Rift Valley fever virus: Entry mechanisms revealed

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, hampering the investigation of RVFV entry mechanisms. Expression of the RVFV glycoproteins on the surface of cells allowed for the development of a cell-cell fusion assay. Using this assay, we demonstrated that RVFV induces syncytia formation when treated with mildly acidic media (below pH 6.2). Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrates) on the host cell surface are needed to support membrane fusion. To investigate the potential cellular factors necessary for RVFV entry, we developed a high-throughput small molecule inhibitor screen in both mammalian and insect cells. We identified 61 drugs that inhibit RVFV infection, of which 14 inhibit RVFV replication in both human and insect cells. Amongst the inhibitors that block in both hosts is a subset that inhibits macropinocytosis, including known inhibitors of Protein Kinase C signaling. Further studies identified the novel PKC isozyme epsilon (PKCϵ) as required for infection in both human and insect cells. Altogether, these data show that RVFV requires a host cell protein and acidic pH to induce fusion, and may utilize PKCϵ signaling and macropinocytosis to migrate to an acidic cellular compartment for productive infection. This information may represent a starting point for the development of anti-RVFV therapeutics.