ABSTRACT
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.
Subject(s)
Influenza A Virus, H3N2 Subtype/chemistry , Viral Matrix Proteins/chemistry , Virion/chemistry , Bromelains/metabolism , Crystallography, X-Ray , Hemagglutinins, Viral/chemistry , Isotope Labeling , Kinetics , Models, Molecular , Surface Plasmon Resonance , Tritium , Virion/metabolismABSTRACT
Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.
Subject(s)
Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H3N2 Subtype/chemistry , Viral Matrix Proteins/chemistry , Virion/chemistry , Amino Acid Sequence , Bromelains/metabolism , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Hemagglutinin Glycoproteins, Influenza Virus , Hydrolysis , Microscopy, Electron, Transmission , Molecular Sequence Data , Neuraminidase , Protein Conformation , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Virion/metabolismABSTRACT
Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-microM compound concentration against influenza strain A/Udorn/72 (H3N2). The "hit" rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-microM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI(50) = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI(50) > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Influenza A Virus, H3N2 Subtype/chemistry , Animals , Automation , Cell Line , Chemistry, Pharmaceutical/methods , Dogs , Drug Design , Inhibitory Concentration 50 , Models, Chemical , Ribavirin/pharmacology , Virus Replication/drug effectsABSTRACT
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.