Viperin-like proteins interfere with RNA viruses in plants

Abstract

Plant viruses cause substantial losses in crop yield and quality; therefore, devising new, robust strategies to counter viral infections has important implications for agriculture. Virus inhibitory protein endoplasmic reticulum-associated interferon-inducible (Viperin) proteins are conserved antiviral proteins. Here, we identified a set of Viperin and Viperin-like proteins from multiple species and tested whether they could interfere with RNA viruses in planta. Our data from transient and stable overexpression of these proteins in Nicotiana benthamiana reveal varying levels of interference against the RNA viruses tobacco mosaic virus (TMV), turnip mosaic virus (TuMV), and potato virus x (PVX). Harnessing the potential of these proteins represents a novel avenue in plant antiviral approaches, offering a broader and more effective spectrum for application in plant biotechnology and agriculture. Identifying these proteins opens new avenues for engineering a broad range of resistance to protect crop plants against viral pathogens.

Keywords: CNX2; MoaA; RNA virus interference; molybdenum cofactor synthesis 1A (MOCS1A); viperin; viperin-like proteins.

Figure 1

Exploration of potential antiviral proteins for RNA virus interference in planta. In the upper panel, an online search unveils reported and predicted VIPERINS and their homologs. The middle panel list potential antiviral proteins and their homologs, identified through a detailed blast search. To optimize coding sequences for expression in eukaryotic systems, they were cloned into a binary vector under the control of a constitutive promoter. Subsequently, the binary vector was utilized to generate stable N. benthamiana lines overexpressing the potential antiviral proteins through tissue culturing. Plants challenged with virus were analyzed under UV light in the dark. Leaf samples were collected and the whole transcriptomic analysis was performed and the data was analyzed.

Figure 2

Assessment of diverse antiviral proteins for viral interference in planta (A) Schematic representation of the screening process of the selected antiviral proteins from different species. All reagents (pJL-TRBO plus Viperine) were delivered agrobacterium-mediated transiently into N.benthamiana leaves. Plants were checked for any green fluorescence three days after infiltration (3dai) under UV light, and photographs were taken in the dark with a Nikon digital camera. (B) GFP monitoring to assess the interference activity of the selected antiviral proteins in Agro-infiltrated wild-type N. benthamiana leaves in transient assays. Images were taken 3 days post-infiltration. EV represents an empty vector. (C) Western blot analysis of the abundance of the virus-expressed GFP protein to confirm the potentially identified antiviral proteins viral interference. Protein blots were developed with anti-HA antibody to detect viperin (upper panel) and anti-GFP antibody (lower panel) to detect the viral abundance due to viperine activity. The gel shows viperine proteins from 1 to 5. (D) The gel image shows the antiviral activity of potential viperin proteins from 6 to 12. The rest of the description remains the same as panel (C). (E) Graphical representation of the quantification of the GFP abundance for protein 1 to 5. The intensity of the GFP-band was measured using image J and the values were plotted in Prism. Error bars indicate SEM (n = 3). (F) Graphical representation of the quantification of the GFP abundance for protein 1 to 6. The intensity of the GFP-band was measured using image J, and the values were plotted in Prism. Error bars indicate SEM (n = 3).

Figure 3

Antiviral role of molybdenum cofactor biosynthetic enzyme from different species against TuMV-GFP in N. benthamiana plants stable lines. (A) Radicle SAM proteins exhibits interference against TuMV-GFP in transgenic tobacco lines. N. benthamiana lines stably expressing Viperin-like proteins were agroinfiltrated with TuMV-GFP virus. Plants were imaged for GFP fluorescence to examine TuMV-GFP systematic spread under UV light in the dark 5-7 dai. The N. benthamiana plants Nb.MOCS1A-Hs-OE overexpresses MOCS1A from Homo sapiens, Nb.MoaA-Zm-OE overexpresses MoaA from Zostera marina and Nb.MoaA-Ds-OE overexpresses MoaA from Dunaliella salina. (B) RT-qPCR analysis to quantify TuMV-GFP RNA in MOCS1A (Homo sapiens) overexpressing plants. For each protein, the interference efficiency is shown compared to the controls. (C) RT-qPCR analysis to quantify TuMV-GFP RNA in MoaA (Zostera marina) overexpressing plants. For each protein, the interference efficiency is shown compared to the controls. (D) RT-qPCR analysis to quantify TuMV-GFP RNA in MoaA (Dunaliella salina) overexpressing plants. For each protein, the interference efficiency is shown compared to the controls. The Student’s t-test analysis indicated a significant difference compared with the WT (*P<0.05). Values are the means of three biological repeats.

Figure 4

Antiviral role of molybdenum cofactor biosynthetic enzyme from different species against PVX-GFP in N. benthamiana plants stable lines. (A) Interference of PVX-GFP by radical SAM proteins expressed in tobacco transgenic lines. Stably transformed N. benthamiana lines expressing Viperin-like proteins were agroinfiltrated with two viral titer (OD 0.03 and 0.05) of PVX-GFP virus. Plants were imaged for GFP fluorescence to examine PVX-GFP systematic spread under UV light in the dark 5 to 7 days post infiltration. The N. benthamiana plants Nb.CNX2-Ns-OE overexpresses CNX2 from Nicotiana Sylvesteris, Nb.MOCS1A-Hs-OE overexpresses MOCS1A from Homo sapiens, and Nb.MoaA-Zm-OE overexpresses MoaA from Xanothomonas oryzae. (B) RT-qPCR analysis to quantify PVX-GFP RNA in cPMP-synthase overexpressing N.benthamiana plants. For each protein, the interference efficiency is shown compared to the controls. (C) RT-qPCR analysis to quantify PVX-GFP RNA in MOCS1 overexpressing N.benthamiana plants. For each protein, the interference efficiency is shown compared to the controls. (D) RT-qPCR analysis to quantify PVX-GFP RNA in MoaA overexpressing N.benthamiana plants. For each protein, the interference efficiency is shown compared to the controls. The Student’s t-test analysis indicated a significant difference compared with the WT (*P<0.05, **P<0.001, ***P<0.0009). Values are the means of three biological repeats.