Cucumber mosaic virus (CMV)
2b protein
plays a key role in the process of CMV infecting plants and symptom
formation and is a potential molecular target for the control of this
important plant virus. The exploitation of antiviral compounds is
one of the strategies with the highest input: output ratio in plant
protection. In this study, the CMV 2b recombinant protein was cloned,
purified, and identified as the target protein by mass spectrometry.
Subsequently, we carried out preliminary functional screening of the
LP series of myricetin derivatives designed and synthesized in our
laboratory and commercial antiviral compounds by microscale thermophoresis
(MST), which showed that LP compounds LP4, LP11, LP13, and LP20 interacted well with CMV
2b, with dissociation constant (K
d) values
of 1.39, 0.88, 1.52, and 1.77 μM, respectively. Among the commercially
available antiviral compounds, ningnanmycin (NNM) was the most active,
with a K
d value of 4.09 μM. Then,
the strongest binding force to CMV 2b was identified to be from LP11 by isothermal titration calorimetry (ITC) experiments,
with a K
d of 1.19 μM. Among the
commercial compounds, NNM had the strongest binding force with CMV
2b, with a K
d of 4.62 μM. Through
the screening of commercial compounds and LP series compounds by MST
and ITC, LP11, NNM (positive control), LP16 (negative control), and the blank control group were selected to
test the in vivo impact of LP11 on CMV. Specifically,
the screened compounds were sprayed onto CMV-inoculated Nicotiana benthamiana plants to determine their impact
on the regulation of CMV pathogenic gene expression, symptoms, and
virus titer. The results showed that LP11 had a strong
ability to inhibit CMV infection of tobacco at the transcriptional
and translational levels. By mutating the CMV 2b protein, the 15th
amino acid leucine and the 18th amino acid methionine at the N-terminal
region were shown to be potential sites for binding to compound LP11. This finding provided a theoretical basis for screening
and developing anti-CMV agents.