Efficient production of Rift Valley fever virus-like particles: The antiviral protein MxA can inhibit primary transcription of bunyaviruses

Abstract

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.

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for G N -G C Glycoprotein Heterodimers

“…UUKV glycoproteins are sufficient to form empty virus-like particles without the nucleocapsid protein (31). In the case of RVFV, glycoproteins alone are not sufficient for generating virus-like particles, but nucleocapsid protein, and potentially also RNA, are required (16). While the interaction between the RVFV N protein and RNA has not been investigated, in members of the genus Orthobunyavirus, the N protein is known to bind viral RNA, cRNA, and, to a lesser extent, also nonspecific cellular RNA (26,27).…”

Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

“…24 h post-transfection cells were infected with RVFV-like particles coding for a firefly luciferase reporter (46). 24 h post-infection cells were lysed and firefly luciferase activity was measured using the SingleLuciferase® Reporter Assay (Promega).…”

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

“…Plasmids expressing HB29-derived minigenomes were created, similar to those previously described for RVFV (37)(38)(39). The M-segmentbased construct contains the humanized Renilla (hRen) gene ORF sequence in negative sense flanked by full-length genomic sense untranslated region (UTR) sequences in TVT7R (0, 0) and was called pTVT7-HB29M:hRen.…”