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Rift Valley fever virus (RVFV) is a highly pathogenic member of the family Bunyaviridae that needs to be handled under biosafety level (BSL) 3 conditions. Here, we describe reverse genetics systems to measure RVFV polymerase activity in mammalian cells and to generate virus-like particles (VLPs). Recombinant polymerase (L) and nucleocapsid protein (N), expressed together with a minireplicon RNA, formed transcriptionally active nucleocapsids. These could be packaged into VLPs by additional expression of viral glycoproteins. The VLPs resembled authentic virus particles and were able to infect new cells. After infection, VLP-associated nucleocapsids autonomously performed primary transcription, and co-expression of L and N in VLP-infected cells allowed subsequent replication and secondary transcription. Bunyaviruses are potently inhibited by a human interferon-induced protein, MxA. However, the affected step in the infection cycle is not entirely characterized. Using the VLP system, we demonstrate that MxA inhibits both primary and secondary transcriptions of RVFV. A set of infection assays distinguishing between virus attachment, entry, and subsequent RNA synthesis confirmed that MxA is able to target immediate early RNA synthesis of incoming RVFV particles. Thus, our reverse genetics systems are useful for dissecting individual steps of RVFV infection under non-BSL3 conditions.

Original publication

DOI

10.1016/j.virol.2008.12.011

Type

Journal article

Journal

Virology

Publication Date

15/03/2009

Volume

385

Pages

400 - 408

Keywords

Animals, Antibodies, Monoclonal, Antiviral Agents, Cell Line, Cercopithecus aethiops, GTP-Binding Proteins, Gene Expression Regulation, Viral, Humans, Mice, Myxovirus Resistance Proteins, Recombinant Proteins, Rift Valley fever virus, Vero Cells, Virion, Virology