Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids

The association of newly synthesized vesicular stomatitis virus proteins into nucleocapsid structures was examined in a cell-free system that supports concurrent viral protein synthesis, transcription, and RNA replication. The vesicular stomatitis virus proteins synthesized by this system associated with the newly replicated RNA to form structures that banded in CsCl gradients with marker nucleocapsids. In reactions lacking nucleocapsid templates to program RNA synthesis, the newly synthesized proteins did not associate into nucleocapsid structures. The newly synthesized proteins associated with nucleocapsids were analyzed by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate after separation from non-associated proteins by chromatography on Bio-Gel A15M agarose columns. The results of this analysis showed that newly synthesized L, NS, and N proteins associated into nucleocapsids in the in vitro system. In addition, a small amount of newly synthesized M protein was stably bound to the nucleocapsids. The molar ratio of the associated, newly synthesized proteins was 2:350:1,000:10 (L:NS:N:M). More than 90% of the newly synthesized NS protein that associated with nucleocapsids in vitro was of the NS2 subspecies, as assayed by DEAE-cellulose column chromatography. The stability of the association of the newly synthesized proteins with nucleocapsids in the system mimicked that of the association of viral proteins with nucleocapsids from infected cells as measured by salt sensitivity. These data indicate that nucleocapsids were assembled from newly synthesized proteins within our in vitro system and that the molar ratio of assembled proteins was similar to that observed for virion nucleocapsids.