No abstract

Transcriptase activity was dissociated from vesicular stomatitis virions by highionic-strength buffer containing Triton X-100. Considerable enzyme activity could be restored by recombining inactive sedimentable and nonsedimentable virion fractions. Reconstituted transcriptase activity was dependent on the presence of all four nucleoside triphosphates and the concentration of heat-labile molecules in both supernatant and pellet fractions. Lower NaCl concentrations removed-46% of virion protein, but did not release transcriptase activity from the pellet fraction, nor could incorporation of 3H-uridine-5'-triphosphate by complete virions be increased by adding soluble transcriptase. Evidence that the virion nucleocapsid is the transcription template was provided by finding that the pellet contained predominantly virion core nucleoprotein, ribonucleic acid, and homogeneous nucleocapsid coils when viewed by electron microscopy. Removal of envelope G and M proteins by Triton and low-salt buffer without decreasing nucleocapsid polymerase activity indicates that neither G nor M protein is necessary for transcription. Additional data are required to determine whether the minor nucleocapsid proteins L or NSl, or both, which are at least partially solubilized in high-salt buffer, are the transcriptase. Preliminary data suggest that the major N nucleoprotein, which was not solubilized by high-salt buffer, is also required for transcription. Defective T virions contained at least as much transcriptase per weight as did B virions, as determined by restoration with T supernatant fluids of transcription function to B nucleocapsid template. However, the T nucleocapsid would not serve as template for B or T transcriptase, a finding which is interpreted as evidence of T template defectiveness. The presence of defective T nucleocapsids did not interfere with B or T transcriptase function reconstituted with B template.

An endogenous transcriptase inhibitor active at high concentrations of vesicular stomatitis (VS) virus was present in trypsinized whole virions but was absent from ribonucleoprotein cores containing only the L, N, and NS proteins. Poly(Lglutamic acid) effectively reversed the transcriptase inhibition. Transcription under noninhibited, inhibited, and poly(L-glutamic acid)-reversed conditions did not appear to greatly affect the nature of the RNA transcription product. The VS virion matrix (M) protein was purified to >98% homogeneity and was found to have an isoelectric point of-9.0. Purified M protein inhibited transcription by ribonucleoprotein cores, an effect that was partially reversed by poly(L-glutamic acid). Two group III temperature-sensitive (ts) mutants of VS virus (tsO23 and tsG31) with lesions in the M protein exhibited little or no endogenous inhibitor activity compared with two wild-type strains and a group V mutant (ts045) with a lesion in the G protein. The data presented strongly suggest that the virion M protein is responsible for the endogenous inhibition of in vitro RNA synthesis seen at high concentrations of VS virus.

composed of a single strand of ribonucleic acid (RNA) and a single peptide of molecular weight 59,500, hereby designated protein N.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.