Analysis of virus-specific RNA species and proteins in Freon-113 preparations of the Borna disease virus.

Treatment of homogenates from Borna disease virus (BDV)-infected brain tissue or cell cultures with Freon-113 yielded infectious particles with a buoyant density of 1.16-1.22 g/ml. Positive- and negative-stranded BDV-specific RNA species as well as three virus-specific proteins, known to be present in BDV-infected cell extracts, were demonstrated in these Freon-treated fractions. When the Freon-purified virus preparations were treated with RNase A prior to RNA extraction, only negative-stranded, genomic RNA was detected in Northern blot hybridizations using sense and antisense RNA probes. These data substantiate that BDV is a negative-stranded RNA virus.

Pneumotropic revertants derived from a pantropic mutant, F1-R, of Sendai virus.

Revertants were isolated from the protease activation mutant of Sendai virus, F1-R, which causes a systemic infection in mice. The fusion (F) glycoprotein of F1-R is susceptible to activation cleavage by ubiquitous cellular proteases and is thus responsible for pantropism in mice (Tashiro et al., 1988. Virology 165, 577-583). The revertants regained several phenotypes of wild-type virus; they required exogenous trypsin for activation of the F protein in cell cultures and in nonpulmonary mouse tissues and they were exclusively pneumotropic in mice. On the other hand, phenotypes of F1-R that remained unchanged by the revertants were bipolar budding in polarized epithelial cells, enhanced electrophoretic migration of the matrix protein, and the lack of a glycosylation site in the F2 subunit of the F protein. Comparative RNA sequence analysis of the F gene of the revertants revealed that the reduced cleavability of the F protein of the revertants was the result of the predicted single amino acid reversion (Pro to Ser) at residue 115 adjacent to the cleavage site. Thus the sequence at the cleavage site of the revertants was Ser-Lys compared with Pro-Lys for F1-R and Ser-Arg for wild-type virus. The results indicate that enhanced cleavability of the glycoprotein, a feature often associated with multiple basic residues within the cleavage site of paramyxovirus F proteins and influenza virus hemagglutinins, can also be determined by a single basic amino acid following proline. Additionally, the revertants were less susceptible to the activator for wild-type virus present in mouse lungs and less pathogenic for this organ than wild-type virus. These results provide further evidence that proteolytic activation of the F protein by host proteases is the primary determinant for organ tropism and pathogenicity of Sendai virus in mice. One of the revertants was also temperature sensitive (ts); the ts lesion in the nucleoprotein gene was identical to that found in ts-f1, the ts host range mutant from which F1-R was derived.

Isolated HE-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity.

Bovine coronavirus (BCV) and hemagglutinating encephalomyelitis virus (HEV) from swine were found to grow to high titers in MDCK I cells, a subline of Madin Darby canine kidney cells. Virus grown in these cells was used to isolate and purify the HE-protein. This protein has been shown recently to have acetylesterase activity and to function as the receptor-destroying enzyme of BCV. Here we show that HEV contains this enzyme, too. The glycoproteins were solubilized by treatment of virions with octylglucoside. Following centrifugation through a sucrose gradient the surface proteins S and HE (hemagglutinin-esterase) were obtained in purified form. After removal of the detergent by dialysis, HE formed rosettes as shown by electron microscopy. The purified HE protein retained acetylesterase activity and was able to function as a receptor-destroying enzyme rendering red blood cells resistant against agglutination by both coronaviruses. HE protein released from the viral membrane failed to agglutinate red blood cells. However, it was found to recognize glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid as indicated by a binding assay with rat serum proteins blotted to nitrocellulose and by its ability to inhibit the hemagglutinating activity of BCV, HEV, and influenza C virus. The purified enzyme provides a useful tool for analyzing the cellular receptors for coronaviruses.

Analysis of the influence of proteolytic cleavage on the structural organization of the surface of the West Nile flavivirus leads to the isolation of a protease-resistant E protein oligomer from the viral surface.

In order to analyze the organization of the membrane proteins pre M, M, and E of the West Nile (WN) flavivirus we have studied the influence of proteolytic cleavage of intact virus on the structure of these proteins. The amino acid sequence of all proteins is known, all six disulfides present in the viral E protein have been identified, and it has been suggested that the E protein contains regions R1, L1, R2, L2, and R3, which together form the E protein ectodomain followed by a carboxyterminal membrane anchor region (Th. Nowak and G. Wengler (1987) Virology 156, 127-137). The results of our analyses can be summarized as follows: (1) The surface of the WN virus contains E protein oligomers; the E protein molecules present in these structures contain two segments which are exposed to proteolytic attack; the segments are located in parts L1 and R3 of the E protein. (2) Proteolytic cleavage of these oligomers in these regions neither destroys nor releases the oligomers from the viral surface. (3) The WN virus surface contains a layer of 7-nm ring-shaped subunits identifiable by electron microscopy which are neither destroyed nor released by proteolytic cleavage. (4) An E protein trimer can be isolated from the surface of protease-treated WN virus. This trimer is morphologically similar to the 7-nm ring-shaped element which can be identified on the surface of native and protease-treated WN virus by electron microscopy.

Establishment and analysis of a system which allows assembly and disassembly of alphavirus core-like particles under physiological conditions in vitro.

Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982) Virology 118, 401-410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 mM K-acetate, 1.7 mM Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun. 71, 393-399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.

Productive infection of chick embryo cells by influenza viruses tightly bound on substratum.

To test whether penetration of influenza viruses could occur at the plasma membrane of host cells, virus particles were tightly bound on Concanavalin A-coated substratum of plastic culture plates and then overlayed with embryo cells. Under these conditions, endocytosis of the viruses was prevented but the cells were found to be effectively infected. The results indicate, that infection by influenza viruses can occur through fusion between the viral membrane and the host cell plasma membrane.

Electron microscope study of cultured cells of the chorioallantoic membrane infected with representative paramyxoviruses.

Chorioallantoic membrane (CAM) tissue cultures were found to be permissive for representative paramyxoviruses. The CAM cells can be used for plaque assay without the presence of trypsin. Ultrastructures of CAM cells infected with paramyxovirus Yucaipa (PMY), Sendai virus, and NDV were different. Nucleocapsids were readily seen in budding structures of cells infected with PMY, and in Sendai virus infected cells, large clusters of nucleocapsids were clearly evident in the cytoplasm. The site of glycoprotein cleavage does not have any effect on the nature of budding. It appears that a factor or factors in addition to the nature of the plasma membrane influences the morphology of cells infected with paramyxoviruses.

[Electron microscopic investigations on encapsulated Staphylococci (author's transl)]. / Elektronenmikroskopische Untersuchungen an bekapselten Staphylokokken

Attempts were made to study the surface of encapsulated staphylococci by electron microscopy. After fixation with osmium tetroxide the encapsulated staphylococci showed a "hazy" cell-surface, possibly because of capsular components (Fig. 1). On the other hand, the nonencapsulated staphylococci exposed a "smooth" surface of the cell wall (Fig 1). Similar findings were obtained after the immuneferritintechnique (Fig 2). The staphylococcal capsules could be clearly demonstrated by the "negative contrasting" method with ferritin (Fig 3). The capsules did not significantly enlarge after reaction of the encapsulated staphylococci with their homologous antiserum.