Bioinformatics Analysis of the Interaction between Coat Protein and Nuclear Shuttle Protein in Babuvirus

Protein and protein interactions play important roles in many biological processes and are responsible for carrying out the function of biological regulatory network in living organisms. Previous study indicated that Banana bunchy top virus (BBTV) coat protein (CP) interacted with BBTV nuclear shuttle protein (NSP). However, the protein and protein interaction and the binding affinity of CP and NSP in Babuvirus are remaining unclear. In this study, the CPs and NSPs proteins of BBTV, Abaca bunchy top virus (ABTV) and Cardamom bushy dwarf virus (CBDV) were used for bioinformatic analysis. The binding free energy and the dissociation constant of the possible interaction proteins were tested in PPA-Pred2, and the results confirmed CP interaction with NSP in Babuvirus. The study will help us to understand the interaction between viral protein and viral protein, and the pathogenesis mechanism of Babuvirus in host plants.

DNA-S encodes a coat protein (CP) which encapsidates each DNA component inside [12]. Meanwhile, CP protein is a viral silencing suppressor that promotes the virus infection in host plants [13]. DNA-N encodes a nuclear shuttle protein (NSP) which can translocate the viral protein or protein and viral DNA complexes out of the cell nucleus [14]. Furthermore, the NSP protein contains an "FNGSF" motif that inhibits plant stress granules (SG) [15]. In vitro experiments showed that both CP and NSP proteins locate in the cytoplasm and nucleus of banana protoplast cell, and movement protein (MP) is able to re-locate NSP protein or NSP-DNA complex around the cell periphery, but not re-locate of the CP protein [14]. In vivo experiments showed the CP locates in the cell nucleus of Nicotiana benthamiana, while the NSP distributes in the cell nucleus and cytoplasm. Co-localization indicated that BBTV NSP interacts and re-distributes BBTV CP in tobacco cells [16].
Protein and protein interactions play important roles in many biological processes and are responsible for carrying out the function of biological regulatory network in living organisms. Previous study indicated that BBTV CP interacted with BBTV NSP [16]. However, the protein and protein interaction and the binding affinity of CP and NSP in Babuvirus are remaining unclear. In this study, the CPs and NSPs proteins of BBTV, ABTV, and CBDV were used for bioinformatic analysis. The study will help us to understand the interaction between viral protein and viral protein, and the pathogenesis mechanism of Babuvirus in host plants.

Nucleotide and Deduced Amino Acid Sequences of CP in Nanoviridae
The nucleotide sequences of BBTV CP (

Nucleotide and Deduced Amino Acid Sequences of NSP in Nanoviridae
The nucleotide sequences of BBTV NSP (

The Prediction of Interaction between CP and NSP Proteins in Babuvirus
Previous study indicated that BBTV CP interacted with BBTV NSP [16]. In order to determine the binding affinity of the two proteins, the amino acid sequences of BBTV CP (AEF97830.1) and BBTV NSP (ACK43793.1) were input into PPA-Pred2 (http://www.iitm.ac.in/bioinfo/PPA_Pred/index.html) for binding affinity analysis using jack-knife test [17]. The relationship between ABTV CP (ACN79534.1) and ABTV NSP (YP_001661656.1) and the relationship between CBDV CP (AGG38924.1) and CBDV NSP (AGG38930.1) were also conducted. The predicted value of Delta G (binding free energy) and the predicted value of American Journal of Plant Sciences Kd (dissociation constant) were used to evaluate the interaction.

Sequences Identity of Nucleotide and Amino Acids of CP in Nanoviridae
The complete ORF of BBTV CP is 513 bp, and its encoded protein is 170 aa with predicted molecular mass of about 19 Figure 1).

Sequences Identity of Nucleotide and Amino Acids of NSP in Nanoviridae
The complete ORF of BBTV NSP is 465 bp, and its encoded protein is 154 aa with predicted molecular mass of about 17.55 kDa in size. The sequence identity analysis of BBTV NSP gene showed that it both shares ~70% homology with ABTV and CBDV, and it was about 50% homology with Nanovirus. Amino acid sequence identity also found that it shares about 70% homology with ABTV and CBDV, while about 45% homology with Nanovirus, a little lower than nucleotide sequence identity (Table 2 and Figure 2). Furthermore, the "FNGSF" motif was conserved in Nanoviridae (Figure 2), which plays the role of inhibiting plant stress granules (SG) formation.

The Prediction of Interaction between CP and NSP Proteins in Babuvirus
PPA-Pred2 analysis indicated that BBTV CP had binding affinity with BBTV NSP. In detail, the predicted value of Delta G (binding free energy) was −11.69 N. T. Yu et al.

Discussion
Protein and protein interactions are crucial bridges for many biological processes involved in cellular signaling, immunity, cellular transport, etc. [18] [19] [20]. Experimentally, protein and protein interactions have been mainly studied with the yeast two-hybrid system, GST pull-down, co-immunoprecipitation (Co-IP), co-localization and so on. In addition, the fluorescence resonance energy transfer (FRET), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and PPA-Pred2 provide the affinity of interacting proteins in terms of binding free energy change and dissociation constant [21] [22] [23], thereby adding a new dimension analysis of the task of protein and protein inte-raction network. Previous study indicated that BBTV CP interacted with BBTV NSP. In this work, the possible interaction of CP protein and NSP protein of BBTV, ABTV and CBDV was further predicted based on their amino acid sequences. The results confirmed CP interaction with NSP in Babuvirus by PPA-Pred2 analysis.
The interaction between CP and NSP in Babuvirus was well predicted by PPA-Pred2 analysis based on the amino acid sequences, not by experiments.
However, this method has a few limitations. For example, the protein whose structure is unclear or the protein which needs further processing modification could not be widely used in this method. In addition, the method cannot be used for prediction the binding affinity of more than two proteins.
In this study, the NSP is a vial nuclear shuttle protein, which would help viral proteins or viral nucleic acid transport outside of the nucleus by interaction with MP [14]. Furthermore, the "FNGSF" motif that inhibits plant stress granules (SG) was conserved in all nanovirids NSPs [15]. It is speculated that Babuvirus NSP proteins have similar functions. The product of the Babuvirus CP gene is an important structural protein that constitutes the virion. Meanwhile, CP protein is a viral silencing suppressor that promotes the virus infection in the host plant.
BBTV NSP affects the BBTV CP distribution by co-localization analysis, suggesting that BBTV NSP interacts and re-locates the BBTV CP in tobacco cells.
During the virus infection, Babuvirus NSP would re-locate the Babuvirus CP, which plays the key role of interaction between virus and host, such as inhibition