Functional Characterization Of Turnip Mosaic Virus P3 And 6K1 Proteins

Viruses in the genus Potyvirus（potyviruses）cause severe diseases on many crops that result in significant economic losses worldwide.The genome of potyviruses consists of a positive-sense,single-stranded RNA,which encodes two polypeptides that is proteolyzed by three viral proteinases,namely,P1,HC-Pro,and Pro,into 11 mature proteins.Among the eleven mature proteins,P3N-PIPO（the N-terminal half of P3 fused to Potyvirus ORF）shares the same N-terminal domain with P3 but a different C-terminal domain.The P3 and 6K1 play important roles in the replication and movement of potyviruses:P3 participates viral replication by colocalizing with NIb in 6K2-induced replication vesicles and 6K1 is essential for the survival of potyviruses;however,lots of important questions are still needed to be answered,e.g.,how P3 participates viral movement,the exact biological function of 6K1.In this study,analyze the functions of P3 and 6K1 using Tu MV as the model virus.Based on the interaction mechanism between P3 and the movement protein P3N-PIPO via the shared P3N domain,the mechanism of P3 in the intercellular movement and revealing the molecular mechanism was speculated and proved.Then analyse the multimers and structure of 6K1protein and the biological function of 6K1 were studied to reveal the pathogenic mechanism of potyviruses,and provide a theoretical basis for the prevention and control of diseases caused.The detailed results are as follows:1.P3 couples viral replication and intercellular movement:Y2H and Bi FC showed that P3directly interacts with P3N-PIPO through the P3N domain.Using defective Tu MV infectious clone that varied domain of P3 or P3N-PIPO were deleted,we found that P3 interacts with 6K2 via the C-terminal domain（P3C）for replication,while P3N-PIPO interacts with P3 via the common N-terminal domain and recruits CI to the plasmodesmata via the PIPO domain for intercellular movement.Further investigations showed that the interaction between P3 and P3N-PIPO is required for the recruitment of P3N-PIPO to 6K2-induced replication vesicles and localization of 6K2-induced replication vesicles to the plasmodesmata.Transiently expression showed that the interaction between P3 and P3N-PIPO is required for the forming of 6K2/P3/P3N-PIPO/CI quadruple complexes.Using defective Tu MV infectious clone,we found that any mutant that disrupt the interaction between P3 and P3N-PIPO will result in lost the localization of 6K2 replication complexes on plasmodesmata and complete failure of intercellular movement.Taken together,these data suggest that P3 uses different domains to interact with P3N-PIPO and 6K2 separately to locate6K2 replication vesicles on plasmodesmata for CI to accomplish the intercellular movement,which results in the coupling of replication and cell-to-cell movement on the plasmodesmata.2.6K1 has an ion channel activity:Transiently expression showed that mature 6K1 protein exists in the cell.Subcellular localization analyses suggest that 6K1 and P3-6K1 locate on the intracellular membranes with different localization models:6K1 mainly exists in a diffuse form with few dotted granules,while P3-6K1 exists as dotted structures.Ultra-centrifugation combined with chemical treatment（Na₂CO₃,urea and Triton X-100）suggest that 6K1 and P3-6K1 are integrate membrane proteins since they can only be dissolved by Triton X-100 treatment,while P3 maybe a peripheral membrane protein since it was dissolved by urea treatment.Subcellular localization showed that 6K1 forms a dotted structures on intracellular membrane,indicating that 6K1 may form multiers.Bi FC and Co-IP confirmed the self-interacting ability of 6K1.Glutaraldehyde-mediated chemical crosslinking assay showed that 6K1 exists as hexamers in plant cell.Alphafold2 prediction showed that the two anti-parallelα-helixes of 6K1 formed a hairpin structure.Alphafold2-Multimer analyses suggest that 6K1 may form hexamers,in which six monomers arranged side by side to form a cylindrical structure with a pore（channel）in the middle resembling the viral ion channel（viroporin）.Toxicity analyses showed that,similar as the hepatitis C virus encoded viroporin p7,6K1 also can inhibit the proliferation of Escherichia coli and increase the cell permeability to allow the entry of SYTOX green（an excellent nuclear counterstain）and Hygromycin B into the epidemical cells of Nicotiana benthamiana.Using K⁺uptake-defective yeast CY162,we found that the expression of 6K1 or p7 but not empty vector allowed the growth of yeast CY162 on medium with low K⁺concentration,confirming that similar to p7,6K1 also has regulating K⁺channel function that able to mediate the entry of K⁺into yeast cells.Using defective Tu MV infection clone that the Pro hydrolysis site between P3 and 6K1 has been mutated we found that 6K1 and P3 have different functions,but also function collaborative in infection.The 6K1 gene is highly conserved in Potyviridae;thus,we analyzed the toxicity of the 6K1 protein encoded by representative species of each genus in this family.All 6K1 proteins had vareid degrees of toxicity,indicating all 6K1 proteins have viroporin activities.The above results indicate that 6K1 of potyvirids is a viroporin and has ion channel activity.Viroporin has been reported in animal viruses,6K1 is the first viroporin found in plant viruses,thus these results are significant in understanding the distribution,structure,and function of viroporins and established solid basis for developing 6K1 inhibitor to control potyvirids.In conclusion,this study reveals the molecular mechanism by which potyviruses use the N-terminal and C-terminal domains of P3 to couple virus replication and intercellular movement with P3N-PIPO and 6K2,respectively,and discovered the ion channel function of 6K1,which deepened our understanding of the pathogenic mechanism of potyviruses and is of great significance for developing new viral control strategies.