Knockdown of a novel ATPase domain of capsid protein inhibits genome packaging in potato leaf roll virus

Potato leaf roll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the ATPase domain of CP exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.


Introduction Transient expression assay
The binary plasmid pART27-CP and the empty vector pART27 were extracted and purified from E. coli cultures and were transformed into Agrobacterium tumefaciens EHA101 using the freeze-thaw transformation method [12]. The transformed cells of A. tumefaciens were plated on Luria-Bertani (LB) agar plates containing 50 g/mL kanamycin, 100 g/mL spectinomycin, and 100 g/mL chloramphenicol for selecting successful transformants. Transformation of the binary plasmid was further confirmed using colony PCR for the CP gene. For agroinfiltration, A. tumefaciens cells harboring the pART27-CP siRNA constructs were grown overnight at 28C in the LB medium supplemented with appropriate antibiotics. The overnight cultures were diluted 1:10 in fresh media containing the above-mentioned antibiotics, 10 mM 2-(Nmorpholino) ethanesulfonic acid (MES), and 200 M acetosyringone to reach an OD 600 of 0.3. The cells were collected by centrifugation at 5000×g for 5 min and were resuspended in the infiltration medium containing 10 mM MES, 10 mM MgCl 2 , and 200 M acetosyringone. The cells were then incubated at room temperature for 2-3 h before agroinfiltration. Solanum tuberosum cv. Khufri Ashoka plants were agroinfiltrated by injecting 2 mL of these cells directly into the phloem and leaves using a syringe. Whole plants were covered with a transparent plastic bag for 3-4 days. All experiments were repeated thrice with five plants in each experiment for each siRNA construct and control.

Northern blotting analysis of siRNA
Leaves of 5-10 dpi agroinfiltrated potato plants and healthy control plants were used for small RNA isolation. Small RNA was extracted using PAX gene Tissue RNA/miRNA Kit (Qiagen, Germany) following the manufacturer's protocol. Denaturing polyacrylamide gels (15%) containing 7 M urea were run at 40 mA (600 V) for approximately 2 h to resolve the total RNA, which contained the siRNA pool. The gels were stained for 10 minutes with 0.5 μg/ml ethidium bromide (EtBr) in DEPC-treated TBE buffer. A Bio-Rad transblot apparatus (Bio-Rad, USA) was used to transfer the RNA onto a Hybond-N + positively charged nylon membrane (GE healthcare, USA) at 200 mA (9-10 V) for 3 h followed by crosslinking for 20 min at 1200 μJ and drying for 30 min at 50 °C to improve sensitivity. Further, the membrane was prehybridized in pre-hybridization buffer (7 % SDS, 200 mM Na 2 HPO 4 (pH 7.0), 5 μg/ml salmon sperm DNA (SSDNA)) at 40 °C for 30 min. The hybridization buffer was replaced with hybridization buffer containing biotin-labeled probes at 50 pmol/mL concentration. The membrane was subsequently hybridized at 40 °C for 12 to 16 h with continuous gentle shaking followed by rinsing with washing buffer (1× SSC, 0.1% SDS) thrice for a total of 15 minutes at room temperature. The membrane was then blocked for 15 minutes with blocking buffer using gentle shaking at room temperature followed by incubation with hybridization buffer containing stabilized streptavidin-HRP conjugate for an additional 15 minutes. The blot was visualized using Amersham typhoon blot imaging systems (GE, Healthcare) after developing by ECL (GE Healthcare).

Insights on the structural details of the PLRV CP ATPase domain
CPs are known as plant virus-encoded factors that nucleate over viral genomes and encapsidate them by deriving energy from ATP [5]. The motifs necessary for ATP binding and hydrolysis are present throughout the polypeptide chain of PLRV CP (Fig 1A & B). Fig 1A presents a multiple sequence alignment of CP sequences from different PLRV variants. Intriguingly, our sequence analysis revealed a unique pattern of ATPase domain, which contained two sets of Walker A, arginine finger, and sensor-like motifs, suggesting divergent evolution within the classical P-loop-containing ATPase superfamily. The Walker-A "P-loop" motif is proposed to coordinate ATP hydrolysis with DNA translocation. The Walker A-like motif (lavender color), with the consensus sequences RGRGSSET, interacts with the β-and γ-phosphates of the bound ATP, whereas the conserved Asp of Walker B (consensus sequence hhhhDG), located next to a β-strand, binds to a metal ion and helps in ATP hydrolysis. The Walker-B (blue) Asp coordinates with the Mg 2+ cation, while another conserved catalytic Gly residue primes a water molecule for the nucleophilic attack on the γ-phosphate of the bound ATP. Our sequence and structure analysis found that all the critical motifs are either present at the tip of the strand or part of the loop, indicating relatedness of CPs to ASCE P-loop ATPases (Fig 1A). Further, we observed that the arginine fingers I and II (red), which are located 9-10 residues downstream of sensor I (green) motif and 5-6 residues downstream of Walker A′ (lavender), and the sensor motif II (green) present about five residues further downstream of the Arginine finger II motif are strictly conserved across various strains of PLRV analyzed in this study (Fig 1A). Sensor motif I (green) is located 48 residues downstream of Walker B and flanking of arginine finger I, II and Walker A′ with sensor motifs I and II represents the uniqueness of this novel P-loopcontaining ATPase. Our structure prediction using I-TASSER revealed that the active site of PLRV CP comprises all the motifs necessary for ATP interaction except Arginine finger I, which is situated far from the rest of the motifs in their folded structure (highlighted in red; Fig   1B). Arginine finger is a classical hallmark of ATPases and is conserved across many ATPases.
It completes the active site from a distinct location, forming contacts with the γ-phosphate of the nucleotide [13]. To the best of our knowledge, this is the first study that presents a novel P-loop containing ATPase fold of CP comprising several repeat motifs and demonstrates that the suppression of CP expression using siRNA constructs can lead to resistance against PLRV.
The inhibition of the expression of CP by gene silencing is an efficient and promising method to introduce resistance to PLRV [2,[4][5]21]. As PLRV causes severe crop yield losses in the potato growing regions worldwide [22][23], our findings will be helpful for developing PLRVresistant potato crops. This approach can also be applied to an extensive range of plant species to develop resistance against various viral diseases. We are developing strategies for the development of PLRV-resistant potato varieties using the RNAi approach by targeting multiple genes.