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...

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

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