Significantly Improved Recovery of Recombinant Sonchus Yellow Net Rhabdovirus by Expressing the Negative-Strand Genomic RNA

Abstract

Generation of recombinant negative-stranded RNA viruses (NSVs) from plasmids involves in vivo reconstitution of biologically active nucleocapsids and faces a unique antisense problem where the negative-sense viral genomic RNAs can hybridize to viral messenger RNAs. To overcome this problem, a positive-sense RNA approach has been devised through expression of viral antigenomic (ag)RNA and core proteins for assembly of antigenomic nucleocapsids. Although this detour strategy works for many NSVs, the process is still inefficient. Using Sonchus yellow net rhabdovirus (SYNV) as a model; here, we develop a negative-sense genomic RNA-based approach that increased rescue efficiency by two orders of magnitude compared to the conventional agRNA approach. The system relied on suppression of double-stranded RNA induced antiviral responses by co-expression of plant viruses-encoded RNA silencing suppressors or animal viruses-encoded double-stranded RNA antagonists. With the improved approach, we were able to recover a highly attenuated SYNV mutant with a deletion in the matrix protein gene which otherwise could not be rescued via the agRNA approach. Reverse genetics analyses of the generated mutant virus provided insights into SYNV virion assembly and morphogenesis. This approach may potentially be applicable to other NSVs of plants or animals.

Rescue of the first Alphanucleorhabdovirus entirely from cloned complementary DNA: an efficient vector for systemic expression of foreign genes in maize and insect vectors

“…We anticipate the introduction of introns for increasing L gene stability can be adapted to construct other plant rhabdovirus infectious clones. In this study, MMV was rescued in N. benthamiana without VSRs, although VSRs were required for the initial rescue of other plant rhabdoviruses (SYNV, BYSMV, and NCMV) to increase the accumulation of core proteins and minireplicon derivatives (Wang et al, 2015, Gao et al, 2019, Ma & Li, 2020, Fang et al, 2022).…”

CRISPR/Cas- and Topical RNAi-Based Technologies for Crop Management and Improvement: Reviewing the Risk Assessment and Challenges Towards a More Sustainable Agriculture

“…Later, it is possible to stably express Cas and gRNA in single-stranded RNA virus negative-sense. For example, Barley yellow striate mosaic virus (BYSMV) and Sonchus yellow net virus (SYNV) were able to efficiently edit Nicotiana benthamiana , but without transgenerational effect due to the inability of these viruses to penetrate the meristematic and reproductive tissues of the plant ( Gao et al, 2019 ; Ma and Li, 2020 ). Currently, the fusion of mobile elements to the gRNA in the infection clone has belonged to the presence of the virus in meristematic tissue, consequently inducing the mutation in the progenies ( Ellison et al, 2021 ; Lei et al, 2021 ).…”

Producing Vaccines against Enveloped Viruses in Plants: Making the Impossible, Difficult

“…As one of the relatively few families of enveloped viruses naturally infecting plants, Rhabdoviruses theoretically present an interesting opportunity for cross-pollination between animal and plant virology. However, the reality so far has been that plant rhabdoviruses have not proven to be of much help in aiding the production of animal/human rhabdovirus VLPs in plants, mostly because plant rhabdoviruses themselves are notoriously low-yielding and difficult to work with [ 85 , 86 , 87 ]. In fact, there is even an example of a plant rhabdovirus G-protein ectodomain being produced in mammalian cell cultures to generate rabbit antiserum to study this virus in plants [ 88 ].…”