Dissociation and Reconstitution of the Transcriptase and Template Activities of Vesicular Stomatitis B and T Virions

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.

Mechanism of Sindbis Virus-Induced Intrinsic Interference with Vesicular Stomatitis Virus Replication

“…Sindbis virus-induced interference does not appear to affect host cell activation of the VSV transcriptase. Although the mechanism of activation of this enzyme is not understood, it probably involves dissolution of' the membrane enveloping the VSV nucleocapsid, since in vitro activation is brought about with carefully controlled detergent concentrations (2) and the transcriptase activity has been shown to purify with the proteins of the nucleocapsid rather than with those of the envelope (4,8,27).…”

Global analysis of polysome-associated mRNA in vesicular stomatitis virus infected cells

“…As VSV RNA synthesis occurs entirely within the cytoplasm, viral RNA synthesis is not subject to the inhibitory effects of M on RNA polymerase II and mRNA export from the nucleus. The VSV RNA synthesis machinery comprises a ribonucleoprotein complex of the negative-sense genomic RNA completely encased by a nucleocapsid protein (N) sheath and associated with the viral polymerase complex [13]. The viral transcriptase copies the N-RNA template into 5 monocistronic mRNAs that are structurally indistinct to those of the host-cell with respect to their 5 0 cap and 3 0 polyadenylate tail [14][15][16][17][18][19][20].…”

Recombinant Isfahan Virus and Vesicular Stomatitis Virus Vaccine Vectors Provide Durable, Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge

“…The VSV particles are bullet-shaped (ϳ80 nm by ϳ200 nm) and comprise a ribonucleoprotein core of genomic RNA and N (nucleocapsid) protein surrounded by a matrix (M) protein layer, enveloped by the host cell plasma membrane containing the transmembrane viral glycoprotein (G) (12). The phosphoprotein (P) and large (L) polymerase protein associate to form the functional viral RNA polymerase, which performs both mRNA transcription and genome replication from the nucleocapsid template (13,14). Viral mRNA transcription initiates at the 3= end of the genome and proceeds with a pronounced 3=-to-5= gradient of gene expression, leading to abundant N protein and successively decreasing levels of P, M, G, and L proteins in the infected cell, providing virus proteins an optimal ratio for subsequent viral genome replication and the assembly of mature virus particles (11,15).…”