Correlation of rotational mobility and flexibility of Sendai virus spike glycoproteins with fusion activity

The rotational mobility of Sendai virus glycoprotein spikes was measured by flash-induced transient dichroism of eosin triplet probes. The possible importance of this molecular motion for function was investigated by parallel assays of hemagglutination and fusion with erythrocytes. For mobility measurements, the glycoproteins were labeled on amino groups with eosin-5-isothiocyanate and on the galactose residues of the oligosaccharide chains with eosin-5-thiosemicarbazide. The decay of the absorption anisotropy of both probes, which has a time constant of about 100-200 musec at 37 degrees is attributed to the rotation of the proteins about an axis normal to the plane of the membrane. This motion was inhibited by crosslinking of the spike proteins with glutaraldehyde or by the specific binding of human erythrocyte glycophorin (a virus receptor) to the HN glycoprotein. Low values of the initial anisotropy for both probes indicate the existence of a second, faster motion. This is attributed to segmental motion of the glycoproteins. Segmental motion is inhibited by crosslinking with glutaraldehyde but appears to be little affected by interaction with glycophorin. The temperature dependence of the segmental and rotational motion of the proteins revealed a pronounced increase in mobility in the range of 30-35 degrees which was not paralleled by the lipid motion of the Sendai virus envelope membrane. Since the temperature dependence of virus-induced hemolysis has a similar characteristic, the mobility of glycoproteins appears to be correlated with the fusion activity. The hemagglutination activity, however, is not dependent on the mobility of the glycoprotein spikes.     

Studies on the interaction of HVJ (Sendai Virus) with liposomal membranes. HVJ-induced permeability increase of liposomes containing glycophorin

Liposomes composed of egg yolk phosphatidylcholine or total lipids of human erythrocytes were found to be damaged by the paramyxovirus HVJ when they were prepared in the presence of a major sialoglycoprotein of human erythrocytes, glycophorin. The permeability increase of liposomes induced by virus was dependent on viral F protein, since trypsinization of HVJ grown in eggs inactivated the activity to cause the permeability increase. HVJ grown in LLC-MK₂ cells was also defective in the activity, confirming that the damage of liposomes is F protein dependent. The activity of HVJ to induce the permeability increase of liposome was affected by freeze-thawing and anti-HVJ serum in a way similar to hemolytic activity. The temperature dependence of marker release from liposomes was also almost the same as that observed in hemolysis. This similarity of the two systems suggests that the events leading to permeability increase of liposomes may mimic cell lysis brought about by the virus.     

Fusion of Semliki forest virus with red cell membranes

The interaction of Semliki Forest virus (SFV) and red cells was studied using biochemical and immunological methods. When [³H]uridine-labeled SFV was adsorbed at 0°, pH 5.8, to ribonuclease-loaded human red cell ghosts, the viral RNA became sensitive to ribonuclease within 2 to 5 min at 20–37°, but not at 0°. Maximal digestion of RNA was achieved within 30 min at 37° suggesting that the nucleocapsids had penetrated the red cell membranes. When the red cells that had been fused with [³⁵S]methionine-labeled SFV were treated with trypsin, about 70% of E2 protein and 50% of E1 protein was digested, demonstrating that the viral envelope proteins remained on the surface of the red cell. This was also shown by radioimmunoassay, by complement-dependent immune hemolysis assay, and by the ability of the red cells to bind Staphylococcus aureus bacteria to the cell surface after treatment with anti-envelope antiserum. When the red cells carrying SFV glycoproteins on their surfaces were transferred to pH 5.8, the cells were readily fused after the temperature had been raised to 42°. This shows that the fusion of virus envelope with red cell membranes precedes the fusion of red cells.     

Size determination of Orgyia pseudotsugata cytoplasmic polyhedrosis virus genomic RNA

Molecular-weight estimates were determined for the Orgyia pseudotsugata cytoplasmic polyhedrosis virus (CPV) genome. The molecular-weight estimates were obtained from measurements of glyoxal denatured RNA molecules following electrophoresis in 1% agarose gels and electron micrographic contour lengths. These estimates were compared to the minimal coding capacity derived from the size of translation products of selected genomic segments. The results obtained from these different experimental approaches were in sufficient agreement to estimate the genome size at 20.4 x 106 (29.8 kbp) with a segmental range from 0.61 to 3.53 x 10(6) (0.89-5.19 kbp). Two other members of the Reoviridae family were examined, the Bombyx mori CPV and the human reovirus type 3 (Dearing strain). The molecular-weight estimates for these two viruses were 20.3 and 19.1 x 10(6), respectively. These results suggest that the previously published molecular-weight estimates for these various double-stranded RNA genomes were underestimated.     

Characterization of the RNA associated with influenza A cytoplasmic inclusions and the interaction of NS1 protein with RNA

Paracrystalline inclusions formed by influenza A virus nonstructural protein (NS₁) in infected BHK21-F cells were found to contain significant amounts of RNA. PAGE analysis and oligonucleotide fingerprint mapping indicated that the inclusion-associated RNA was primarily of cellular origin. Analysis of the RNA by oligodeoxythymidylic acid—cellulose chromatography and in vitro translation indicated that the RNA was not host or viral messenger RNA. Purified inclusions were solubilized with 6 M urea and 1 M KCl, and the NS₁ protein was separated from the RNA by sucrose gradient centrifugation. When urea and KCl were removed by dialysis, the NS₁ protein reassociated with the RNA. The resulting complexes obtained in the absence of Mg²⁺ appeared to be smaller and more heterogeneous in size than the original NS, inclusions, whereas complexes obtained in the presence of 10 mM Mg²⁺ were similar in size to the original NS₁ inclusions. RNA-free NS₁ protein also formed tightly packed structures similar to the tightly packed NS₁-RNA complexes, indicating that RNA is not necessary for this interaction. The buoyant densities in CsCl of the original NS₁ inclusions, the reassociated NS₁-RNA complexes, and the RNA-free NS₁ protein complexes were determined to be 1.347, 1.341, and 1.270 g/ml, respectively. The results indicate that influenza A NS₁ protein has binding activity for RNA, and forms inclusions containing bound RNA molecules in the cytoplasm during virus replication.     

Fusion of Sendai virus with liposomes: dependence on the viral fusion protein (F) and the lipid composition of liposomes

The characteristics of fusion of the membrane of Sendai virus with that of liposomes has been investigated using two different methods to monitor the fusion reaction. The first method, which permits quantitation of lipid fused with virus, depends on separation by centrifugation of unfused liposomes from those fused with virus. The second involves the digestion after fusion of internal viral proteins by trypsin contained in liposomes; this assay is completely independent of exchange of lipid between liposomal and viral membranes in the absence of fusion. A fusion-inactive mutant virus, pa-cl, with an uncleaved F protein served as the appropriate control in these experiments. It was found that fusion of the virus with liposomes that contained no protein required cleavage of the F protein; such cleavage was previously shown to be required for fusion of the virus with cell membranes. This indicates the relevance of this model system for studies of fusion. Kinetic studies indicated that at neutral pH fusion was 88% complete in 10 min at 37 degrees. Investigation of the effects of liposomal lipid composition indicated that the presence of cholesterol in the liposomal membrane was required for fusion; a 0.3-0.4-mole fraction of cholesterol was optimal. The presence of neuraminic acid in the membrane was not essential for fusion. The results obtained are compatible with previous evidence suggesting a hydrophobic interaction between the cleaved F protein and the target membrane during fusion.     

Low-molecular-weight RNAs associated with tobacco ringspot virus are satellites

The low-molecular-weight RNA associated with an isolate of tobacco ringspot virus [TRSV(F)] lacks polyadenylic acid [poly(A)] sequences. Complementary DNA (cDNA) specific to this RNA was made with an oligo(dT) primer after tailing the RNA with poly(A). However, a significant amount of cDNA was made only when the RNA was treated with alkaline phosphatase prior to tailing. Hybridization of RNA in solution and by a Northern blot technique showed that there was no detectable nucleotide sequence homology between TRSV(F) RNA and two of the low-molecular-weight RNAs associated with this virus. These associated RNAs are therefore, satellites of TRSV(F). The relationship of these satellites to a previously reported satellite of TRSV is discussed.     

Identification of a satellite RNA associated with turnip crinkle virus

Turnip plants infected with turnip crinkle virus (TCV) contain four major RNA species which are not found in uninfected plants (A = 1.3 x 10(6) MW, B = 0.28 x 10(6) MW, C = 0.17 x 10(6) MW, and D = 0.13 x 10(6) MW). At least two of these RNAs, RNA A and RNA C, are packaged in the mature virion, but only the large RNA A is required for infection. Plants infected with RNA A alone produce neither the small virion RNA C nor the small nonvirion RNAs B and D. The small virion RNA C is not infective by itself, but requires coinfection with RNA A to replicate in plants. RNA C increases the severity of symptoms in plants infected with RNA A and restores the production of the nonvirion RNAs B and D. T1 RNase oligonucleotide mapping and copy DNA hybridization analysis indicate that the virion RNAs A and C do not have extensive homology. These data suggest that the large virion RNA A contains the full TCV genome and that the smaller virion RNA C is a dispensible satellite, designated S-TCV.     

Characterization of a satellite RNA associated with cucumber mosaic virus

Two types of RNA, each with a molecular weight of approximately 0.12 × 10⁶, designated RNA 5 and satellite RNA, have been found in purified cucumber mosaic virus (CMV) preparations and have been characterized by molecular hybridization analysis using ³²P-labeled complementary DNA probes transcribed from these RNAs. RNA 5 usually makes up about 5% or less by weight of the total viral RNA and was shown to consist of specific cleavage products of CMV RNAs 1–4. Its nucleotide complexity was equivalent to about three times its molecular weight. By contrast, satellite RNA could form up to about 50% by weight of virion RNA and had the following properties: (1) It contained a unique nucleotide sequence with no homology with CMV RNAs, (2) CMV and tomato aspermy virus, but not alfalfa mosaic virus or tobacco ringspot virus, could function as helper viruses for its replication and encapsidation, (3) its nucleotide sequence was independent of the host plant and the helper virus used for its propagation and it was not derived from a host plant RNA, and (4) it was not a negative copy of any of the CMV RNA species. We concluded that this RNA is a true satellite RNA and has no relationship to the RNA found in defective interfering particles of animal viruses.     

Comparative study of host capsule depolymerases associated with Klebsiella bacteriophages

Using 74 serological test strains for different Klebsiella K antigens as propective hosts, 50 new bacteriophages were isolated, the particles of which catalyze host capsular glycan depolymerization. As seen under the electron microscope, these viruses most often belong to Bradley's morphology group C (36 cases), occasionally to group B (11), and rarely to group A (3); they all carry tail spikes (or thick fibers) with which, presumably, the glycanase activity is associated. Employing the same set of Klebsiella strains, the host range of a total of 55 such bacteriophages was tested and 28 cross-reactions (host capsule depolymerization and plaque formation at a relative efficiency of plating ? 10⁵) were detected; in addition, one abortive infection (host capsule depolymerization without plaque formation) was found. The viral depolymerases thus proved to be very specific (33 cross-reacting with no, 18 with one, 2 with two, and 1 each with three or four heterologous polysaccharides). For further characterization of the over 80 phage-catalyzed depolymerization reactions thus identified, the purified virus particles were incubated with the isolated bacterial capsular polysaccharides, and the oligosaccharides produced were analyzed for reducing end sugar (glucose, galactose, mannose, fucose, or rhamnose), and for size (mainly one to three repeating unit oligosaccharides in about 85% of the cases). In those 49 phage-host systems, where the primary structures of the substrate polysaccharides were known, the reducing end sugar analyses led to unequivocal (36 reactions) or tentative (13) identifications of the phage enzyme susceptible glycosidic linkages, and hence to the following general conclusions: (I) in most cases cleavage occurs on either side of the (a) sugar unit carrying the negative charge(s), but reducing glycuronic acids are not produced; (II) most often, the reducing end sugar formed is substituted at position 3; (III) in the majority of cases, β-glycosidic linkages are hydrolyzed; (IV) in most polysaccharides which are acted upon by several phage enzymes, the same glycosidic bonds are split by the different agents.     

Protease activity associated with the capsule protein of Estigmene acres granulosis virus

The polypeptide composition of a granulosis virus of Estigmene acrea was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enveloped nucleocapsids are made up of 24 polypeptides with molecular weights ranging from 11,000 to 98,000. Virus capsule protein contains protease activity which degrades capsule protein from 30,000 to 25,000 through two intermediates of 29,000 and 27,000. The putative endoprotease is active at alkaline pH, sensitive to heat and diethylpyrocarbonate inhibition, and resistant to 10(-3) M phenylmethylsulfonyl fluoride. No alteration in the electrophoretic pattern of viral structural proteins was detected in virus preparations containing endoproteolytic activity. A low level of alkaline exoprotease activity associated with capsule protein was also detected.     

Expression of altered ribonucleotide reductase activity associated with the replication of the Epstein-Barr virus.

The expression of the Epstein-Barr virus (EBV) genome can be regulated in the epithelial/Burkitt hybrid (D98/HR-1) cell line and Raji lymphoblastoid nonproducer cell line by induction with 5-iododeoxyuridine (IUdR) or by superinfection with EBV. Extracts of control and induced D98/HR-1 and Raji cells were assayed for ribonucleotide reductase activity in the presence and absence of hydroxyurea (HU). Enzyme activity of control D98/HR-1 and Raji cells was inhibited by greater than 70%, by HU at both low (2 × 10^?4 M) bot high (5 × 10^?4 M) concentrations; however, the reductase activities of IUdR-induced D98/HR-1 cells, superinfected Raji cells, and IUdR-induced Raji cells were resistant to both levels of HU, and enzyme activities of 85 to +100% of control values were obtained in all cases. Under conditions that allowed only partial expression of the EBV genome (before removal of IUdR; early time after superinfection), very significant levels (70–85°k) of HU-resistant enzyme activity were obtained in the presence of 2 × 10^?4 M HU, whereas only 30 to 45% of control reductase activity was observed at the high HU concentration. Mixing experiments employing combinations of various D98/HR-1 control and induced cell extracts indicated that the HU-resistant reductase activity present in induced 1)98/HR-1 cells was due to the presence of an altered enzyme activity and not due to some nonenzymatic factor(s). Additional experiments, in which HU was preincubated with control and induced cell extracts, showed that the HU-resistant ribonucleotide reductase in extracts of induced D98/HR-1 cells was not due to inactivation of the inhibitor by an enzyme or factor present in these cells. These findings of an altered ribonucleotide reductase activity associated with EBV replication, considered in light of similar findings for equine herpesvirus type 1 and herpes simplex virus types 1 and 2, suggest that alteration of this enzyme activity may be a feature of herpesvirus replication.     

Antigenic reactivity of a soluble glycoprotein associated with bovine leukemia virus

A glycoprotein associated with bovine leukemia virus was treated with exoglycosidases, trypsin, and a specific protease from Staphylococcus aureus V8. Antigenic activity of the glycoprotein was destroyed by the exoglycosidases within 1 hr. Trypsin and S. aureus protease required longer periods of time to destroy antigenic reactivity. We concluded that the antibody produced in infected animals is directed against the carbohydrate moiety of the glycoprotein.     

A temperature-sensitive mutant of Sendai virus which establishes persistent infection in Vero cells without cell crisis

Eleven temperature-sensitive (ts) mutants of Sendai virus were examined for the ability to establish persistent infection in Vero cells at 37°. Only one mutant, ts-23, readily established persistent infection, while some of the mutants tested, like the parental wild virus, required long-term subcultivation for the establishment of persistent infection because of repeated cell crisis, and the remaining mutants failed to establish persistent infection. The ts-23 mutant replicated well in Vero cells, but infected cells showed no cytopathic effect and were readily subcultured. The fluid of these cultures contained neither defective interfering particles nor interferon, indicating that these factors are not involved in the establishment of persistent infection. In mixed infections, ts-23 virus markedly inhibited the development of cytopathic effect by the wild-type virus or the other ts mutants, and all or a part of the cells in the cultures beame persistently infected without showing cell crisis.     

Polypeptides associated with inclusion bodies from leaves of turnip infected with cauliflower mosaic virus

Inclusion bodies, the major intracellular site of accumulation of cauliflower mosaic virus (CaMV), have been partially purified by centrifugation onto a saturated sucrose cushion, followed by detergent treatment to lyse chloroplasts, then centrifugation on a saturated sucrose cushion again. The resulting preparation was enriched for inclusion bodies as judged by light and electron microscopic observation and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides in the preparation. Two major polypeptides of 61,000 and 43,000 apparent molecular weights were found in inclusion body preparations from leaves infected separately with each of three different isolates of CaMV. Polypeptides of apparent molecular weights 56,000, 50,000, 39,000, 37,000, and 34,000 were also detected in preparations from infected leaves, but not in comparable preparations from healthy leaves. The mobilities of all but the 61,000 and 34,000 molecular weight polypeptides were slightly different from the CM4-184 isolate, as compared with Cabbage B Davis or NY8153. The 61,000 molecular weight polypeptide did not correspond in mobility to any of the virion polypeptides. The relative intensities of polypeptide bands common to inclusion bodies and isolated virions were different in these two preparations, suggesting that considerable degradation took place during virion isolation.     

Complete sequence of the Sendai virus NP gene from a cloned insert

A DNA molecule representing all but the three terminal bases of the Sendai virus nucleoprotein (NP) gene, copied from viral mRNA, was inserted into pBR322. The NP insert comprised 1673 bases. The first AUG protein initiation codon, at position 65, began an open reading frame of 1551 bases, encoding a protein of 517 amino acids with an amino acid composition corresponding to previously published data. The NP gene sequence determined in the present work is similar to that described by Shioda et al. [ Nucl . Acids Res. 11, 7317 (1983)], but there are 14 amino acid differences that probably reflect differences in virus strains. The predicted secondary structure of the NP molecule and the locations within that structure of potential protease cleavage sites are in accord with structural domains previously defined by controlled protease digestion.