No particles were visualized by electron microscopy when the genome, N, and RdRp, which form the viral ribonucleoprotein complex, were expressed without the envelope glycoproteins and there was no expression of the RLuc reporter above background levels in target cells. Our results corroborate the results of previous findings that ribonucleoprotein complexes are not MDV3100 released from the cell in the absence of glycoproteins. We next determined which viral components are necessary for efficient RVF-VLP release. For the purpose of this analysis we equated RVF-VLP release with Gn/Gc signal on immunoblots of isolated RVF-VLPs. Gn/Gc expression levels were measured and normalized to expression levels in transfected cells. The experimental condition that included all structural proteins and genome was designated as 100% release efficiency and the condition in which both envelope glycoproteins were omitted from the transfection was considered background. The samples lacking N or the genome exhibited average release efficiencies of only 15.6 and 18.1%, respectively. These efficiencies were similar to when the entire ribonucleoprotein complex was absent. Our results demonstrate that efficient release requires both N and the genome, presumably in the form of encapsidated genome. Conversely, the absence of RdRp did not adversely affect the efficiency of release of the glycoproteins or the packaging of N, indicating that RdRp does not play a critical role in viral budding or release. Particles can be generated at low levels when either Gn or Gc is absent, however the amount of glycoproteins released was at or below the limit of detection by immunoblot. Release efficiencies were decreased,2-fold when either GnK48 or GcW1 was expressed, however this decrease was only significant for GnK48. Since N is packaged under both conditions, the cytoplasmic tails of these glycoproteins may perform additional functions in the release process. The presence of genome in RVF-VLPs can be inferred from RLuc activity in infected cells. However, many of the experimental CYT387 conditions used in this study do not produce RLuc in target cells at levels significantly different from a negative control. Since RVFV can package both sense and anti-sense genomic RNA, the requisite packaging signals must be present on both senses of genomic RNA. Therefore cells used to make RVF-VLPs in the absence of replication contain genomic RNA that is competent for packaging. The presence or absence of both senses of genomic RNA in RVF-VLPs was assayed by RT-PCR. BSR-T7/5 cells were transfected with minigenome, pN, pRdRp, pGn, pGc or one or more of the components were replaced with an equivalent amount of empty vector, pGnK48 or pGcW1.