Construction of a Sonchus Yellow Net Virus Minireplicon: a Step toward Reverse Genetic Analysis of Plant Negative-Strand RNA Viruses

Abstract

e Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.A revolution in understanding of RNA viruses was initiated more than 25 years ago by the discovery that plus-strand viral RNA genomes could be reverse transcribed into cDNAs, and that purified plasmids containing these cDNAs could be faithfully transcribed in vitro to yield infectious "genomic" RNAs (gRNAs) (1). This finding permitted genetic manipulation of cDNAs and recovery of mutant RNAs whose effects on virus biology and pathology could be assessed. However, direct application of this strategy to negative-strand RNA (NSR) viruses was not possible because the minimal infectious unit of NSR viruses is the viral nucleocapsid (vNC), rather than naked viral gRNA (2, 3). Moreover, the development of methods for reconstitution of nucleocapsids (NCs) from cloned NSR cDNAs proved to be an enormous challenge that required nearly a decade to resolve (4, 5).Generation of biologically active NCs was eventually solved with animal NSR viruses by cotransfecting permissive cell lines with plasmids designed to transcribe positive-strand or "antigenomic" RNA (agRNA) derivatives, along with plasmids able to express the three NC core proteins (6,7,8,9). These approaches resulted in in vivo assembly of NCs that were able to replicate and jump-start the replication process. In seminal experiments to recover full-length recombinant Rabies virus and Vesicular stomatitis virus (VSV), ectopic expression of viral core pr...

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

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Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

“…More detailed electron microscopy studies and a reverse genetics system based on further developments of plant rhabdovirus minireplicons (Ganesan et al, 2013) will be needed to confirm the site(s) of ADV replication and morphogenesis.…”

The family Rhabdoviridae: mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins

“…The recent development of the first-ever reverse genetics system for a plant rhabdovirus provided a major technical breakthrough and a guide for the future study of other plant negative-sense ssRNA viruses (Ganesan et al, 2013; Wang et al, 2015; Jackson and Li, 2016). It allowed the recovery of infectious recombinant virus (rSYNV) from agroinfiltrated plants and the generation of rSYNV stably expressing a green fluorescent protein (GFP) reporter.…”