A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication

Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication

Efficient Cellular Release of Rift Valley Fever Virus Requires Genomic RNA

The Rift Valley fever virus is responsible for periodic, explosive epizootics throughout sub-Saharan Africa. The development of therapeutics targeting this virus is difficult due to a limited understanding of the viral replicative cycle. Utilizing a virus-like particle system, we have established roles for each of the viral structural components in assembly, release, and virus infectivity. The envelope glycoprotein, Gn, was discovered to be necessary and sufficient for packaging of the genome, nucleocapsid protein and the RNA-dependent RNA polymerase into virus particles. Additionally, packaging of the genome was found to be necessary for the efficient release of particles, revealing a novel mechanism for the efficient generation of infectious virus. Our results identify possible conserved targets for development of anti-phlebovirus therapies

Characterization of the Golgi Retention Motif of Rift Valley Fever Virus G(N) Glycoprotein

As Rift Valley fever (RVF) virus, and probably all members of the family Bunyaviridae, matures in the Golgi apparatus, the targeting of the virus glycoproteins to the Golgi apparatus plays a pivotal role in the virus replication cycle. No consensus Golgi localization motif appears to be shared among the glycoproteins of these viruses. The viruses of the family Bunyaviridae synthesize their glycoproteins, G(N) and G(C), as a polyprotein. The Golgi localization signal of RVF virus has been shown to reside within the G(N) protein by use of a plasmid-based transient expression system to synthesize individual G(N) and G(C) proteins. While the distribution of individually expressed G(N) significantly overlaps with cellular Golgi proteins such as β-COP and GS-28, G(C) expressed in the absence of G(N) localizes to the endoplasmic reticulum. Further analysis of expressed G(N) truncated proteins and green fluorescent protein/G(N) chimeric proteins demonstrated that the RVF virus Golgi localization signal mapped to a 48-amino-acid region of G(N) encompassing the 20-amino-acid transmembrane domain and the adjacent 28 amino acids of the cytosolic tail

VPS21 Controls Entry of Endocytosed and Biosynthetic Proteins into the Yeast Prevacuolar Compartment

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment

Encapsidated genome required for infectivity.

<p>*Value is significantly different from -Gn/-Gc, p<0.001.</p

Gn recruits RdRp.

<p><b>A.</b> BSR-T7/5 cells were transfected with pRdRp or pGn, and the proteins were visualized with anti-RdRp and anti-Gn, respectively (green channel). Cellular resident Golgi apparatus proteins, GS-28 or β-COP were also labeled (red channel). Percentage of cells displaying co-localization of viral proteins with resident Golgi proteins is indicated with the number of cells counted in parentheses. <b>B.</b> BSR-T7/5 cells were transfected with pRdRp and either pGn/pGc, pGc, pGn, or pGnK48. Cells were incubated with anti-RdRp (green channel) and anti-Gn or anti-Gc (red channel), and then analyzed by immunofluorescence microscopy. Percentage of cells displaying co-localization of RdRp with Gn or Gc is indicated with the number of cells counted in parentheses.</p

Viral components required for efficient RVF-VLP release.

<p>BSR-T7/5 cells were transfected with genome and all of the structural proteins (WT), or one or more of the components was replaced with an equivalent amount of empty vector (-RNPCs, -Gn/Gc, -N, -genome, -RdRp). RNPCs refer to ribonucleoprotein complexes and are defined as genome, N, and RdRp. Transfected cells were analyzed for protein expression by immunoblot. RVF-VLPs were immune precipitated from the clarified media from transfected cells and analyzed by immunoblot. The numbers below the immunoblots indicate the ratio of glycoprotein to N signal.</p

Packaged, catalytically active RdRp is necessary for an early event in the replicative cycle.

<p><b>A.</b> BSR-T7/5 cells were transfected with genome and all of the structural proteins (WT), or one or more of the components was replaced with an equivalent amount of empty vector (-Gn/Gc and –RdRp) or with plasmids expressing mutant alleles of Gn or RdRp (GnK48 or pRdRp<i>^cat1</i>). Transfected cells were analyzed for protein expression by immunoblot. RVF-VLPs were immune precipitated from the clarified media from transfected cells and analyzed by immunoblot. The numbers below the immunoblots indicate the ratio of glycoprotein to N signal. <b>B.</b> BSR-T7/5 cells were transfected with pGn and either pRdRp or RdRp catalytic domain mutants, pRdRp<i>^cat1</i> or pRdRp<i>^cat2</i>. Cells were incubated with anti-Gn (red channel) and anti-RdRp (green channel), and then analyzed by immunofluorescence microscopy. Percentage of cells displaying co-localization of RdRp alleles with Gn is indicated with the number of cells counted in parentheses.</p

Gn packages N.

<p><b>A.</b> BSR-T7/5 cells were transfected with genome and all of the structural proteins (WT), or one or more of the components was replaced with an equivalent amount of empty vector, (-Gn/-Gc, -Gn, or -Gc) or a plasmid expressing an allele of Gc that lacks the entire cytoplasmic tail (GcW1). Transfected cells were analyzed for protein expression by immunoblot. RVF-VLPs were immune precipitated from the clarified media from transfected cells and analyzed by immunoblot. The numbers below the immunoblots indicate the ratio of glycoprotein to N signal. <b>B</b>. BSR-T7/5 cells were transfected with the indicated plasmids and proteins were cross-linked at 48 h post-transfection. Mouse monoclonal anti-Gn antibodies were used to immune-precipitate Gn containing complexes. The cross-links were cleaved and then Gn-containing complexes were identified by immunoblot using rabbit anti-Gn or rabbit anti-N antibodies.</p