Mechanisms Of Tomato Yellow Mottle-Associated Virus Viroplasm Formation And Its Interaction With Stress Granules

Plant rhabdoviruses are a group of enveloped negative-strand RNA viruses(NSVs),which have a wide range of hosts including a variety of cereal and economic crops and cause serious crop yield losses.The cellular replication sites of NSVs are called viroplasms,inclusion bodies or viral factories.Their formation is particularly important for replication and transcription.Also,they are considered as important targets for the development of antiviral strategies.However,due to the lag in the development of reverse genetics technology and the lack of effective labeling methods in plant cells,the research on the biology of plant NSVs has been delayed.In this study,we focused on a cytorhabdovirus,tomato yellow mottle-associated virus(TYMaV).Based on the successful construction of TYMaV reverse genetics before,we studied the biophysical characteristics and formation mechanisms of its viroplasms.Meanwhile,we further explored the interplay between TYMaV infection and the stress granule(SG),a cellular membraneless organelle in the cytoplasm.This study found that TYMaV N and P proteins were the smallest units for the formation of viroplasms like multiple animal NSVs.We first employed TYMaV minireplicon(MR)reporter assay to ascertain whether N or P protein tolerate tagging and confirmed that P tagged with the small epitopes at its C termini could permit replication at comparable levels.We next attempted to fluorescently label the P protein in virus-infected cells using the ‘superfolder’ GFP(sf GFP)-based split-GFP system,the obtained TYMaV-Psf GFP infectious clone was viable and behaved similarly to TYMaV-GFP in terms of the infectivity,and the purpose for real-time monitoring of viroplasms was successfully achieved.By tracking and observing TYMaV-Psf GFPinfected cells under a laser confocal microscopy,we found TYMaV N,P and viral RNA were indeed presented in the viroplasms,suggesting they were sites of viral replication and transcription.Furthermore,the high dynamic,spherical/irregular nature and fusion/fission activities suggested that TYMaV viroplasms shared the properties of biomolecular condensates driven by LLPS.At the same time,in vivo and in vitro experiments have confirmed that the cytoplasmic inclusion bodies(IBs)formed by NP co-expression had the same characteristics of liquid droplets as viroplasms.The deletion mutants of P demonstrated the intrinsically disordered regions(IDRs)and Cternimal domain(CTD)of P collectively contributed to inclusion formation with N.The rapid movement of TYMaV viroplasms or N-P inclusions suggested an involvement of cytoskeletal transport systems.By co-expression of IBs with actin microfilament and microtubule marker proteins,followed by the treatment of corresponding depolymerization agents,we ensured that the intracellular rapid movement of TYMaV viroplasms was actin-dependent.The destruction of actin microfilaments resulted in the significant inhibition of IBs movement and formation,manifesting as an increase in the number of small particles and the inability to form large particles.These results showed the actin-dependent motility was essential for the coalescence of viroplasms.Expression of the C-terminal tails of class Ⅺ myosins drastically decreased the IB motility and formation,by contrast,the expression of the class Ⅷ myosin tails has no discernible effects.TYMaV P specifically interacted with the class Ⅺ myosins and the C-terminal cargo-binding GTD domain had a major contribution to their interaction.Overexpression of myosin Ⅺ tails inhibited TYMaV replication.Myosin Ⅺs were further confirmed to participate in viroplasm formation via TRV VIGS and overexpression of tails with TYMaV vectors.In conclusion,the small liquid droplets formed by N and P recruited the actin-myosin Ⅺs complexes for their intracellular movement and fusion into viroplasms to promote TYMaV replication.Stress granules(SGs)are cytoplasmic membraneless organelles formed when eukaryotic cells stimulated by external pressures such as heat shock,oxidative stress,nutritional deficiency or viral infection,and play important roles in responding to biotic and abiotic stress.The core component G3BP of SGs was identified as the interactional protein of TYMaV P via IP-MS and the interplay between TYMaV infection and SGs were further explored.The cloned G3BP in N.benthamiana was firstly confirmed to be the component of SGs and the interaction between P and NbG3BP was verified.NbG3BP and SGs were proved to negatively regulate TYMaV infection via gene silencing,CRISPR knockout and overexpression of NbG3BP by TYMaV vectors.Further study found that NbG3BP could bind TYMaV m RNA and then inhibit the expression of viral proteins.No SGs were found to form during TYMaV infection.However,the formation of SGs induced by heat shock and oxygen stress were suppressed during TYMaV infection.By identifying the interaction domains between P and NbG3BP,it was found that the N-terminal IDR1 domain of P and the N-terminal NTF2 L domain of NbG3BP mediated their interactions.NTF2L-mediated NbG3BP dimerization was important for SGs formation,while P could disturb SGs formation by inhibiting the dimerization of NbG3BP via its interaction with NTF2 L domain.In summary,this study realized real-time monitoring of the dynamic of viroplasms during TYMaV infection through labeling methods of viral proteins and live-cell imaging.The roles of actin cytoskeleton in viroplasm formation were further uncovered.We also preliminarily found that SGs negatively infected TYMaV replication,and TYMaV P could inhibit SGs formation by disturbing G3BP self-interaction to suppress their antiviral abilities.All these studies promote the understanding of plant rhabdovirus replication mechanism and the interactions between hosts,and will provide new ideas for the development of effective antiviral strategies.