Development of Rice Stripe Tenuivirus Minireplicon Reverse Genetics Systems Suitable for Analyses of Viral Replication and Intercellular Movement

Abstract

Rice stripe virus (RSV), a tenuivirus with four negative-sense/ambisense genome segments, is one of the most devastating viral pathogens affecting rice production in many Asian countries. Despite extensive research, our understanding of RSV infection cycles and pathogenesis has been severely impaired by the lack of reverse genetics tools. In this study, we have engineered RSV minireplicon (MR)/minigenome cassettes with reporter genes substituted for the viral open reading frames in the negative-sense RNA1 or the ambisense RNA2-4 segments. After delivery to Nicotiana benthamiana leaves via agroinfiltration, MR reporter gene expression was detected only when the codon-optimized large viral RNA polymerase protein (L) was coexpressed with the nucleocapsid (N) protein. MR activity was also critically dependent on the coexpressed viral suppressors of RNA silencing, but ectopic expression of the RSV-encoded NS3 silencing suppressor drastically decreased reporter gene expression. We also developed intercellular movement-competent MR systems with the movement protein expressed either in cis from an RNA4-based MR or in trans from a binary plasmid. Finally, we generated multicomponent replicon systems by expressing the N and L proteins directly from complementary-sense RNA1 and RNA3 derivatives, which enhanced reporter gene expression, permitted autonomous replication and intercellular movement, and reduced the number of plasmids required for delivery. In summary, this work enables reverse genetics analyses of RSV replication, transcription, and cell-to-cell movement and provides a platform for engineering more complex recombinant systems.

The Bunyavirales: The Plant-Infecting Counterparts

“…However, in contrast to RRV, rescue of TSWV relies on a codon-optimized RdRp and occurs in the presence of various viral suppressors of RNAi, but in which ectopic expression TSWV NSs seemed to interfere negatively. Very recently, a minireplicon system has been established for the RSV tenuivirus in human cells and in planta [ 75 , 277 , 278 ]. Moreover, with RSV, mini-replicon reporter gene expression was only achieved with a codon-optimized RdRp and was critically dependent on the presence of a viral suppressor of RNAi, but wherein the RSV p3/NSs drastically reduced reporter gene expression.…”

Highly adaptive Phenuiviridae with biomedical importance in multiple fields

“…p4 is a disease-specific protein whose expression is associated with the severity of RSV symptoms [56]. MP of fungiinfecting phenuiviruses and plant-infecting phenuiviruses is vital for them to spread from infected cells to neighboring cells [39,57,58] Terminal sequences of phenuivirus genomic segments are conserved and complementary, likely in a genus-specific manner [39,47,48]. For phleboviruses, they are 5-ACACAAAG……CUUUGUGU-3.…”

“…47 MP of fungi-infecting phenuiviruses and plant-infecting phenuiviruses is vital for them to spread from infected cells to neighboring cells. 34,48,49 Insertion and deletion of nucleotides occur more frequently in the intergenic regions and the 5′ or 3′ noncoding regions of RSV genomes. 50 RSV replicating in vector insects may enrich 15-and 16nt extensions in the 3′-termini of the viral RNA segments.…”

“…p4 is a disease‐specific protein whose expression is associated with the severity of RSV symptoms 47 . MP of fungi‐infecting phenuiviruses and plant‐infecting phenuiviruses is vital for them to spread from infected cells to neighboring cells 34,48,49 …”

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

“…Rescue of plant rhabdoviruses has been achieved for sonchus yellow net virus (SYNV) in Nicotiana benthamiana (Ganesan et al, 2013 ; Wang et al, 2015 ) and barley yellow striate mosaic virus (BYSMV) (Gao et al, 2019 ) and northern cereal mosaic virus (NCMV) in barley (Fang et al, 2022 ) by co‐expressing RNPs and VSRs. More recently, reverse genetics were developed for segmented NSRs, including tomato spotted wilt virus (TSWV) (Feng et al, 2020 ), rose rosette virus (RRV) (Verchot et al, 2020 ), and a mini‐replicon system for rice stripe virus (RSV) (Feng et al, 2021 ; Zhang et al, 2021 ).…”