Utilizing a Rift Valley fever virus-like particle system, we have determined that encapsidated genome acts as the primary stimulus for RVFV release from the cell. The driving of virus release by encapsidated genome is an elegant mechanism for LY2109761 ensuring that infectious particles are the dominant specie released from cells. We demonstrate that Gn is necessary and sufficient for packaging of the RdRp and N. Furthermore, we show that distinct regions of the Gn cytosolic tail are required for binding RdRp and N. These data provide the most complete description of RVFV assembly and release to date, and suggest novel targets of the development of anti-phlebovirus drugs. Hybridomas that secrete neutralizing monoclonal antibodies recognizing Gn and Gc were a generous gift of Dr. G. Ludwig. Polyclonal antibodies that were generated against RVFV in mice were a generous gift of Dr. P. Rollin. The N-terminal 150 amino acids of the RdRp and full-length N were expressed with N-terminal histidine tags and purified under denaturing conditions on Ni-NTA agarose columns. RdRp and N polyclonal antibodies were generated in rabbits using these purified proteins as antigens. Monoclonal antibodies recognizing GS-28 and b-COP were purchased from Transduction Labs and ABR, respectively. Horseradish peroxidase-conjugated secondary antibodies, goat anti-rabbit and goat anti-mouse, were acquired from GSK2118436 1195765-45-7 Amersham and MP Biomedical, respectively. AlexaFluor 488-labelled goat anti-rabbit and AlexaFluor 594-labelled goat anti-mouse were purchased from Invitrogen. Efficiency of cellular release was determined through quantitation of Gn/Gc levels in the cell lysates and within the RVF-VLPs. RVF-VLPs were purified through high-speed ultracentrifugation or immune precipitation. Both methods generated similar results for the release efficiencies, therefore immunoblots from both types of purification were combined to calculate the average release efficiencies with standard deviation and to perform the statistics. Immunoblots were scanned on a PhosphoImager and analyzed using ImageQuant 5.2 to determine the signal intensity. Glycoprotein signal volume from the cell lysates was divided by background volume to attain the normalized glycoprotein expression levels in the cell lysates. The glycoprotein signal volume for RVF-VLPs was divided by the normalized glycoprotein signal from the corresponding cell lysate. Normalizing the glycoprotein signal for RVF-VLPs had little to no effect on the calculated release efficiencies for conditions lacking genome, N, RdRp or with the RdRpcat1 allele since glycoprotein expression levels were similar across these conditions. Replication and transcription of the viral genome by RdRp occurs in the cytoplasm and assembly of virus particles takes place at the Golgi apparatus.