Toyozo Maeda

Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes

Although myxoviruses share many ~~~~~~erni~~~ and morphological features with ~a~myxo~ruses, their infection mechanism is not completely elucidated It is established that paramywviruses penetrate the plasma membrane via envelope fusion [ 11, eventually i~d~~~g lysis and cell fusion, These ~~ti~ties have not been reported for influenza v&s, In t2] rnor~~oIo~i~a1 evidence appeared for e-nvelope fusion of influenza virus with &e cell rne~~~~~~e, but in [3] an ~~f~~ti~~ me~han~m via Tiropexis was suggested. fn [4,5 ] infectivity #f influensla virus harvested from tissue culture cells was greatly e~I~~~~ed when i~~~e~~a virus HA protein was split into two fragments by trypsin. In [6] the amino~termlnal region formed by proteolysis was found to be similar to the hydrophobic sequence of the F protein of hemagglue ti~~t~~

Membrane fusion. Transfer of phospholipid molecules between phospholipid bilayer membranes

virus of Japan (HVJ) (also known as Sendai arums), The F protein is involved in ,the envelope fu&n, hemolytic, and fusion actions of HVJ f7], These activities may be caused by direct i~te~a~tio~ of the. am~o-te~~al hydrophobic s~q~e~~~ with ~~d~~~~obi~ regions of the target cell ~emb~ne. The HA ~~~~ei~ of influenza virus may f~~t~o~ as the F protein of HVJ does in certain conditions, causing hemolysis and cell fusion. Here, we found so&at& virus inlcubated with cells in acidic medium of pH 5.2 caused high hemolytic and fusion activities, (I+3 &&i Co,, T-A-4201) with 2 setting of I mA. Bu%rs contained 085% (w/v) NaCl in ~~d~t~o~~ to 20 mM sodium acetate for pH 4.5-X8, IO n&l sodium phosph&te far pH 5.0-8.0 and 20 mM: gly tine-NaOH for pH 9.0-10.0. Protein concentration was determined by Lowry’s method [9] and hema~luti~~ti~~ assay was carried out using

Membrane receptor mobility changes by sendai virus

alk”s pattern method [la]. Hema~utination acti~ty~m~ virus protein was 1.3 X IO5 for AePR8,3.0 X 10’ for WSN and 6.3 X 103 for HYJ(z). Hemolysis was assr~ed ~e~~~~~~t~~~etri~~l~ at 540 nm_ The s~n~I~~e~ assay method for phosph~l~~d transfer was describes in [S].

Transfer of phosphatidylcholine between different membranes in Tetrahymena as studied by spin labeling.

Abstract Fusion of phosphatidylcholine (PC) vesicles and of PC-phosphatidylserine (PS) vesicles has been studied using spin-labeled PC and PS. Analysis of ESR spectra indicated transfer of phospholipid molecules between phospholipid vesicles at the instant of membrane contact by vesicular collision. The transfer rate of PC was not greatly affected by the presence of the anionic lipid in the membranes. The rate of PC transfer between PS-PC vesicles was nearly the same as that of PS transfer. Calcium ion greatly enhanced the transfer of phospholipid molecules between PS-PC vesicles. The enhancement of PS transfer occurred instantaneously. The phospholipid transfer is related to the fusion of vesicles.

Interaction between Sendai virus and KB cell lines with altered cell fusion ability: a study employing electron spin resonance.

Treatment with Sendai virus does affect the lateral mobility of cell surface components on cultured human cells (KB) and cultured mouse cells (3T3). Diffusion coefficients and mobile fractions of a fluorescent lipid analog, tetramethyl rhodamine-succinyl concanavalin A (ConA) and tetramethyl rhodamine-anti human β2 microglobulin antibodies on the cell surface were measured by fluorescence photobleaching recovery (FPR). Diffusion coefficients of receptors for ConA and for antibodies to β2 microglobulin (presumably histocompatibility antigens) were increased 2- to 3-fold by exposure to the ultraviolet inactivated virus at virus doses from 250 to 2 500 HAU/ml, regardless of whether the particular cells measured had been fused. No mobility enhancement was induced with trypsinized Sendai virus, nor by influenza virus, indicating a probable requirement for the Sendai virus F protein. Virus treatment reduced the diffusion coefficient of a lipid analog in 3T3 cells. The results are compatible with the hypothesis that the Sendai virus disrupts a mobility restraint system, perhaps cytoskeleton-membrane connections.

A kinetic study on conformational change of deoxyhemoglobin induced by a group of effectors

Abstract Transfer of phosphatidylcholine molecules between different membrane fractions of Tetrahymena pyriformis cells grown at 15, 27 and 39.5°C was studied by electron spin resonance (ESR). Microsomes were labeled densely with a phosphatidylcholine spin label and the spin-labeled microsomes were incubated with non-labeled cilia, pellicles or microsomes. The transfer of the phosphatidylcholine spin labels was measured by decrease in the exchange broadening of the electron spin resonance spectrum. In one experiment, the lipid transfer was measured between 32P-labeled microsomes and non-labeled pellicles by use of their radioactivity. The result was in good agreement with that by ESR. The fluidity of the membrane was estimated using a fatty-acid spin label incorporated into the membranes. Transfer between lipid vesicles was also studied. The results obtained were as follows: (1) The transfer between sonicated vesicles of egg- or dipalmitoyl phosphatidylcholine occurred rapidly in the liquid crystalline phase, with an activation energy of 20 kcal/mol, whereas it hardly occurred in the solid crystalline phase. (2) The transfer rate between microsomal membranes increased with temperature, and an activation energy of the reaction was 17.8 kcal/mol. (3) The transfer from the spin-labeled microsomes to subcellular membranes of the cells grown at 15°C was larger than that to the membranes of the cells grown at 39.5°C. The membrane fluidity was larger for the cells grown at lower temperature. (4) Similar tendency was observed for the transfer between microsomal lipid vesicles prepared from the cells grown at 15°C and at 39.5°C. (5) The transfer from microsomes to various membrane fractions increased in the order, cilia

Interaction between haemagglutinating virus of Japan and human En (a—) erythrocytes lacking major sialoglycoprotein

The nature of the interaction between Sendai virus and Sil mutant cells was examined by measuring a change in ESR spectrum of spin-labeled phosphatidylcholine molecules on the viral envelope. When spin-labeled virus was incubated with the Sil cells that had a reduced ability to respond to virus-induced cell fusion, interchange of the phospholipid molecules between viral envelope and cell surface membrane occurred to a smaller extent than that observed with parental cells. Moreover, the degree of the interchanging correlated with the degree of the fusion capacity of the mutant lines. The results show that the mutant cells carry such a lesion(s) on their surface membranes that the viral envelopes can hardly fuse into them.

Infection‐triggered release of tempocholine from bacteriophage T4 studied by electron spin resonance

Abstract The rates of reactions of two different reagents, BTB and DTNB, with hemoglobin were measured to examine the effects of organic phosphates and other compounds on hemoglobin. DPG, ATP and K4Fe(CN)6, which are known to influence the oxygen affinity, greatly affected the rates for Hb, but showed almost no effects on the rates for HbO2. By contrast, other compounds with no effects on the oxygen affinity did not change the rates of BTB with Hb and HbO2. The effectors are considered to induce conformational changes on binding to Hb and such conformational changes are responsible for the control of the oxygen affinity by the effectors.

Infectious Cell Entry Mechanism of Influenza Virus

Haemagglutinating virus of Japan (HVJ, synonym of Sendai virus) has been an useful tool to investigate the molecular mechanism of membrane fusion. The virus envelope is composed of two membrane proteins, HANA and F proteins [I]. The HANA protein binds sialic acid or oligosaccharides with sialic acid on erythrocyte surface, causes aggregation of cells at 0°C and also expresses neuraminidase activity at 37’C! [I]. The F protein is responsible for lipid stewing between the envelope and cell membrane and also between the fusing cell membranes [23 as well as haemolysis and fusion (31. It is important to identify the membrane components on erythrocyte surface with which the two envelope proteins interact and to know how the F protein causes chemical or structural modifications of cell membrane to effect these phenomena. In this regard the use of a genetic variant of erythrocyte with defmed alterations in membrane components provides a particularly interesting system. In this paper we studied the ~temction between HVJ and human erythrocytes of blood group En(a-) compared with normal erythrocytes En(a+). The rare &(a-) completely lacks the major sialoglycoprotein (glycophorin) and have an altered sugar composition of band 3 [4]. The major sialoglycoprotein represents -75% of total sialoproteins of normal erythrocytes and carries 30-50% of total cell surface carbohydrates [4-61. The results have shown that the virus binding, virusinduced lipid intermixing, haemolysis and fusion occurred with both En(a-) and normal En(a+) cells to similar extent.

A spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan.

The complex infection process of bacteriophage T4 involves a step of DNA ejaculation into the host bacterium,Escherichi coli [l] . DNA 50 I.cm in length packaged inside the head shell (1100 A length, 850 A diam.) passes through the cylindrical tail tube. Accompanying the DNA, internal proteins are also released into the bacterium [2] , We have found that a spin label tempocholine (N,N-dimethyl-N-(2’,2’,6’6’tetramethyl-4’-piperidyl)-2-hydroxyethylammor~ium chloride) can be incorporated in the heads of phage T4 and is rapidly released when phage are mixed with bacteria or bacterial envelopes. This infection-triggered ‘release of tempocholine was monitored by the electron spin resonance (ESR) method and its kinetics seem to parallel those of DNA ejaculation.