Functional Analysis Of Heat Shock Proteins Hsc70-2 And Hsp17.6 In Beet Black Scorch Virus Infection

Heat shock proteins(Hsp)are commonly found in plant and animal cells and are known to be induced by various stresses.As the key components of the cellular chaperones,Hsp are responsible for protein folding,assembly,translocation,and degradation during normal cellular growth and development.Hsp also function in maintaining cell homeostasis under stress conditions.In recent years,Hsp70 was shown to be tightly associated with multiple aspects of viral life cycle,including viral entry,virion assembly and disassembly,replication and viral gene expression.However,how the interactions between Hsp70 and different viral components coordinately regulate viral infection remains to be fully understood.Furthermore,little information is avaible concerning the function of small Hsp(sHsp)in plant virus infection.In this study,we used Beet black scorch virus(BBSV)to explore the relationship between CP,p23 and Hsc70-2 and investigate the mechanisms underlying their interactions.The functional role of Hsp17.6 in BBSV infection was also preliminarily analyzed.Our previous studies found that Hsc70-2 was co-precipitated with BBSV coat protein(CP)and auxiliary replication protein p23.Based upon this,the Hsc70-2 gene was cloned from Nicotiana benthamiana.Direct interactions of Hsc70-2 with CP and p23 were demonstrated by using pull-down,bimolecular fluorescence complementation(BiFC),co-immunoprecipitation(Co-IP),and firefly luciferase complementation imaging(LCI)experiments,whereas CP and p23 did not interact with each other and they cannot form complexes through Hsc70-2.BiFC assay showed that the CP-Hsc70-2 interaction occurred in the cytoplasm and formed larger aggregates,while the interaction of p23-Hsc70-2 localized to the endoplasmic reticulum-derived aggregates.Pull-down and BiFC analysis of Hsc70-2 truncation mutants indicated that both CP and p23 bind to the nucleotide binding domain(NBD)of Hsc70-2.Hsc70-2 was mildly induced by BBSV infection.Overexpression of Hsc70-2 enhanced BBSV infection of N.benthamiana,whereas silencing Hsc70-2 severely impaired viral replication,indicating the indispensable role of Hsc70-2 in BBSV replication.Further experiments revealed that CP negatively regulates BBSV replication and disruption of RNA binding activity of the CP only has marginal effects on the CP-mediated suppression of BBSV replication,suggesting that there may be other mechanisms respobsible for the inhibitory effect of CP on BBSV replication.Overexpression of Hsc70-2 mitagates the inhibition effect of CP on BBSV replication.BiFC assay revealed that CP interferes with the interaction between Hsc70-2 and p23 in a dose-dependent manner,implying the impairment of viral replication complexes formation with the increase of CP accumulation during BBSV infection.Taken together,our studies indicate that BBSV can manipulate Hsc70-2 using different viral components to orchestrate the BBSV infection,which provides new insght into the multifunctional roles of Hsp70 family proteins in virus infection.Our previous studies showed that BBSV infection induces the expression of Hsp17.6 and silencing Hsp17.6 impacts virus infection in N.benthamiana.To further investigate the mechanisms underlying the role of Hsp 17.6 in BBSV infection,thermal stability experiment was carried out and results showed that Hspl7.6 does have chaperone activity.Analysis of Hsp17.6 tissue-specific expression showed that Hsp17.6 is highly expressed in seeds followed by roots and leaves.Subcellular localization analyses showed that Hsp17.6 localizes to the cytoplasm and forms aggregates with variable sizes,some of the aggregates can move in the cell.BBSV infection leads to the absence of Hsp17.6 aggregates.Considering previous results that Hsp90 overexpression has similar effects on the localization pattern of Hsp17.6,we proposed that the changes of Hsp17.6 localization may be associated with Hsp90,whose expression is augmented during BBSV infection.Overexpression of Hspl7.6 has little effect on BBSV infection,whereras Hspl7.6 silencing attenuates BBSV accumulation but does not affect viral replication level,suggesting that Hsp17.6 may function in the movement of BBSV.Further experiments showed that Hspl7.6 interacts with both BBSV movement protein p7a and Actin,suggesting that Hsp17.6 may play an important role in mediating the efficient intracellular movement of BBSV along actin cytoskeleton.