[An atomic force microscopy study on the images of para influenza virus under different treatment conditions].

Using atomic force microscope (AFM), we investigated the images of Pars influenza virus (PIV) under different treatment conditions and observed the different appearances of the virus and its ultra-microstructure from the exterior to the interior. From the 2D images under transmission electron microscope (TEM), we could see that the surfaces of PIV particles exhibited spherical and band-shaped 'tufts'; from the 3D images under AFM, we could further observe the whole spherical virus particles and their detailed surfaces, which exhibited round and band-shaped 'tufts'. Comparing the images under TEM with those under AFM, we found that the latter could reveal the surface topograph and ultramicrostructure of viruses more truly than did the former. The samples of viruses were treated by Tritonx-100, the lipid envelopes of virions were partly or completely resolved, and then most of their capsids were exposed. We could observe the different appearances of the virions under AFM, the lipid envelopes of which were gradually removed. The samples of viruses were also treated by SDS, and the RNA was released from the virions. From the AFM images, we could see the structure of the RNA. It was thus clear that AFM could be used to investigate the different appearances and ultramicrostructure of viruses rapidly and efficiently.

Loss of the N-linked glycan at residue 173 of human parainfluenza virus type 1 hemagglutinin-neuraminidase exposes a second receptor-binding site.

BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes.

Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'-tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++-independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259).

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

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.

Asymmetric fusion between synthetic di-n-dodecylphosphate vesicles and virus membranes.

The interaction between vesicles, prepared from the synthetic amphiphile di-n-dodecylphosphate (DDP), with Sendai virus membranes was investigated. DDP vesicles fuse in the presence of Ca2+ ('symmetric' fusion). However, in the absence of Ca2+, DDP vesicles and Sendai virus, both displaying a high intrinsic fusion capacity with various target membranes, can also readily fuse with each other ('asymmetric' fusion). Under these conditions, fusion was found not to depend on specific viral proteins. Thus fusion occurs over a broad pH range (3.0-9.0) and is not affected by perturbation of viral protein structure. The overall interaction process was further analyzed with a mass action kinetic model. The analysis reveals that the destabilization and reorganization of the synthetic and viral bilayers are as fast as in pure phospholipid systems. Furthermore, the drastic effect of temperature on the overall reaction appears to be related to an effect of this parameter on fusion itself rather than on vesicle-virus aggregation. This could suggest that protein mobility constraints modulate the fusion reaction. The morphology of the fusion products, which consist of a single virus particle and several DDP vesicles, indicates a bilayer stabilization of the fusion product, rather than formation of tubular structures, as observed for symmetric DDP fusion products. The present results further emphasize the high susceptibility of vesicles composed of synthetic amphiphiles to engage in (protein-mediated) membrane fusion. This bears relevance to their potential application as carriers for biomolecules.

Electron microscopic study on the interaction of Sendai virus with liposomes containing glycophorin.

The interaction of liposomes containing glycophorin, a major sialoglycoprotein of human erythrocytes, with Sendai virus was studied by freeze-fractures and negative staining electron-microscopy. Viral envelopes were absorbed on liposomal membranes at 0 degrees C. When the temperature was shifted up to 37 degrees C, the viral envelopes fused with the liposomal membranes (envelope fusion). Particles representing viral membrane components formed clusters on liposomal membranes after incubation for more than 1 h at 37 degrees C.

Use of reconstituted Sendai virus envelopes for fusion-mediated microinjection of double-stranded RNA: inhibition of protein synthesis in interferon-treated cells.

Poly(I).poly(C) molecules were trapped with reconstituted Sendai virus envelopes when added to the reconstitution system. A quantitative estimation indicated that about 10% of the added poly(I).poly(C) remained associated with the fusogenic viral envelopes. About 50% of the associated poly(I).poly(C) were found to be RNAase A resistant, enclosed within the viral envelopes. Incubation of loaded viral envelopes with HeLa or L-cells resulted in strong inhibition of protein synthesis, indicating fusion-mediated microinjection of the enclosed poly(I).poly(C). Introduction of poly(I).poly(C) into cultured cells by the use of reconstituted Sendai virus envelopes was as efficient as the introduction of these polynucleotides using the calcium phosphate coprecipitation technique. The inhibition of protein synthesis in L-cells but not in HeLa cells was dependent upon pretreatment with interferon. Incubation of poly(I).poly(C)-loaded viral envelopes with interferon-treated variant cells of the NIH 3T3 line, which possess a very low amount of RNAase L, resulted in only 25% inhibition of protein synthesis, compared to 85% inhibition observed in L-cells.

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies.

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.

Experimental panencephalitis induced in suckling mice by parainfluenza I (6/94) virus. III. Ultrastructural studies.

Intracerebral inoculation of newborn mice with Parainfluenza I (6/94) virus produces a chronic panencephalitis. Electron microscopic studies were carried out over 125 days of the infection. Productive infection of choroidal and ependymal epithelial cells was seen from postinoculation days 2nd to the 8th. Fusion of adjacent choroid and ependymal cells resulted in giant cell formation. Completed virions were seen adsorbed to circulating macrophages and these cells replicated intracytoplasmic nucleocapsids. Neuronal infection was evident on the 3rd postinoculation day, was widespread by the 6th day postinoculation and persisted to the 35th day postinoculation. Nucleocapsid alignment and budding from neuronal plasma membranes was never seen. An initially intense mononuclear cell infiltrate subsided by the 35th day but residual inflammation persisted throughout the study. Late in the course of the infection, vacuolation of the neuropil and a periventricular and deep cerebral spongiform change was seen which could not be directly associated with local viral replication. These ultrastructural findings are correlated with prior light microscopic, virological and immunofluorescent studies of the infection and compared to other experimental models of myxovirus central nervous system infections.

Targeted delivery of hygromycin B using reconstituted Sendai viral envelopes lacking hemagglutinin-neuraminidase.

Hygromycin B was encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F-virosomes). Incubation of loaded F-virosomes with cultured HepG2 cells resulted in fusion mediated delivery of hygromycin B to the cell cytoplasm, as was inferred from inhibition of DNA synthesis. Binding of the F-virosomes to HepG2 cells was mediated by the interaction of terminal beta-galactose residues of fusion protein with asialoglycoprotein receptor on HepG2 cells, subsequently leading to fusion between the two membranes. The cytotoxic effect of hygromycin B enclosed in F-virosomes was comparable with that of F,HN-virosomes containing both hemagglutinin-neuraminidase (HN) and F protein and F,HNred-virosomes containing HN whose disulfide bonds were irreversibly reduced (HNred). Hygromycin B loaded fusogenic liposomes were prepared by coreconstituting the viral envelope containing only fusion protein with exogenous lipids. These fusogenic liposomes were found to be more active than F-virosomes at the same fusion protein concentrations.

Sendai virus M protein is found in two distinct isoforms defined by monoclonal antibodies.

The use of a monoclonal antibody defines a subset of Sendai virus M protein representing about 30% of total. This M protein acquires, during the hour following synthesis, an epitope not present on the bulk of M. This epitope maturation is observed in acutely as well as in persistently infected cells. It takes place in vivo in absence of other viral proteins, but it is not observed when the protein is synthesized in a reticulocyte lysate. Epitope maturation does not appear to result from phosphorylation, acylation or disulfide bond formation. If immunofluorescent staining seems to indicate a preferential association of this subset of M protein with nucleocapsids, this is not confirmed by immunogold staining or by nucleocapsid isolation. Incubation of cytoplasmic extracts or of purified M protein in conditions which do not favor M to M protein association results in a relative increase of M protein carrying the maturing epitope. It is concluded that M protein exists in two distinct isoforms.

Cryoelectron microscopy of vitrified Sendai virions.

Morphology of vitrified Sendai virions was studied by transmission type electron microscopy. Almost all the virions appeared to be completely spherical, although their diameters differed. A possibly continuously long nucleocapsid was seen running helically in an envelope. Spikes were seen on the virion surfaces. The results indicate pleomorphism of Sendai virions in size but not in shape.

Immunoelectron microscopic study of the detection of the glycoproteins of influenza and Sendai viruses in infected cells by the immunoperoxidase method.

The envelope glycoproteins of influenza virus (HA and NA) and paramyxovirus (HN and F) were visualized on the surface of infected cells by immunoelectron microscopy using the indirect immunoperoxidase technique. In X7 influenza virus-infected fibroblasts, the hemagglutinin (HA) and the neuraminidase (NA) were observed on the cell membrane respectively 2 and 3--4 h after infection. The antigens were initially seen as discrete patches and later evenly distributed along the plasma membrane prior to budding. Antibody induction of HA and NA was observed as cytoplasmic inclusions, with peroxidase-positive activity, attributed to endocytosis. The redistribution of HA and NA supports the hypothesis of lateral mobility of the viral glycoproteins in cellular membranes as visualized by the immunoperoxidase method. The glycoproteins of Sendai virus, in infected Madin--Darby bovine kidney cells, were found to be evenly distributed along the plasma membrane and endoplasmic reticulum, the latter by the indirect microperoxidase method. The immunoperoxidase methods may be useful for investigating the polarized distribution of envelope glycoproteins.

Evaluation of reconstituted Sendai virus envelopes as intra-articular drug vectors: effects on normal and experimentally arthritic rabbit knee joints.

Fusogenic vesicles reconstituted from the envelopes of Sendai virus particles were injected into rabbit knee joints (both normal and experimentally arthritic) to evaluate the in-vivo biocompatibility of these putative drug carriers. The reconstituted Sendai virus envelopes (RSVE) were greater than 80% retained within the arthritic knee joints after 24 h and studies with 125I- and fluorescein-labelled RSVE both showed association of the vesicles with the synovia of arthritic and healthy joints. However, RSVE were found to cause inflammation after intra-articular injection, as judged by joint swelling and histological assessment, and these effects were exacerbated by successive administrations. RSVE-entrapped methotrexate, whether free or conjugated to human serum albumin, was ineffective in preventing the irritancy of RSVE or in reducing the chronic inflammation in joints affected by an experimentally induced arthritis.

Scanning electron microscope study of Sendai virus fusion between HeLa cells and chick erythrocytes.

Chick erythrocytes were fused with HeLa cells by Sendai virus and preparations examined by scanning electron microscopy at different times after fusion. Heterokaryons were usually formed by fusion of erythrocyte ghosts with HeLa cells. Occasionally whole erythrocytes were engulfed but there was no evidence that free nuclei fused. Initial inter-cell attachments were usually, and possibly always, made at the site of an attached virus particle. This study helps to correlate topographical findings with previous two-dimensional studies with the transmission electron microscope and may also provide a model system for the fusion of parasitised erythrocytes with eukaryotic cells.

[Investigation of myxovirus structure by spin probes. I. Thermostability of the lipid membrane of influenza and Sendai viruses]. / Issledovanie struktury miksovirusov metodom spinovykh zondov. I. Termostabil'nost' lipidnoi obolochki virusa grippa i virusa Sendai.

Electron spin resonance (ESR) spectra of the spin probes C5 and C16, the iminoxyl derivatives of stearic acids, have been investigated after their incorporation into lipid membranes of influenza and Sendai viruses. At room temperature the spectra of both probes coincided indicating the similar composition and origin of lipid viral membranes. Similar to influenza virus, Sendai virus was shown to possess the external lipid bilayer. Investigation of the rigidity of the lipid phase of myxoviruses as a function temperature has shown that the structure of the lipid external layer (no less than 8 A in thickness) is due to surface glycoproteins incorporated into the viral membrane. The structural transition of the lipid phase of influenza and Sendai viruses 8 A apart from the viral surface was observed at 50 degrees C. The surface glycoproteins do not probably penetrate deep in the lipid membrane of myxoviruses and do not condition its structure at long distances (22 A) from the viral surface. The completely reversible structural transition of lipid phase at this distance was observed at temperatures 17--20 degrees C.

[Myxovirus structural investigations by spin probe methods. II. Influence of rimantadine on the structure of viral and artificial lipid membranes]. / Issledovanie struktury miksovirusov metodom spinovykh zondov. II. Vliianie rimantadina na strukturu virusnykh i iskusstvenno sformirovannykh lipidnykh membran.

The lipid membrane structure of two myxoviruses (influenza and Sendai viruses), red blood cells and liposomes (prepared from total lipids of Sendai virus) have been investigated in the presence of rimantadine by means of two spin probes C5 and C6--the iminoxyl derivatives of stearic acids. Rimantadine was shown to penetrate the lipid membranes of myxoviruses and red blood sells and change the structure of the lipid phase no less than 0.8 nm apart from the viral surface. In the depth of 2.2 nm the structure of these lipid membranes remains unchanged after addition of rimantadine. Rimantadine also displaces the phase transition points of cell and viral lipid membranes to lower temperatures. Rimantadine does not change the lipid bilayer structure of liposomes 0.8 nm apart from its surface. These finding allow to suggest that rimantadine affects the cell and viral lipoprotein membranes.

[Dispersion analysis of viral suspensions by the technic of optical mixing spectroscopy]. / Dispersionnyi analiz virusnykh suspenzii metodom spektroskopii opticheskogo smesheniia.

To evaluate the heterogeneity of virus populations and determinations of virus monoparticle size in suspensions, a dispersion analysis by the method of spectroscopy of optic mixing was developed. The parameters of influenza USSR/Khabarovsk and parainfluenza Sendai virus suspensions were measured in correlation spectrometer and the results were computerized.

[Effect of formaldehyde on the ribonucleoprotein structure of Sendai virus]. / Issledovanie vliianiia formal'degida na strukturu ribonukleoproteida virusa Sendai.

The effect of formaldehyde on the conformation of Sendai virus ribonucleoprotein (RNP) was studied by electron microscopy and a spectrophotometric method. The effect of formaldehyde on RNP conformation was found to depend strongly on the ionic strength of the solution. Under conditions of a low ionic strength in the presence of 1.5% formaldehyde the tightly coiled rods of RNP stained with uranyl acetate become loosely coiled or almost completely extended. At the same time, formaldehyde reaction with RNP results in a decrease in extinction of the RNP suspension in the 250 to 290 spectral region. However, RNP incubation with formaldehyde in the presence of 0.5M NaCl caused no changes in the morphology and spectral properties of the RNP under study. The results indicate that formaldehyde cannot be used for fixation of Sendai virus nucleocapsids before negative staining for electron microscopy examinations.

[Morphological and structural studies of Sendai virus ribonucleoprotein]. / Morfologicheskie i strukturnye issledovaniia ribonukleoproteida virusa Sendai.

The structure and morphology of Sendai virus ribonucleoprotein (RNP) with native and split protein subunits were studied by the method of circular dichroism and electron microscopy. Cleavage of the polypeptide fragment from RNP protein subunits was shown to cause changes in the secondary structure of RNP in situ but not to affect the RNP morphology; when stained with uranyl acetate both RNP types had an appearance of spiral strands.