Co-Infection of Cucumber Mosaic Virus Stabilized Its Recombinant Coat Protein Expressed by Zucchini Yellow Mosaic Virus

Background: Plant viral proteins can be part of the virus particle or its components. Some viral proteins are detectable only under specific conditions, such as during co-infection with another virus, which may lead to their stabilization. Understanding the mechanisms behind protein stabilization could help develop effective strategies for managing viral diseases. Objective: This study aimed to investigate why the recombinant coat protein (CP) of cucumber mosaic virus (CMV) is undetectable when expressed by zucchini yellow mosaic virus (ZYMV) but becomes highly detectable during mixed infections of both viruses. Materials and Methods: Three open reading frames (ORFs) of the CMV genome-encoding the coat protein (CP), movement protein (MP), and 2b protein-were amplified by RT-PCR, cloned into a ZYMV-based vector, and expressed in squash plants (Cucurbita pepo L.) to determine the stabilization mechanism of CMV-CP. Squash plants were inoculated with the recombinant viruses alone or in combination with wild-type CMV. Immunoblotting and indirect enzyme-linked immunosorbent assays (ELISA) were used to quantitatively detect ZYMV-expressed proteins, CMV CP, and ZYMV CP in infected plants at 3, 6, 9, and 12 days post-inoculation (dpi). Fresh leaf tissues harvested at 12 dpi were analyzed using immuno-gold electron microscopy (IGEM). Results: The expression of CMV CP by the ZYMV vector altered symptom development but was undetectable by immunoassays and immunoblotting in all treatments except in the presence of wild-type CMV. Immuno-gold electron microscopy revealed that the recombinant CPs were incorporated into virus particles, suggesting that their stabilization occurred through binding to RNA. Conclusion: Our results indicate that CMV-MP and-2b do not contribute to CP stabilization. Instead, we propose that the recombinant CP is stabilized by participating in virus particle assembly, as it is an RNA-binding protein. The IGEM results, showing gold particles attached to CMV particles, confirm that the recombinant CMV-CPs bind to RNA and integrate into CMV particles, leading to their stabilization.