[Changes in cell membrane fluidity evoked by removal of influenza virus receptors with neuraminidase]. / Zmeny vo fluidite bunkovej membrány vyvolané odstránením receptora vírusu chrípky neuraminidázou.

The effect of the removal of the influenza virus receptor (N-acetylneuraminic acid) from the erythrocyte membrane by neuraminidase (RDE = receptor destroying enzyme) on the biophysical properties of the cell membrane has been examined using a fluorescent polarization technique. Diphenylhexatriene (DPH) was used as a fluorescent probe for structural ordering in the core of the membrane. A statistically significant decrease in the steady state values of DPH anisotropy was observed in membranes treated with RDE as compared to control. The anisotropy decrease reflected a reduced ordering of the hydrocarbon region of the erythrocyte membrane as well as an increase of its fluidity. An increase of membrane fluidity evoked by the action of influenza virus neuraminidase may play a significant role in the process of influenza virus penetration into the susceptible cell by endocytosis.

Infection of the central nervous system of mice by standard Sendai virus, defective interfering Sendai virus and the mixture of both: comparison of virus multiplication and pathogenicity.

The intracerebral (i.c.) infection of newborn mice with standard Sendai virus (SV), defective interfering Sendai virus (DV) and their mixture (SV + DV) has been used as a model for the possible role of defective interfering particles of paramyxoviruses in several chronic degenerative diseases of central nervous system (CNS). The dynamics of Sendai virus multiplication and virus distribution in CNS of mice, as well as the histological changes and the clinical symptoms were evaluated for up to 112 days post-infection (p.i.). The infectious virus was detected in the brains of animals inoculated i.c. either with SV, or DV, or SV + DV as soon as by 5 hr p.i., with maximum infectivity titre at 24 hr p.i. In brains of animals inoculated with SV, the virus was detected until 5th day p.i.; nevertheless in those, inoculated with SV + DV or DV, low infectious titres could be detected even at later intervals. In mice inoculated i.c. with DV, traces of Sendai virus were detected in subpassages, as late as 3 months p.i.

Voltammetry of tobacco mosaic virus and its isolated protein at the graphite electrode.

The electrochemical behaviour of tobacco mosaic virus (TMV) and its isolated protein was studied using differential pulse (DP) voltammetry at a graphite electrode and by direct current (DC) polarography in Brdicka solution. TMV and its isolated protein were found to be electrooxidized at the graphite electrode in the adsorbed state. Both species yielded two oxidation peaks on DP voltammograms. The first, more negative peak, corresponded to electrooxidation of tyrosine residues, whereas the other, more positive, peak corresponded to electrooxidation of tryptophan residues. DC polarography was used to detect degradation of TMV and denaturation of TMV-protein induced by an increased pH and by the addition of urea, respectively. These structural transformations resulted in increased DP voltammetric oxidation currents as recorded using a graphite working electrode. It has been suggested that the higher oxidation currents were due to an increase in the number of tyrosine and tryptophan residues accessible to the reaction at the graphite electrode. The results of these electrochemical investigations were in a good agreement with the estimation of the accessibility of tyrosine and tryptophan residues based on the well-explored three-dimensional structure of TMV and its isolated protein.

Differential pulse-polarographic analysis of tobacco mosaic virus.

The tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483 (with glutamine-9 replaced by histidine) and the denatured protein of TMV vulgare were analysed by direct current (d. c.) and differential (derivative) pulse polarography (DPP) in the basic electrolyte composed of 0.001 M Co(NH3)6Cl3, 0.1 M NH4Cl and 0.1 M NH3 at 0 degrees C. The DPP method gave a substantially better resolution of the polarographic catalytic maxima A and B, but a much lower resolution of the maxima B and C, as compared with the d. c. polarographic method. The clear differentiation of the maximum A from maximum B by DPP permitted to study the variation of maximum A in the course of alkaline degradation of TMV. But for the study of TMV protein denaturation the d. c. polarography is preferable, because the denaturation is accompanied by the appearance and rise of maximum C which can be clearly differentiated from maximum B by d. c. polarography rather than by DPP. The DPP method was more, sensitive than d. c. polarography. The denatured TMV protein can be determined by DPP at concentrations around 0.1 microgram/ml.

Marked difference in electrophoretic migration rates between two influenza A viruses.

Comparative moving boundary electrophoresis revealed that influenza virus A/PR/8/34 (H0N1) has a 2.5 times higher electrophoretic migration rate at pH 7 than influenza virus A/Singpore/1/57 (H2N2). This difference was the same whether the compared viruses were purified first by either ammonium sulphate precipitation or adsorption onto and elution from red blood cells and then by density gradient centrifugation. The same electrophoretic methods was used for testing the homogeneity of influenza virus preparations purified by either method.

Effect of amino acid replacement on the stability of the tobacco mosaic virus protein structure.

A comparative polarographic study on the alkaline degradation of tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483, having histidine instead of glutamine at position 9 in the polypeptide chain, was performed. In the course of alkaline degradation and subsequent incubation in the supporting electrolyte at 0 degrees C TMV 483, unlike TMV vulgare, showed a polarographic effect indicating the unfolding of the TMV polypeptide. It was concluded that the replacement of glutamine-9 by histidine causes a decrease in the stability of the three-dimensional structure of the TMV protein subunit. A polarographic study of untreated virions as well as denatured proteins of both TMV strains showed that histidine, when incorporated into the polypeptide chain, is not active polarographically at the conditions used.

Electrophoretic separation and characterization of subunits released from influenza virus by detergents.

Subunits released from influenza A/Singapore/1/57 (H2N2) virus by either Triton-X-100 (T-X-100); or sodium lauryl sarcosinate (SLS) or ether were separated by electrophoresis in agarose suspension into a rapidly migrating fraction (I) and a slowly migrating fraction (II). Fraction I obtained after T-X-100 treatment contained the viral ribonucleoprotein (RNP) in a form indistinguishable from the obtained after ether treatment. SLS treatment of the virus resulted in a rapidly migrating fraction containing only the protein part of the viral RNP. Fraction II obtained after T-X-100 or SLS treatment contained both haemagglutinin (HA) and neuraminidase (NA), mostly dissociated from each other, in contrast to fraction II obtained after ether treatment which contained mixed aggregates of HA and NA. The yields of electrophoretically isolated RNP and HA-NA were essentially the same irrespective of whether T-X-100 or ether was used for virus disruption. Treatment of virus by T-X-100 and subsequent removal of the latter resulted in a 10-20-fold increase of the HA activity. After sodium dodecyl sulphate (SDS) treatment of the virus, the NA activity was found in a heterogeneous fraction with surprisingly high migration rate towards the anode, indicating that NA remained active despite its extensive SDS binding.