Protein-protein Interactions Of Citrus Tristeza Virus Viral Proteins

Citrus fruit trees belonging to the Rutaceae family of flowering plant is grown throughout the world where ever there is sufficient rainfall or irrigation to sustain this plant species. Citrus annual production all over the world today measures up to230million metric tons. The most economically important viral disease of this plant is "tristeza" disease caused by Citrus tristeza virus (CTV).Citrus tristeza virus is a member of the genus Closterovirus within the family Closterovirideae. CTV virions are flexuous filament of about2000x11nm in size and contain a plus-sense single-stranded genomic RNA of19.2-19.3kb, organized into12open reading frames (ORFs) that encode19potential proteins with two untranslated regions (UTRs) at the5’and3’termini. Two of the ORFs, CP and CPm code for capsid proteins (CPs) that encapsidate～97%and3%of the genomic RNA, respectively. CTV genome has two large conserved gene modules. One is responsable for genome replication (replication gene block), while the other functions in genome packaging and intercellular transport (quintuple gene block). The coat protein (CP) gene of Citrus tristeza virus (CTV) acts as intracellular silencing suppressor and in a collective manner suppresses viral RNA silencing and forms part of CTV conversed gene block responsible for virion assembly and movement. Protein of approximately20KD (p20) is known to be a major characteristic of viral amorphous inclusion bodies and takes part in viral RNA silencing. These two proteins are reported to show strong self-interaction for CTV isolate T36.Protein-protein interaction is essential for all biological processes for cells to carry out their normal physiological functions. Pathogens infect their host at different level, pathogenesis is due to a series of interaction within and between viral/host and viral/viral proteins. In this work we used a yeast two-hybrid analysis to construct a partial protein interaction map for CTV3terminal encoded proteins and used a truncation based analysis to map the region(s) responsible for CTV-CP and CTV-p20self-interaction. The interactions among three proteins (CP, CPm and p20) from four CTV isolates:S4, N4, S45and HB1were studied by yeast two-hybridization (Y2H) and bimolecular fluorescence complementation (BiFC) methods. Consistent strong self interactions were noticed between the pair of CP-CP and p20-p20irrespective of the CTV isolate. In this study, the results revealed a unique interaction between the CPm of S4and CP of N4isolates. Analysis of the deletion mutants delineated the domains of CP-CP and p20-p20interaction. The domains consisting of N-terminal conserved amino acids of CP (aa41-84) and the main part of p20(aal-21) were responsible for CP and p20self-interactions, respectively. This study implicated interactions among ten CTV proteins, three proteins (CP, p20and CPm) of two CTV isolates and provided interaction site information for CTV self-interacting proteins. Protein-protein interaction and identification of actual interacting domain may promote a better understanding of biological pathways during CTV infection.Yeast two-hybrid, CTV, protein-protein interaction, Bimolecular Fluorescence Complementation...