Recently, the Functional Genomics of Crop Pathogens Research Innovation Team at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), published a study titled  Cucumber green mottle mosaic virus encodes additional small proteins with specific subcellular localizations and virulence function  in  Science China-Life Sciences  (with an impact factor of 9.463). Using cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, as a model, the study revealed novel protein-coding strategies in tobamoviruses. It further validated the hypothesis that negative-sense RNA (-RNA) of positive-sense single-stranded RNA (+ssRNA) viruses can encode functional proteins (Gong et al., Molecular Plant, 2023). 

CGMMV, classified as a nationally regulated agricultural plant quarantine pest in China, primarily infects cucurbit crops such as watermelon, cucumber, and melon, causing severe global economic losses. As a +ssRNA virus, CGMMV synthesizes negative-sense RNA (-RNA) as a replication intermediate during its lifecycle. While -RNA has traditionally been considered non-coding, emerging evidence suggests potential coding capabilities in +ssRNA viruses. However, whether Tobamovirus members employ similar strategies remained unconfirmed. 

The study analyzed multiple tobamoviruses and identified conserved small open reading frames (rORFs) on their negative-strand RNAs. Using CGMMV and tobacco mosaic virus (TMV) as models, the researchers demonstrated that these rORFs encode proteins with distinct subcellular localizations, such as peroxisomes and nucleoli. These rORFs likely utilize internal ribosome entry sites (IRES) for translation. Functional analyses revealed critical roles for these novel rORFs in viral infection. For example, CGMMV rORF1, localized to peroxisomes, interacts with viral replication-related proteins and is essential for full virulence. Structural predictions via ColabFold showed that CGMMV rORF1 interacts with peroxisomal protein PEX3, which directs rORF1 to peroxisomes to facilitate viral infection. These findings expand the known proteome of tobamoviruses and deepen understanding of +ssRNA viral coding strategies. The study also highlights peroxisomes as novel viral targets in the evolutionary arms race between viruses and plants, offering new insights for developing plant antiviral strategies.  

Postdoctoral researcher Pan Gong (now a professor at Yangtze University), master’s student Mengxin Gao, and Ph.D. graduate Yalin Chen (now an associate professor at Hebei Agricultural University) are co-first authors of the paper. Professor Xueping Zhou and Researcher Fangfang Li from CAAS are the corresponding authors. The research involved collaborations with Researcher Hui Zhang (Shanghai Academy of Agricultural Sciences), Associate Researcher Qingtang Shen (Fujian Medical University), Researcher Yong Liu (Hunan Academy of Agricultural Sciences), Professor Rosa Lozano-Duran (University of Tübingen), and Dr. Aiming Wang (Agriculture and Agri-Food Canada). The study was supported by the National Natural Science Foundation of China (32320103010, 32172385), the Postdoctoral Innovative Talent Support Program (BX20220345), and the Yunnan Provincial Science and Technology Project (202202AE090022). 

Figure 1: Identification of rORFs with protein-coding functions in the negative-strand RNA of Tobamoviruses.

Figure 2: IRES activity in upstream sequences of CGMMV rORFs.

Figure 3: CGMMV rORF-encoded proteins influence viral infection.

Figure 4: Subcellular localization of rORF-encoded proteins in CGMMV and TMV.

Figure 5: Interaction between CGMMV rORF1 and viral RNA-dependent RNA polymerase (RP-S) in peroxisomes.

Figure 6: PEX3-mediated targeting of CGMMV rORF1 to peroxisomes.

Source: https://www.sciengine.com/SCLS/doi/10.1007/s11427-024-2892-1

Related Research:  

Molecular Plant: IPPCAAS Reveals Novel Small Protein-Coding Strategy via Negative-Strand RNA in +ssRNA Viruses  

(http://www.ippcaas.cn/kyjz/123d016b30ab4085b9f2edca1163ebd9.htm)