[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.

Human parainfluenza virus type 1 matrix and nucleoprotein genes transiently expressed in mammalian cells induce the release of virus-like particles containing nucleocapsid-like structures.

The matrix (M) protein plays an essential role in the assembly and budding of some enveloped RNA viruses. We expressed the human parainfluenza virus type 1 (hPIV-1) M and/or NP genes into 293T cells using the mammalian expression vector pCAGGS. Biochemical and electron microscopic analyses of transfected cells showed that the M protein alone can induce the budding of virus-like particles (vesicles) from the plasma membrane and that the NP protein can assemble into intracellular nucleocapsid-like (NC-like) structures. Furthermore, the coexpression of both the M and NP genes resulted in the production of vesicles enclosing NC-like structures, suggesting that the hPIV-1 M protein has the intrinsic ability to induce membrane vesiculation and to incorporate NC-like structures into these budding vesicles.

Elevated expression of the human parainfluenza virus type 1 F gene downregulates HN expression.

Interactions involved in the expression of parainfluenza glycoproteins were examined by expressing cDNA clones of the HN and F genes from human parainfluenza virus type-1 (hPIV1) or Sendai virus (SV) in recombinant Semliki Forest virus (recSFV) or vaccinia-T7 expression vectors. We found that expression of a cloned F protein gene of hPIV1 resulted in downregulation of the HN proteins of hPIV1 or SV. Compared to the amount of HN expressed in the absence of F, coexpression of HN and F led to about 70% reduction in HN. This reduction of HN was observed in both total cell lysates and in protein localized on the cell surface. In contrast to hPIV1 F, SV F did not suppress the expression of HN. Northern blot analysis indicated that similar levels of HN mRNA accumulated in the absence or presence of hPIV1 F. The reduction of HN protein expression by hPIV1 F was detectable after as little as a 10-min labeling period, suggesting that downregulation occurred at the level of translation or at an early stage of protein folding. In hPIV1-infected cells, the amount of F protein synthesized was only about 15% of that of HN, whereas SV F is expressed at high levels. When the level of F in hPIV1-infected cells was artificially increased by recSFV, HN expression was suppressed. The reduction of F protein production in hPIV1-infected cells was regulated at the level of transcription. Characterization of mRNAs produced in hPIV1-infected cells showed that only 20% of the hPIV1 F mRNAs were monocistronic transcripts; 80% were bicistronic M-F readthrough mRNAs. Because proteins are suggested to be synthesized from only the first cistron of bicistronic mRNA in paramyxovirus (T. C. Wong and A. Hirano (1987) J. Virol. 61, 584-589), production of F protein is likely suppressed by transcriptional regulation in hPIV1-infected cells. These results suggest that F is capable of downregulating the synthesis of HN, but that this is normally prevented in hPIV1-infected cells by suppression of F protein synthesis by transcriptional regulation.

Interaction of reconstituted Sendai viral envelopes with sperm cells: reconstituted Sendai virus envelope-induced fusion-mediated introduction of foreign material into bull sperm cells.

Reconstituted Sendai virus envelopes (RSVE), i.e. membrane vesicles bearing the viral envelope glycoproteins and phospholipids, are able to fuse with bull sperm cells. This was inferred from the increase in the degree of fluorescence dequenching (DQ) obtained following incubation of fluorescently labeled (R18 labeled) RSVE with bull sperm cells and from electron microscopy studies of RSVE-sperm interaction. Only a low degree of DQ was observed, under the same conditions, with non-fusogenic fluorescently labeled RSVE. This, and electron microscopy results, show that binding and membrane fusion events occur between RSVE and sperm cells. In addition, DQ was observed following incubation of RSVE that had been pre-loaded with the self-quenched fluorochrome Calcein, with bull sperm cells, indicating fusion-mediated injection of the dye from the RSVE space into the sperm cells.

The effects of membrane physical properties on the fusion of Sendai virus with human erythrocyte ghosts and liposomes. Analysis of kinetics and extent of fusion.

A number of amphiphiles which raise the bilayer to hexagonal phase transition temperature (TH) of phosphatidylethanolamine (PE) have been shown to inhibit viral fusion. In this study we have further evaluated the mechanism of this inhibition. Several anionic amphiphiles, including cholesterol sulfate, a component of mammalian plasma membranes, lower the final extent of Sendai virus fusion with both human erythrocyte ghosts and liposomes composed of PE and 5% of the ganglioside, GD1a. A cationic amphiphile slightly increased the final extent of fusion. The fusion rate constant is not greatly affected by the presence of as much as 20% cholesterol sulfate or other charged amphiphiles. The zwitterionic amphiphile, cholesterol phosphorylcholine has no effect on the final extent of fusion but it lowers the fusion rate constant. This amphiphile is potent in raising TH. The amphiphile cholesterol hemisuccinate (CHEMS) stabilizes the bilayer relative to the hexagonal phase at neutral pH, while at acidic pH the formation of the hexagonal phase is promoted. When CHEMS is added to vesicles of egg PE containing 5% GD1a, the rate of Sendai virus fusion is little affected at neutral pH but the rate is significantly enhanced at pH 5.0. These results demonstrate that viral fusion can be modulated, in part, by the tendency of the membrane to convert to the hexagonal phase.

Immunoelectron microscopy of parainfluenza virion glycoproteins with polyclonal and monoclonal antibodies: movement of glycoprotein with monoclonal antibody.

Two glycoproteins (HN and F) of parainfluenza virus were immunogold-labeled with polyclonal and monoclonal antibodies, respectively, and their labeling patterns were compared. Both glycoproteins HN and F were efficiently and homogeneously labeled with polyclonal antibodies, whereas they were labeled much less and heterogeneously with monoclonal antibodies. When either protein was initially labeled with monoclonal antibody, and then the other one, with polyclonal antibody, immunolabels of two glycoproteins were almost completely segregated. Although this segregation deformed virion morphology, it supported the concept of monoclonal antibody-mediated movements of glycoproteins.

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.

Fusogenic properties of Sendai virosome envelopes in rat brain preparations.

Sendai virosomes were characterized with respect to their ability to bind to, fuse with, and introduce substances into several rat brain preparations. Encapsulation efficiency for Sendai virosomes was enhanced but binding to cerebral cortical P2 preparations was attenuated by addition of bovine brain phosphatidylcholine during reconstitution. A higher percentage of Sendai virosomes than phosphatidylcholine liposomes appeared to bind to, fuse with and subsequently deliver [14C]sucrose into osmotically labile pools of the P2 preparation. Fusogenic activity was estimated by measuring dequenching of fluorescently labelled N-NBD-phosphatidylethanolamine. More virosomally encapsulated [14C]sucrose was bound to the P2 fraction than introduced into osmotically labile organelles, and the fraction of vesicles undergoing fusion was intermediate between these two values. Non-encapsulated [14C]sucrose did not bind to and was not taken up by the P2 fraction in a quantifiable manner. Virosomal envelopes also bound to primary cultures of rat brain neurons and glia in an apparently saturable manner. Addition of increasing amounts of the adenoassociated virus-derived vector pJDT95 increased encapsulation efficiency, and virosomes reconstituted in the presence of 60 micrograms DNA retained most of their binding activity (5.4% of total label) compared to those containing [14C]sucrose alone (8.4%). These data indicate that Sendai virosomes may be useful in the delivery of substances into brain-derived tissues, potentially for the modulation of gene expression and neurotransmission.

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.

Fusion of enveloped viruses with sperm cells: interaction of Sendai, influenza, and Semliki Forest viruses with bull spermatozoa.

Incubation of fluorescently labeled Sendai, influenza, as well as Semliki Forest viruses with bull sperm cells resulted in fluorescence dequenching. Fluorescence dequenching was observed with Sendai virus at pH 7.4 while with influenza and Semliki Forest viruses at pH 5.0, a pH value which is required for triggering their fusogenic activity. Control experiments performed with nonfusogenic Sendai and influenza viruses, or with bull sperm cells from which the viral receptors have been removed by treatment with neuraminidase, showed little fluorescence dequenching. These results clearly indicate that animal enveloped viruses are able to interact and to fuse with bull sperm cells. The possibility that following virus-sperm fusion spermatozoa can serve as a carrier of the virus genome and introduce it into recipient eggs during fertilization is discussed.

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.

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.

Significance of basolateral domain of polarized MDCK cells for Sendai virus-induced cell fusion.

Fusion (fusion from within) of polarized MDCK monolayer cells grown on porous membranes was examined after infection with Sendai viruses. Wild-type virus, that buds at the apical membrane domain, did not induce cell fusion even when the F glycoprotein expressed at the apical domain was activated with trypsin. On the other hand, a protease activation mutant, F1-R, with F protein in the activated form and that buds bipolarly at the apical and basolateral domains, caused syncytia formation in the absence of exogenous protease. Anti-Sendai virus antibodies added to the basolateral side, but not at the apical side, inhibited cell fusion induced by F1-R. In addition, T-9, a mutant with bipolar budding phenotype of F1-R but with an uncleavable F protein phenotype like wild-type virus, induced cell fusion exclusively when trypsin was added to the basolateral medium. By electron microscopy, cell-to-cell fusion was shown to occur at the lateral domain of the plasma membrane. These results indicate that in addition to proteolytic activation of the F protein, basolateral expression of Sendai virus envelope glycoproteins is required to induce cell fusion.

Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides.

Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18), a lipid mixing assay for fusion. This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. 31P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not. One possible interpretation of these 31P NMR results was that the inhibitory peptide stabilized a membrane structure with a large radius of curvature, when the fusion pathway demanded a membrane defect with a small radius of curvature. This hypothesis was tested by determining the influence of an inhibitory and a noninhibitory peptide on the formation of membraneous structures with small radii of curvature, through ultrasonic irradiation of phospholipid dispersions. The inhibitory peptide prevented the formation of membrane structures with small radii of curvature, while the noninhibitory peptide did not prevent the formation of such structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural localization of gold particles within neural grafts labeled with gold-filled Sendai viral envelopes.

The ability to discriminate between host and donor cells is required to interpret the organization of neural grafts at the electron microscopic (EM) level. Using light microscopy, Ardizzoni et al. (Ardizzoni, S.C., Michaels A., and Arendash, G.W. [1988] Science 239:635-637) described a method, using gold-filled Sendai viral envelopes, for labeling cell suspensions prior to grafting. As the colloidal gold used in this procedure is especially attractive for use with EM, we have examined the ultrastructural distribution and character of this label with transplanted cells. Cell suspensions taken from the nucleus basalis of fetal rats were labeled using gold-filled Sendai viral envelopes and grafted into the dorsal neocortex of adult host rats with nucleus basalis lesions. After varying survival times ranging from 1 to 14 months, grafts and surrounding host tissue were examined using standard EM techniques. Within the graft site, gold particles ranging from 10-200 nm were found associated with various membranes throughout the cytoplasm of both neurons and glia. Gold particles of similar size were also found within the nuclei of neuronal and non-neuronal cells. Host cells near the graft site contained some small gold particles (10-40 nm). Control injections of non-viable, gold-labeled cells or colloidal gold alone resulted in similar patterns of small gold particles which were readily discriminable from the larger virally inserted gold particles found in viable labeled donor cells. We conclude that this method allows discrimination between closely associated host and donor cells.

Organ tropism of Sendai virus in mice: proteolytic activation of the fusion glycoprotein in mouse organs and budding site at the bronchial epithelium.

Wild-type Sendai virus is exclusively pneumotropic in mice, while a host range mutant, F1-R, is pantropic. The latter was attributed to structural changes in the fusion (F) glycoprotein, which was cleaved by ubiquitous proteases present in many organs (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). These studies were extended by investigating, by use of an organ block culture system of mice, whether differences exist in the susceptibility of the lung and the other organs to the viruses and in proteolytic activation of the F protein of the viruses. Block cultures of mouse organs were shown to synthesize the viral polypeptides and to support productive infections by the viruses. These findings ruled out the possibility that pneumotropism of wild-type virus results because only the respiratory organs are susceptible to the virus. Progeny virus of F1-R was produced in the activated form as shown by infectivity assays and proteolytic cleavage of the F protein in the infected organ cultures. On the other hand, much of wild-type virus produced in cultures of organs other than lung remained nonactivated. The findings indicate that the F protein of wild-type virus was poorly activated by ubiquitous proteases which efficiently activated the F protein of F1-R. Thus, the activating protease for wild-type F protein is present only in the respiratory organs. These results, taken together with a comparison of the predicted amino acid substitutions between the viruses, strongly suggest that the different efficiencies among mouse organs in the proteolytic activation of F protein must be the primary determinant for organ tropism of Sendai virus. Additionally, immunoelectron microscopic examination of the mouse bronchus indicated that the budding site of wild-type virus was restricted to the apical domain of the epithelium, whereas budding by F1-R occurred at the apical and basal domains. Bipolar budding was also observed in MDCK monolayers infected with F1-R. The differential budding site at the primary target of infection may be an additional determinant for organ tropism of Sendai virus in mice.

The effect of virus particle size on chemiluminescence induction by influenza and Sendai viruses in mouse spleen cells.

Suspensions of orthomyxo- and paramyxoviruses are composed of pleomorphic particles ranging from large filaments to small spheres. Influenza and Sendai viruses were separated according to size by gel filtration and the induction of luminol-dependent chemiluminescence (CL) by particles of similar size was studied in suspensions of mouse spleen cells known to contain phagocytes. CL reflects the generation by the cells of reactive oxygen species. CL induction decreased with particle size for both viruses. Compared with small spheres, large influenza filaments were approximately 10 times as efficient in activating cellular light emission while the ratio between large and small Sendai viruses was 3:1. Small Sendai virus particles were also less efficient in lysing red cells and had lower neuraminidase activity. By contrast, with influenza virus, only neuraminidase and not the hemolytic activity decreased with the virus size. When influenza virus filaments were broken into smaller particles by sonication, the capacity to induce chemiluminescence dropped markedly while the hemolytic and hemagglutinating activities increased and neuraminidase activity remained unaltered. These results suggest that the presentation of influenza virus hemagglutinin and neuraminidase glycoproteins in a large particle, leading to extensive receptor crosslinking, may be an important factor in the efficient activation of CL by filamentous influenza virus. We suggest that radical generation as reflected in cellular CL may relate to the toxic in vivo effects that contribute to the pathogenesis of influenza and infections with paramyxoviruses.

Replication of parainfluenza (Sendai) virus in isolated rat pulmonary type II alveolar epithelial cells.

The major objectives of this study were to determine whether alveolar type II epithelial cells isolated from rat lung and maintained in tissue culture would support productive replication of parainfluenza type 1 (Sendai) virus and to determine whether isolated type II cells from neonatal (5-day-old) rats that are more susceptible to viral-induced alveolar dysplasia supported viral replication to a greater extent than those from weanling (25-day-old) rats. Isolated and cultured type II cells from neonatal and weanling rats that were inoculated with Sendai virus supported productive replication as indicated by ultrastructural identification of budding virions and viral nucleocapsids in type II cells and by demonstration of rising titers of infectious virus from inoculated type II cell cultures. Alveolar macrophages from neonatal and weanling rats also supported viral replication, although infectious viral titers in macrophage cultures were lower than those from type II cell cultures. Only minor differences were detected between viral titers from neonatal and weanling type II epithelial cell cultures. Higher densities of viral nucleocapsids were observed in neonatal type II cells than in those from weanling rats. The results indicate that isolated type II alveolar epithelial cells support productive replication of parainfluenza virus and that type II cells are probably more efficient in supporting productive viral replication than are alveolar macrophages.

The Sendai virus nucleocapsid exists in at least four different helical states.

Sendai virus nucleocapsids have been observed by electron microscopy to coexist in three different helical pitch conformations, 5.3, 6.8, and 37.5 nm. The 5.3- and 6.8-nm conformations are present both in uranyl acetate negatively stained preparations and in tantalum-tungsten metal-shadowed preparations, whereas the 37.5-nm conformation, which has not been previously reported, is present only in the shadowed preparations. The 5.3-nm pitch conformation appears to be a mixture of two discrete structural states, with a small difference in the twist of the structure between the two. We have used image reconstruction techniques on an averaged data set from eight negatively stained nucleocapsids to produce a three-dimensional reconstruction at 2.4-nm resolution of the structure in one of the 5.3-nm pitch states. There are 13.07 nucleocapsid protein (NP) subunits in each turn of the helix in this state. The helical repeat is 79.5 nm, containing 196 subunits in 15 turns of the left-handed 5.3-nm helix. The arrangement of subunits produces a 5.0-nm-diameter hollow core which forms an internal helical groove. The RNA accounts for about 3% of the mass of the nucleocapsid, and so its location is not conspicuous in the reconstruction. Because of the RNA remains associated with the NP subunits during mRNA transcription and genome replication, structural transitions in the nucleocapsid may determine the accessibility of the genome to polymerases. Alternatively, the large hollow core and internal helical groove we have reconstructed may allow access to the RNA even in the tightly coiled 5.3-nm pitch conformation.