Immediate persistent infection by human parainfluenza virus 3: unique fusion properties of the persistently infected cells.

We describe here a persistently infected cell system with unique properties. Cells infected with human parainfluenza virus type 3 (PF3) at high multiplicities of infection showed little or no cytopathic effects (cell fusion). Unlike other paramyxovirus persistent infections that require a long development time, the majority of the cells survived the initial infection and formed persistently infected cell cultures that were immediately available for study. In addition, unlike other paramyxovirus persistent infections, the PF3 system described here produced high levels of infectious virus and did not undergo periodic crises. Although cells persistently infected with PF3 contained large amounts of the cleaved, active form of the viral fusion protein, F1, the persistently infected cells did not fuse with each other. However, they did fuse with uninfected cells within minutes of cell-to-cell contact. Other persistent paramyxovirus infections do not have this property. Fusion occurred with all cells tested, including red blood cells, and was not dependent on protein synthesis. The unique fusion properties of these PF3 persistently infected cells make this an interesting system for the study of mechanisms of viral fusion and mechanisms of inhibition of viral fusion.

A simple method for increased recovery of purified paramyxovirus virions.

A simple method involving vigorous agitation of infected cell monolayers prior to collection of culture medium is described to greatly increase the recovery of purified virions of measles virus, respiratory syncytial virus and human parainfluenza virus. Vigorous agitation of the flasks containing monolayers of infected cells increased the recovery of purified virions by at least 3- to 10-fold as judged by the intensity of [35S]methionine labeled viral proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). These protein profiles also indicated that these virions were as clean as those purified from gently collected medium. Analysis of titers of infectious virus recovered from medium of agitated and non-agitated flasks showed similar increases. These results suggest that the cell associated nature of these viruses may be at least partly due to either partially budded virions or mature virions sticking to the cell membrane, since these both might be expected to be freed from the cell by mechanical shearing.

Improvement of respiratory syncytial virus replication in actively growing HEp-2 cells.

The growth of respiratory syncytial (RS) and parainfluenza type III (PI3) viruses has been studied in actively growing versus relatively stationary HEp-2 cells. There was no effect on PI3 virus growth. RS virus synthesis was stimulated from 20- to 60-fold by growth in actively growing cultures when the cell density was approximately 1/3 that of a confluent culture. This stimulation was manifested by a greater yield of virus per cell, more virus-specific mRNA produced per cell, and a 50% decrease in the time before cytopathic changes appeared.

On the role of substrate and GTP in the regulation of argininosuccinase activity.

As determined by equilibrium dialysis, bovine liver argininosuccinase of molecular weight 202,000 binds 4 mol of argininosuccinate or arginine/mol of enzyme. Negative homotropic interactions occur in the binding of both ligands at 0.15 ionic strength in the presence of phosphate. Argininosuccinate binds to two sites (Kdiss 1.6 times 10(-5) M) and four sites (Kdiss 2.9 times 10(-4) M) at low and high substrate concentration. Similarly, arginine binds to two sites (Kdiss 4.9 times 10(-4) M), and four sites (Kdiss 1.6 times 10(-3) M). At 0.05 ionic strength in Tris-HCl buffer, the four enzyme sites bind argininosuccinate independently and arginine binding remains negatively cooperative. Kinetic analysis gave double reciprocal plots that showed negative cooperatively also. The changes in Km were analogous to changes in Kdiss, thus indicating that the substrate binding sites correspond to catalytic sites. Since the catalytically active enzyme is a tetramer composed of four identical or closely similar subunits (Lusty, C.J., and Ratner, S. (1972) J. Biol. Chem. 247, 7010-7022), the present results show that each subunit contains one catalytic site. Ionic strength, phosphate ions, and GTP have each been found to influence negative cooperatively through a change in the affinity for argininosuccinate. The significance of the negative homotropic interactions and of the specific stimulation of activity by GTP is discussed with respect to different conformational forms of the enzyme and the in vivo regulation of argininosuccinase activity.