Functional Characterization of Pepper Vein Banding Virus-Encoded Proteins and Their Interactions: Implications in Potyvirus Infection.

Pepper vein banding virus (PVBV) is a distinct species in the Potyvirus genus which infects economically important plants in several parts of India. Like other potyviruses, PVBV encodes multifunctional proteins, with several interaction partners, having implications at different stages of the potyviral infection. In this review, we summarize the functional characterization of different PVBV-encoded proteins with an emphasis on their interaction partners governing the multifunctionality of potyviral proteins. Intrinsically disordered domains/regions of these proteins play an important role in their interactions with other proteins. Deciphering the function of PVBV-encoded proteins and their interactions with cognitive partners will help in understanding the putative mechanisms by which the potyviral proteins are regulated at different stages of the viral life-cycle. This review also discusses PVBV virus-like particles (VLPs) and their potential applications in nanotechnology. Further, virus-like nanoparticle-cell interactions and intracellular fate of PVBV VLPs are also discussed.

Development of pepper vein banding virus chimeric virus-like particles for potential diagnostic and therapeutic applications.

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.

Intracellular trafficking and endocytic uptake pathway of Pepper vein banding virus-like particles in epithelial cells.

Aim: Plant virus-like particles (VLPs) have emerged as a novel platform for delivery of drugs/antibodies. The aim of the present investigation is to establish the entry mechanism of flexuous rod-shaped virus particles into mammalian cells. Methods: Far-Western blot analysis, pull-down and ELISA were used to characterize vimentin and Hsp60 interaction with VLPs. The mode/kinetics of internalization of VLPs was deciphered using pharmacological inhibitors/endosomal markers. Results & discussion: The flexuous rod-shaped VLPs of Pepper vein banding virus (PVBV) enter HeLa and HepG2 cells via cell-surface proteins: vimentin and Hsp60, respectively. VLPs internalize via different modes of endocytosis in HeLa, HepG2 cells and are biodegradable. Vimentin and Hsp60 could be potential epithelial ligands that facilitate targeting of nanoparticles to tumor cells.

Structural and functional studies on Salmonella typhimurium pyridoxal kinase: the first structural evidence for the formation of Schiff base with the substrate.

A large number of enzymes depend on the ubiquitous cofactor pyridoxal 5' phosphate (PLP) for their activity. Pyridoxal kinase (PLK) is the key enzyme involved in the synthesis of PLP from the three forms of vitamin B6 via the salvage pathway. In the present work, we determined the unliganded structure of StPLK in a monoclinic form and its ternary complex with bound pyridoxal (PL), ADP and Mg2+ in two different tetragonal crystal forms (Form I and Form II). We found that, in the ternary complex structure of StPLK, the active site Lys233 forms a Schiff base linkage with the substrate (PL). Although formation of a Schiff base with the active site Lys229 was demonstrated in the Escherichia coli enzyme based on biochemical studies, the ternary complex of StPLK represents the first crystal structure where the Schiff bond formation has been observed. We also identified an additional site for PLP binding away from the active site in one of the ternary complexes (crystal Form I), suggesting a probable route for the product release. This is the first ternary complex structure where the modeled γ-phosphate of ATP is close enough to PL for the phosphorylation of the substrate. StPLK prefers PL over pyridoxamine as its substrate and follows a sequential mechanism of catalysis. Surface plasmon resonance studies suggest that StPLK interacts with apo-PLP-dependent enzymes with µm affinity supporting the earlier proposed direct transfer mechanism of PLP from PLK to PLP-dependent enzymes.

Mapping the domain of interaction of PVBV VPg with NIa-Pro: Role of N-terminal disordered region of VPg in the modulation of structure and function.

VPg-Pro is involved in polyprotein processing, therefore its regulation is important for a successful potyviral infection. We report here that the N-terminal disordered region of VPg forms the domain of interaction with NIa-Pro. This region is also demonstrated to be responsible for modulating the protease activity of VPg-Pro, both in cis and trans. The disordered nature of VPg is elicited by the N-terminal 22 residues as removal of these residues (∆N22 VPg) brought about gross structural and conformational changes in the protein. Interestingly, ∆N22 VPg gained ATPase activity which suggested the presence of autoinhibitory motif within the N-terminal region of VPg. The autoinhibition gets relieved upon interaction of VPg with NIa-Pro or removal of the inhibitory motif. Thus, the N-terminal 22 residues of VPg qualify as molecular recognition feature (MoRF), regulating both protease and ATPase activity of VPg-Pro as well as forming the domain of interaction with other viral/host proteins.