Frantisek Sokol

Phosphoproteins, structural components of rhabdoviruses

Abstract Polypeptides derived from purified rabies, vesicular stomatitis, and Kern Canyon viruses, which were labeled with 3H-amino acids and 32P-phosphate, were subjected to electrophoretic fractionation in order to determine which of the structural proteins is phosphorylated. In the rabies virion, the nucleocapsid protein (N protein) was found to be phosphorylated, whereas the other three virus proteins were not. N protein of free viral nucleocapsids isolated from rabies virus-infected cells was also phosphorylated. The phosphorylation of the N protein of rabies virus is confined to a relatively small terminal segment of the polypeptide chain which can be specifically cleaved off by treatment of the nucleocapsid with trypsin. In vesicular stomatitis virus the minor NS protein component is the only phosphoprotein. In Kern Canyon virus the envelope glycoprotein and the N protein are both phosphorylated, but to a lesser extent than either the N protein of rabies virus or the NS protein of vesicular stomatitis virus. All three rhabdoviruses contain a virion-bound protein kinase. Free nucleocapsids derived from rabies virus-infected cells and purified by equilibrium centrifugation in CsCl solution also contain a protein kinase. Intracellular nucleocapsids of vesicular stomatitis or Kern Canyon viruses do not exhibit protein kinase activity.

Biochemical and biophysical studies on the nucleocapsid and on the RNA of rabies virus.

Abstract Nucleocapsids containing the viral ribonucleic acid (RNA) were isolated from deoxycholate (DOC)-disrupted rabies virions by rate zonal centrifugation in sucrose gradient followed by equilibrium centrifugation in CsCl gradient. The sedimentation coefficient of the nucleocapsid is approximately 200 S and its buoyant density in CsCl is 1.32. The nucleocapsid is composed of 96% protein and 4% RNA. It is a single-stranded, right-handed helix. The helix is about 1.0 μ long, 100 A in diameter, and has a periodicity of 75 A. The helical structure of the nucleocapsid is extremely labile. The uncoiled strand of the helix is about 4.2 μ long, and its width varies from 20 to 65 A depending on the angle of viewing. The nucleocapsid exhibits complement fixing, but not hemagglutinating (HA), activity. It is noninfectious. The viral RNA isolated from virions or nucleocapsids is single-stranded and has a sedimentation coefficient of 45 S. The envelope of the virion exhibiting HA activity is disintegrated by DOC into slowly sedimenting subunits. They possess hemagglutination-inhibition activity, but not HA activity.

Structural phosphoproteins associated with ten rhabdoviruses.

Summary The phosphorylation of structural proteins of ten rhabdoviruses has been studied. Each virus preparation has been found to possess at least one phosphoprotein which, in the case of Chandipura, Cocal, Piry viruses or vesicular stomatitis virus (VSV), Indiana and New Jersey serotypes, is the minor NS protein. In rabies virus the sole phosphoprotein appears to be the N protein of the nucleocapsid. For Mokola and Lagos bat viruses, two or three phosphoproteins have been observed, one of which is the N protein. Spring viraemia of carp virus (SVCV) preparations contain two phosphoproteins one of which is, or has a mobility close to, that of the N protein. Kern Canyon virus preparations also possess two phosphoproteins, one with the same electrophoretic mobility as (and possible identity to), the virus glycoprotein while the other has a mobility slightly slower than the N protein. Antigenically the core proteins of rabies, Mokola and Lagos bat viruses have been shown to be related to each other but distinct from those of SVCV or VSV.

Heterogeneity in the phospholipid content of purified rabies virus (ERA strain) particles.

Summary When rabies virus (era strain) was purified by precipitation with zinc acetate, Sephadex filtration, and centrifuging in a sucrose density gradient, usually about half of the virus particles spontaneously lost a portion of their envelope phospholipids. Because of differences in buoyant density, partially delipidized virus particles (about 1.16 g./cm.3) can be separated from intact virus (about 1.14 g./cm.3) by centrifuging in a sucrose density gradient. High egg passage virus (flury strain) purified by the same procedure, did not exhibit this type of heterogeneity. The release of phospholipids from era strain virus particles did not cause a marked decrease in the infectivity of the virus, but the morphology of the virus envelope changed from bullet- to bag-shaped. The protein (glycoprotein) composition and the RNA contents of intact and partially delipidized virus forms were similar.