenza virion as as here the area occupied by the surface glycoproteins could be calculated. It appeared to be equal to approximately 1.4 yen 10 nm that is about 40% of the total viral surface. This is consistent with the cryoelectron tomography data published for the influenza virus (strain A/X-31). The developed approach could be applied for other enveloped high pathogenic viruses such as HIV and Ebola.

[Internal epidemic influenza virus proteins: isolation and investigation].

The internal influenza virus proteins M1 and RNP free from surface protein impurities were isolated from subviral particles (virions free from HA and NA ectomenes). The spikeless particles had no propensity to aggregate in the solution at pH 5.0 as compared with native viruses. The subviral particles of B/Hong Kong/330/01 influenza virus, which belonged to B/Victoria/2/87-lineage, were obtained by proteolytic treatment with the enzyme bromelain under the same conditions as in cases of influenza B viruses of B/Jamagata/16/88 lineage. A chromatographic analysis of the tryptic hydrolyzates obtained for matrix (M1) proteins of A(H1N1) and A(H3N2) influenza viruses revealed differences that were greatest between the protein M1 molecules isolated from influenza viruses of different subtypes of hemagglutinine. These findings suggest there are variations in the structure of this conservative internal viral protein M1 during evolution.

[The use of bromelain in obtaining the subviral particles of influenza A and B viruses]. / Ispol'zovanie bromelaina dlia polucheniia subvirusnykh chastits virusov grippa A i B.

Subviral particles of modern strains of influenza A viruses, i.e. A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2), reassortant X-31 (subtype H3N2) and B/Sichuan/379/99, were obtained by using two preparations of bromeline ("Sigma Co., Catalogues' Nos. B2252 and B5144). A selective ability of bromeline B5144 was detected to the proteolytic splitting of hemagglutinin of influenza A and B viruses. An influence of enzyme B5144 produced on influenza B viruses brought about an appearance of subviral particles. As for influenza A(H1N1) virus, the above enzyme did not have any impact on it under the similar experimental conditions. An incomplete effect was noted for the influenza A(H3N2) virus with particles (with intact external coatings) being found in the reaction mixture. Enzyme B2252 was found to be effective in respect to all viruses selected for testing, however, the highest effect was noted for influenza A(H1N1) and B viruses.