Importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention of spread of infection.

The effects of monospecific antibodies to the viral glycoprotein with hemagglutinating and neuraminidase activity (HN) and the viral glycoprotein with membrane-fusing activity (F) of the paramyxovirus simian virus 5 (SV5) on the spread of infection in two cell types have been investigated. In CV-1 cells, infection can spread by either released progeny virus adsorbing to and infecting other cells, or by fusion of an infected cell with an adjacent cell as a result of the cell-fusing activity of the F glycoprotein. In these cells, antibodies specific for the HN glycoprotein prevented the dissemination of infection by released infectious virus, but spread by cell fusion was not inhibited. Antibodies to the F glycoprotein completely prevented the spread of infection in these cells. In Madin-Darby bovine kidney cells, which are relatively resistant to SV5-induced fusion, antibodies to either the HN or F glycoproteins were capable of preventing the dissemination of infection. These results indicate that effective immunological prevention of the spread of paramyxovirus infection requires the presence of antibodies that inactivate the F glycoprotein. This requirement for anti-F antibodies has obvious implications for the design of effective paramyxovirus vaccines and provides an explanation for previous failures of formalin-inactivated paramyxovirus vaccines as well as additional insight into the possible immunopathological mechanisms involved in the atypical and severe infections that have occurred in individuals who received inactivated paramyxovirus vaccines and were subsequently infected by the virus.

Studies on pneumonia virus of mice (PVM) in cell culture. I. Replication in baby hamster kidney cells and properties of the virus.

Pneumonia virus of mice (PVM) has been serially propagated in a line of baby hamster kidney (BHK21) cells. A maximum titer of 6.3 x 10(6) TCID(50) per ml was obtained, and there was little variation in yield on serial passage. PVM grown in BHK21 cells was antigenically similar to virus obtained from the mouse lung, but was somewhat less virulent for the mouse after 10 serial passages in these cells. Virus produced by BHK21 cells agglutinated mouse erythrocytes without prior heating or other treatment. Sedimentation of PVM in the ultracentrifuge or precipitation by ammonium sulfate resulted in a loss in infectivity but an increase in hemagglutinating activity, presumably due to disruption of the virus particle. In a potassium tartrate density gradient, the major portion of infective virus sedimented at a density of approximately 1.15, and noninfective hemagglutinin, at a density of approximately 1.13. Stock virus preparations appear to contain a large amount of noninfective hemagglutinin. The replication of PVM was not inhibited by 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. Infected cells contained eosinophilic cytoplasmic inclusions which showed the acridine orange staining characteristic of single-stranded RNA. Foci of viral antigen were observed in the cytoplasm of infected cells by fluorescent antibody staining. The results suggest that PVM is an RNA virus that replicates in the cytoplasm.

On the role of the response of the cell membrane in determining virus virulence. Contrasting effects of the parainfluenza virus SV5 in two cell types.

The simian myxovirus SV5 multiplies in a continuous line of baby hamster kidney (BHK21-F) cells causing extensive cell fusion, followed by cell death. After inoculation of 15 PFU/cell, the latent period was 7 hr, the doubling time approximately 60 min, and the yield 7 PFU per cell. Giant cell formation began about 6 hr after infection and rapidly progressed to the formation by 14 to 18 hr of a single syncytium which disintegrated by 24 to 36 hr. In contrast, SV5 multiplies in primary rhesus monkey kidney cells for long periods of time producing high yields of virus with little cytopathic effect. High multiplicities of SV5 induced cell fusion in BHK21-F cells within 1 hr in the absence of virus multiplication but had no visible effect on monkey kidney cells. Time-lapse photomicrography has demonstrated that giant cells form by fusion of infected cells, and that some polykaryocytes divide. During aberrant division of polykaryocytes giant nuclei are formed from the nuclear material of several parent nuclei. The cytoplasmic development of viral antigens as demonstrated by immunofluorescence is similar in BHK21-F and monkey kidney cells. Synthesis of cellular DNA, RNA, and protein in monkey kidney cells is not shut off by SV5-infection, and in BHK21-F cells synthesis of these macromolecules is not inhibited until after extensive cell fusion has occurred 12 to 15 hr after infection. Persistently infected BHK21-F and monkey kidney cells have been serially carried through 11 and 28 cell passages, respectively. The results suggest that whether SV5 acts as a moderate virus, as in monkey kidney cells, or a virulent virus, as in BHK21-F cells, depends on the response of the cell membrane to the virus.

Studies on pneumonia virus of mice (PVM) in cell culture. II. Structure and morphogenesis of the virus particle.

Pneumonia virus of mice (PVM) particles are spheres 80-120 mmicro in diameter, or filaments of similar diameter with lengths up to 3 micro. The particles possess an outer spike-covered envelope and helical internal component 120-150 A in diameter. Virus particles acquire their envelope by a budding process at the cell membrane; mature particles are seen only extracellularly. Dense inclusions are prominent in the cytoplasm of PVM-infected BHK21 cells by 48 hr after inoculation. The inclusions appear to consist of aggregates of the internal component of PVM, and the helical component has been isolated in a cesium chloride gradient from extracts of osmotically shocked cells. Murine erythrocytes, which are agglutinated by PVM, adsorb to the surface of infected cells and to budding and extracellular PVM particles. On the basis of its structure and morphogenesis, PVM appears to be a myxovirus; however, it does not fit into either of the established subgroups of myxoviruses. The 120-150 A diameter of the PVM internal component differs from the diameters of the internal components of the two established subgroups of myxoviruses, and suggests that a third subgroup of these viruses may exist.

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia.

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501-520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.

On the role of microtubules in movement and alignment of nuclei in virus-induced syncytia.

Infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes rapid and extensive cell fusion. Time-lapse cinematography shows that when cells fuse, their nuclei migrate straight to the center of the syncytium at rates of 1-2 micro/min. Nuclei are often arranged in long, tightly packed, parallel rows in syncytia derived from the fibroblastic BHK21-F cells. Polarization microscopy shows birefringent material between and parallel to these rows of nuclei, and electron microscopy shows bundles of cytoplasmic microtubules, approximately 250 A in diameter, and filaments, approximately 80 A in diameter, parallel to and between the rows of nuclei. Colchicine treatment causes disappearance of microtubules from BHK21-F cells and an apparent increase in the number of 80-A filaments. Although colchicine-treated, SV5-infected cells fuse, their nuclei do not migrate or form rows but remain randomly scattered through the syncytial cytoplasm. Incubation at 4 degrees C does not disrupt microtubules in BHK21-F cells. Rows of nuclei have been isolated from SV5-induced syncytia, and the nuclei in them have been found to be intimately associated with microtubules but not with other cytoplasmic structures. These results suggest that microtubules demarcate cytoplasmic channels through which nuclei migrate and that they may also be involved in the mechanism of nuclear movement.

Biochemical studies on cell fusion. II. Control of fusion response by lipid alteration.

The preceding communication (Roos, D.S. and P.W. Choppin, 1985, J. Cell Biol. 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.

Biochemical studies on cell fusion. I. Lipid composition of fusion-resistant cells.

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.

Enhanced yields of measles virus from cultured cells.

Conditions for increasing the yield of measles virus from cultured cells have been investigated. Important factors for high virus yields were found to be cell type, incubation at 33 degrees C, concentration of serum in the medium, use of virus as inoculum within five passages of plaque purification, and repeated harvesting of the virus-containing medium at 12 h intervals after extensive virus-induced cell fusion had occurred. Under these conditions, Vero cells infected with the Edmonston strain of measles virus yielded a total of 2.98 X 10(9) plaque-forming units (p.f.u.) of released virus, which is equivalent to 270 p.f.u./cell. A similar amount of cell-associated virus was produced.

The J.I.M. interview. Purnell W. Choppin, MD.

The Howard Hughes Medical Institute (HHMI) is known worldwide for its support of high calibre research in the basic biomedical sciences. At present, 332 men and women at 72 institutions are HHMI investigators, putting them in the ranks of the best and the brightest (and certainly the most generously supported) researchers in America. But the Howard Hughes Medical Institute's portfolio goes well beyond its core program as a medical research organization. Purnell W. Choppin, MD, discussed the breadth of Hughes' activities during a recent interview in his office at the Institute's main campus in Chevy Chase, Maryland, a few blocks away from the National Institutes of Health. Among other things, Choppin talked about US medical schools, clinical research, postgraduate education, and HHMI support of research abroad.

Influenza: the agent.

Less perplexing than its epidemic behavior is the nature of the influenza virion as such, which is now one of the best understood viruses in terms of morphology and replication. Among the aspects of the virus discussed in this article are the composition of its genome, the components of its envelope, and the probable molecular structure of its two surface proteins as well as their relative roles in antigenicity.

Mice immunized with measles virus develop antibodies to a cell surface receptor for binding virus.

Mice were immunized with measles virus to determine whether an auto-anti-idiotypic antireceptor response could be generated as a probe for measles virus receptors. Mice initially responded to viral antigens (days 11 to 18) and subsequently developed antibodies to a putative measles virus receptor (peak at day 30 to 35) by three criteria: the sera (1) agglutinated erythrocytes which virus agglutinates, (2) reacted with Vero cells, and (3) inhibited virus attachment to Vero cells. Additionally, select sera inhibited virus infection of Vero cells. The cell-reactive activity was identified as immunoglobulin G antibody and was neutralized by sera reacting with virus (idiotype). The application of this anti-idiotypic antibody to identify measles virus-binding sites on Vero cells was revealed by the ability of sera to immunoprecipitate 20- and 30.5-kilodalton proteins from metabolically labeled ([35S]methionine) Vero cells.

Selective inhibition of WSN influenza virus haemolysis by pea lectin.

The capacity of lectins to inhibit viral haemolysis of chicken erythrocytes was tested, to evaluate the role of carbohydrate in the fusion reaction. Pretreatment of cells with pea lectin provided a 70% to 85% haemolysis inhibition with WSN influenza virus, but only 10% to 14% with PR8 influenza virus. Pea lectin did not detectably bind to virus, nor did it inhibit virus binding to cells, but it did inhibit WSN influenza virus elution. Additionally, pea lectin was active against Sendai virus and B/Lee influenza virus, but inactive against Newcastle disease virus. Haemolysis by WSN and PR8 influenza viruses was unaffected in cells pretreated with concanavalin A, peanut, wheatgerm or soybean lectins. A possible role of cellular carbohydrate in virus-cell fusion is discussed.