Many notable viruses encode a protease with deubiquitinating (DUB) activity, which is essential for viral replication and evasion of ubiquitin (Ub)-mediated host antiviral responses. The activity of viral DUBs thus promotes viral replication by protecting viral proteins from the Ub-dependent antiviral system in the host and participating directly in virus replication through polyprotein processing. Therefore, viral proteases with DUB activity are potential therapeutic targets for small-molecule antiviral inhibitor compounds. The recent development of protein-based inhibitors based on variants of Ub (known as UbVs) is promising due to the remarkable target specificity and potency of UbVs for their target DUB. Several cell culture-based studies demonstrated that cellular expression of UbVs suppresses virus replication in animal cells, including the Middle East Respiratory Syndrome Coronavirus and Severe Acute Respiratory Syndrome Coronavirus 2. However, the delivery of UbVs to the cytosol of cells as a protein-based therapeutic remains a significant challenge. Interestingly, several economically important plant species are susceptible to infection by pathogenic viruses that depend on DUB proteases, whose replication could potentially be blocked by transgenic expression of UbVs. This research explored the potential of UbVs as antiviral agents against the plant virus, Turnip Yellow Mosaic Virus (TYMV). A strong binding UbV was identified as explicitly targeting the DUB protease (PRO) and inhibiting the protease and deubiquitinase activity. In addition, Structural studies revealed the mechanism of inhibition and demonstrated the high affinity of the UbV for TYMV PRO. Also, the binding specificities of the UbVs for TYMV PRO and a panel of recombinant plant DUBs from Arabidopsis thaliana demonstrated that none of the UbVs were found to have significant affinity for any of the plant DUBs. To study further, strong binding UbV was introduced to model plant A. thaliana and the infection study was shown to reduce TYMV replication in transgenic plants, suggesting that this approach could be effective against other economically important plant viruses.