Plant viruses, discovered over a century ago, cause significant agricultural losses. While significant advancements have been made in virology and molecular biology through the study of plant viruses (e.g., the discovery that viral RNA itself is infectious, and the elucidation of RNA interference as an antiviral defense), effective antiviral drugs for treating already infected plants are lacking. Current prevention methods rely on disease-resistant cultivars and insecticides targeting insect vectors. Understanding the structure and function of plant virus replication machinery, particularly RNA-dependent RNA polymerases (RdRps), is crucial for developing effective antiviral treatments. RdRps from Articulavirales and Bunyavirales, including influenza, Crimean-Congo hemorrhagic fever, Rift Valley fever, and Lassa viruses, have been extensively studied to inform antiviral drug design. However, despite the existence of over 2,100 plant virus species, the structure of their replication machinery remained largely unknown, hindering the development of effective plant antiviral pesticides. Tomato spotted wilt orthotospovirus (TSWV), a globally impactful negative-sense RNA virus, presents an ideal model due to its large RdRp (L protein, ~330 kDa), significantly larger than most animal-infecting viral polymerases. This study aimed to resolve the cryo-EM structures of the full-length TSWV RdRp to elucidate its activation mechanism and identify potential targets for antiviral drugs.

The study extensively reviews existing literature on polymerase structures and replication mechanisms of human and animal viruses. The RNA-dependent RNA polymerases (RdRps) from the orders Articulavirales and Bunyavirales have received particular attention due to the epidemic potential of some of their members and the absence of effective antivirals or vaccines. Previous studies have provided structures for RdRps of several viruses, including influenza virus, La Crosse virus (LACV), Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Hantaan virus (HTNV), Sin Nombre virus (SNV), Lassa virus (LASV), and Machupo virus (MACV), some also showing the structures in the presence of their cognate regulatory Z proteins. This work contrasts with the lack of structural information for any plant virus replication machinery, despite the significant economic impact of plant viruses.

The researchers successfully expressed and purified the full-length TSWV L protein in High Five insect cells. Cryo-EM structures were determined for both apo L (without RNA) and RNA-bound L (complex with viral genomic RNA). The apo-L structure, resolving the polymerase core at 3.9 Å resolution, lacked density for the N-terminal and C-terminal domains (CTD) due to flexibility. Addition of the vRNA promoter (5′ and 3′ vRNA, 1-17 nucleotides) improved the resolution to 3.7 Å and ordered the endonuclease region. A structure with the CTD was also resolved (4.0 Å resolution) upon addition of 10 nucleotides of the 5′ vRNA. The TSWV L structure was divided into three regions analogous to influenza virus polymerase subunits: N-terminal PA-like, central PB1-like (RdRp), and C-terminal PB2-like regions. Despite sequence and molecular weight differences, the overall architecture showed similarity to other sNSVs polymerases. The endonuclease domain was larger than in other polymerases and showed structural similarity to an iron-binding protein. The CTD was considerably larger than in other sNSVs, comprising three distinct domains: cap-binding, middle, and C-terminal extension. The study further investigated the conformational changes upon vRNA promoter binding, revealing significant rearrangements, including the movement of subdomains leading to a more compact core and ordered endonuclease. The RNA binding regions, fingertips (motif F), and the priming loop, disordered in apo-L, were stabilized in the RNA-bound state. The researchers also determined the structure of the complex with the 5′ vRNA (1-10 nts), showing that the hook structure alone is sufficient to activate the L protein. Site-directed mutagenesis was performed on the TSWV infectious clone to study the functional impact of key residues and RNA elements identified in the structural analysis, particularly Lys1291 in motif F and cytosine at position 5 in the 5' hook. Three antiviral drugs (remdesivir, ribavirin, favipiravir) were screened for their inhibitory effects on TSWV replication using infectious clones, with ribavirin demonstrating strong efficacy. The effect of ribavirin on a normal viral infection was confirmed in planta, showcasing ribavirin's ability to inhibit TSWV symptoms and viral presence. Cryo-EM structures of TSWV L complexes with ribavirin and ribavirin triphosphate were determined, revealing dual binding sites: the active site and the RNA binding site. Electrophoretic mobility shift assays (EMSAs) and in vitro RdRp activity assays were performed to confirm the inhibitory effects of ribavirin on polymerase activity.

The study's key findings include the elucidation of five cryo-EM structures of TSWV RdRp in different states, providing atomic-level details of its architecture and conformational changes upon RNA binding. Crucially, the research identified a flexible loop within motif F acting as both a 'sensor' detecting the viral RNA 'hook' structure and an 'adaptor' promoting the formation of the active catalytic center. A ten-base RNA hook structure was shown to be sufficient to activate the polymerase. The study also revealed that ribavirin, a nucleoside analog, inhibits TSWV replication by dual-targeted mechanisms, binding to both the active site and the RNA entry channel, thereby blocking both RNA recognition and the formation of a complete catalytic center. The discovered dual-targeted inhibition of TSWV RdRp by ribavirin, and specifically the interaction between Lys1291 and cytosine 5 of the RNA hook, offers promising targets for designing plant-specific antiviral drugs. The identification of a new conserved motif (I) in the active site of various sNSV polymerases expands our understanding of polymerase structure and function. The experiments using infectious clones and in planta experiments strongly support the conclusion that ribavirin inhibits viral replication by binding to these two sites. Further mutagenesis experiments validated the importance of specific amino acid residues and RNA elements in viral replication, demonstrating their essential roles in the activation and function of the polymerase.

The results directly address the research question by providing a structural mechanism for plant bunyavirus RdRp activation and its dual-targeted inhibition by ribavirin. The findings significantly advance our understanding of plant virus replication and provide novel targets for antiviral drug development. The large size of TSWV L, being the largest polymerase structure to date, indicates a possible evolutionary relationship between plant and animal sNSV RNA polymerases, with similarities in overall architecture. The study clarifies the structure of the viral promoter, showing a proximal double-stranded region alongside the hook structure. The detailed structural insights and functional analyses provide a strong foundation for the rational design of novel antiviral pesticides targeting plant viruses, particularly orthotospoviruses, by focusing on the interaction between Lys1291 and the 5’ RNA hook or the polymerase hook formation tunnel. This work addresses a critical gap in agricultural antiviral strategies, paving the way for much-needed improvements in plant disease control.

This research presents a comprehensive structural and functional analysis of the TSWV RdRp, revealing the intricate mechanisms underlying its activation and inhibition by ribavirin. The identification of dual binding sites for ribavirin offers promising targets for developing novel antiviral pesticides. The discovery of a new conserved motif further enhances our understanding of sNSV polymerases. Future research could focus on developing specific inhibitors targeting the identified interaction site (Lys1291-C5), exploring the potential of ribavirin derivatives or other nucleoside analogs, and investigating the structure and function of RdRps from other plant viruses to identify conserved features applicable to broad-spectrum antiviral drug development. The work highlights the potential of structural biology in addressing significant challenges in plant disease management.

While the study provides a detailed mechanistic understanding of TSWV RdRp, the findings may not be directly generalizable to all plant viruses. Further research is necessary to determine the extent to which the identified mechanisms are conserved across different plant virus families. The in vitro experiments and those conducted in planta should be validated using alternative methods to confirm the specificity and reproducibility of the results, further strengthening the implications of the study. The use of ribavirin is limited by current restrictions on its use as a pesticide, so alternative drugs are needed. While the study proposes several specific points of intervention, the creation of such an antiviral drug requires further development and testing.