Yasuhiro Hosaka

Artificial assembly of envelope particles of HVJ (Sendai virus): I. Assembly of hemolytic and fusion factors from envelopes solubilized by Nonidet P40

Abstract Treatment of HVJ (Sendai virus) with Nonidet P40(NP40) solubilized some of viral lipids and envelope proteins. Subunit(s) of the solubilized envelopes had hemagglutinating (HA), HI blocking, and neuraminidase activities, and a sedimentation coefficient of about 9 S or less, and they contained two main polypeptide bands. After equilibrium centrifugation of the solubilized envelopes, the lipids were found at the top of the tube and the viral protein occurred in two visible bands, both with HA, HI blocking, and neuraminidase activities, but they were different in subunit morphology, in protein content and relative content of two immunological components. Interactions of the two defatted band fractions and the viral lipid fraction containing NP40, separated as described above, were investigated by mixing and dialyzing the mixtures to remove the detergent: (1) dialysis of the unfractionated solubilized envelopes resulted in assembly of membranous particles with spikes, which had hemolytic and fusion activities; (2) the interaction of the upper band and the lipid fraction resulted in assembly of similar particles as seen in (1); but (3) the interaction of the lower band and the lipid fraction resulted in assembly of membranous particles without spikes, which had neither hemolytic or fusion activities. The spikes of assembled particles were seen on both the inner and outer surfaces, unlike those of virion envelopes. The phospholipid composition of assembled particles was similar to that of the virions. Interactions of the bands of paramyxoviruses (HVJ, NDV) with lipids resulted in formation of active hemolysins, but similar mixtures made with influenza virus did not. These findings suggested that lipids act as an activator for formation of the active hemolysin. Nonviral lipids were also available for it.

Studies on the Pleomorphism of HVJ Virions.

Abstract Partially purified preparations of HVJ were fractionated by zone centrifugation in sucrose gradients, and several properties of the separated classes of virions were compared. The most slowly sedimenting band of HVJ consists of the smallest virions, which contain the shortest nucleocapsids, enclosed by the smallest envelopes. The lengths of the nucleocapsids distribute regularly. The minimum length associated with infectivity is 1.0 μ. This nucleocapsid is designated the mononucleocapsid: its multiples are called polynucleocapsids. The maximum length of polynucleocapsids was more than 20 μ. Usually the unfractionated viral population contains in the highest proportion the virions with mononucleocapsids. The separated classes of virions are all infectious. Their progenies are again heterogeneous. Experiments on inactivation of HVJ virions with polynucleocapsids by Co60 show that they contain multiply infectious units. The pleomorphism of HVJ virions can be explained in terms of heterogeneity in length and flexible conformation of nucleocapsids, enclosed by flexible envelopes.

Isolation and structure of the nucleocapsid of HVJ

Abstract A method for isolation of the nucleocapsids of HVJ from virions was developed: HVJ virions suspended in Na 2 CO 3 ue5f8NaHCO 3 buffer (0.02 M , 1:1) were disintegrated with Emasol 1130 (a Tween 20-like surfactant) and banded by CsCl equilibrium centrifugation. The nucleocapsids of HVJ formed a band at a buoyant density of 1.31, and most of the envelopes and partially disintegrated virions formed a top layer. The nucleocapsids thus obtained retain their full length and have a sedimentation constant S 20, w , of about 250S, but no detectable infectivity for eggs. The nucleocapsids contain about 3.7% RNA, but no detectable phospholipid or DNA. The nucleocapsids mainly consist of one antigenic component, named S, in gel diffusion. Treatment of the nucleocapsids with trypsin or RNase has no significant effect on its main morphology or on sedimentation or antigenicity of the S component. The nucleocapsid is a single helix, probably right-handed and with 15 subunits per turn. Some of the characteristics of the disintegration of HVJ virions by the present alkali-Emasol treatment are also described.

Electron microscopic study of cell fusion by HVJ virions.

Abstract The process of cell fusion of Ehrlich ascites tumor cells by a high concentration of HVJ virions in vitro was studied by electron microscopy to clarify the role of the virions in cell fusion. The study showed that the cell fusion proceeds along the following morphologically distinct steps: (1) agglutination of cells by virions in the cold; (2) close contact of adjacent cell membranes near adsorbed virions of agglutinated cells; (3) degradation of the cell membranes in contact with virions and communication of the cytoplasms of adjacent cells through the breaks; (4) connection of broken cell membranes between adjacent cells and formation of cytoplasmic bridges; (5) enlargement of the bridges and engulfing of virions within vesicles; (6) rounding of fused cells and alteration of the virions within vesicles, followed by the appearance of free nucleocapsids in cytoplasm. Steps 1 and 3 depend directly on the virions; the virions agglutinate cells in step 1 and have a degrading effect on cell membranes in step 3. These results imply that the site of cytoplasmic bridges is determined by adsorbed virions and that the phagocytosis of virions occurs later than formation of the bridges.

Artificial assembly of envelope particles of HVJ (Sendai virus): II. Lipid components for formation of the active hemolysin

Abstract The upper HA band fraction of HVJ was isolated by the method as described in the previous report ( Hosaka and Shimizu, 1972 ) and mixed with lipids in the presence of Nonidet P40 and dialyzed against PBS. In this system, lipid components permitting formation of the active hemolysins were investigated. When the four major phospholipid components of HVJ were used separately, phosphatidylethanolamine was the most effective, phosphatidylcholine and sphingomyelin were effective only at high concentrations, and phosphatidylserine was ineffective. Phosphatidylethanolamine from commercial sources was as effective as that from viral sources. In mixed viral phospholipids, something like cooperative effectiveness was found. A low concentration of cholesterol stimulated the formation of active hemolysins, but a high concentration inhibited it. Cholesterol together with phospholipids seemed to contribute to the rigidity of the membranes. Thus formed active hemolysins appeared large membranous particles of more than 600 A with spikes. No phospholipase activity was detected in the active hemolysins.

Artificial Assembly of Envelope Particles of HVJ (Sendai Virus). Fusion Activity of Envelope Particles

SummarynAn assay system for fusion activity of envelope particles of Sendai virus, reassembled from NP40-solubilized envelopes, was established and conditions for the artificial assembly of NP40-solubilized Sendai virus envelope particles with haemolytic and fusion activities were investigated. Large (GP1) and small (GP2) glycoproteins and lipids seemed to be required for the expression of haemolytic and fusion activities of envelope particles. Potential haemolysin activity was associated with GP2. A relatively high proportion of GP1 was required for formation of envelope particles with a high fusion activity.nWhen the top lipid fraction (Hosaka & Shimizu, 1972a) was used for reassembly, the envelope particles usually exhibited both fusion and haemolytic activities but the optimal concentrations of the lipid for the two activities were different. An unidentified factor extractable with NP40 seemed to be necessary for fusion activity but not for haemolytic activity.

Biological activities of sonically treated Sendai virus.

Summary Sendai virus induced fusion of Ehrlich ascites tumour cells but this capacity was decreased by sonic treatment and the product then interfered with virus-induced fusion. Sucrose density gradient fractionation of the sonic product showed that the capacity for fusion resided in intact particles and the interfering effect in fragments of the virus envelope. Such fractionation also showed that haemolytic activity was restricted to intact particles or to large envelope fragments, while haemagglutination is found with large or small fragments. However, envelope fragments, with haemolytic activity, induced fusion in cell monolayers with little or no inhibitory effect. Envelope fragments complexed with antibody lose their capacity to inhibit fusion and show no capacity for fusion of Ehrlich ascites tumour cells. These findings, and studies by electron microscopy on interactions of envelope fragments to cells, support the hypothesis that the fusion of suspended cells depends on the strength of contact between cells induced by virus components with haemolytic activity.

Cell-mediated lysis of heat-inactivated influenza virus-coated murine targets

The involvement of inoculated virus antigens in the induction of target susceptibility to cytotoxic T lymphocyte (CTL)-mediated lysis was investigated using heat-inactivated influenza virus, PR8 strain, and various inhibitors in comparison to the cases for live or ultraviolet (u.v.)-irradiated influenza and Sendai viruses. Induction of target susceptibility with heated PR8 was not inhibited by cycloheximide and actinomycin D as in the case of u.v.-irradiated Sendai virus, whereas live virus and u.v.-irradiated PR8 were inhibited under conditions which suppress protein synthesis. Induction of target susceptibility with the live and inactivated PR8 tested was suppressed in the presence of chloroquine, contrary to the case of Sendai virus, and was dependent on the cleavage type of influenza virus haemagglutinin. These findings suggest that the viral target antigens recognized by CTL in heated PR8-coated targets came from inoculated virus proteins, whereas those in PR8-infected or u.v.-irradiated PR8-coated targets involved newly synthesized viral proteins. The former viral target antigens seem to be transferred or processed from the endosome, depending on low pH fusion in the endosomes into which they were engulfed. In this point, the induction of viral target antigens with heated PR8 was different from that induced by u.v.-inactivated Sendai virus. Targets made with heated PR8 were recognized by cross-reactive CTL over the HA subtype.

Study of negatively stained images of sendai virus nucleocapsids using minimum-dose system

Using minimum-dose system and optical diffraction, effects of electron irradiation on negatively stained images of trypsin-straightened nucleocapsids of Sendai virus were semiquantitatively compared for uranyl-acetate (UA) and phosphotungstic acid (PTA). The results confirmed the superiority of UA in display of fine structures and showed that both UA- and PTA-stained images tended to turn from a one-sided to a two-sided image during irradiation, the general contrast of the picture increased in the UA-stained images but not in the PTA-stained ones, and furthermore the electron doses for the richest information were 18 000 to 30 000 e-/nm2 for UA, but 1000 e-/nm2 for PTA, under the condition used. The optical diffraction patterns of the UA-stained nucleocapsids, its analysis by the superposition method, and rotational harmonics of end-on views of nucleocapsids, together indicated that the most probable arrangement of subunits was 13 per turn of a helix with 5-nm periodicity. This helix also had an arrangement of subunits parallel to the axis. The occurrence of 2.5 nm periodicity was probably produced by an arrangement of a UA-penetrable concave substructure of the subunit.

Electron microscopic study on RNA of cytoplasmic polyhedrosis virus of the silkworm

Abstract Cytoplasmic polyhedrosis virus preparations of the silkworm were treated with urea or sodium perchlorate. The double-stranded viral RNA released when spread on protein monolayers according to the Kleinschmidt technique was examined by electron microscopy. CPV-RNA contains subunits of 0.36 μ and 1.12 μ RNA molecules. The longest RNA observed attained 6.8 μ. On the assumption that this RNA is the viral genome, the calculated molecular weight of the genome RNA is between 14 and 18 × 10 6 daltons. This value is consistent with that obtained from chemical determinations. Morphological evidence is presented that the viral RNA of CPV is released through the substructure at the vertex of the icosahedral capsid on these treatments.