Molecular characterization of a novel cryptic virus infecting pigeonpea plants

Abstract

A new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5' non-coding region (5'-GATAATGATCCAAGGA-3'). The largest dsRNA (dsRNA-1) was identified as the viral RNA dependent RNA polymerase (replicase), predicted to encode a putative 55.34 kDa protein (P1). The two other smaller dsRNAs (dsRNA-2 and dsRNA-3) predicted to encode for putative capsid proteins of 38.50kDa (P2) and 38.51kDa (P3), respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A), not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb) encodes a protein (P4), with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.

Fig 1. Resolution of dsRNA species isolated from symptom free pigeonpea plants and SISPA-PCR amplified dsRNAs fractionated on 1.5% agarose gel.

(A) Lane-2, dsRNAs isolated from symptomless pigeonpea field collected plants; (B) Lane-2, amplified dsRNAs. The ArCV-1, dsRNA-2 and 3 were almost similar in size. Lane 1 (A, B) contains 1kb DNA marker (Fermentas, Thermo scientific, USA).

Fig 2. Schematic representation of genome organization of Arhar cryptic virus-1(ArCV-1) and associated CP-like dsRNA-2A.

(A) ArCV-1, dsRNAs were denoted (RNA1, RNA2 and RNA3) are represented by solid black lines. The larger RNA was identified putative RdRp encoding a single peptide of P1, marked as grey box. The two smaller RNAs encode two individual peptides of P2 and P3 and were identified as putative capsid proteins, marked as grey boxes. (B) Schematic representation ofa fourth dsRNA isolated along with the ArCV-1 genome with an unusually long 3’NTR, referred as RNA-2A (Black solid line) is predicted to encode a putative coat protein P4 marked as grey box.

Fig 3. Alignment of the nucleotide sequences of 5’ non-coding regions of dsRNAs-1, 2 and 3 of ArCV-1, Fragaria chiloensis cryptic virus (FcCV), Rose cryptic virus-1 (RoCV-1) and Raphanus sativus cryptic virus-2 (RsCV-2).

Conserved residues are marked as in grey box.

Fig 4. Molecular graphics of ArCV-1 RdRp 3D structure at 7.9Å resolution.

Molecular graphic of ArCV-1, RdRp 3D structure of 7.9Å resolution with central cavity flanked by N-terminus and C-terminus structures and the folding topology resemble the RdRps of picornaviruses: (A) Cartoon representation of the molecule in a conventional orientation of the closed right hand depicted with sub-domains, fingers (shades of blue- cyan), thumb (shades of red) and palm. (B) Cartoon representations of ArCV-1 3Dpol Motifs A, B, C, D, E and F are represented in blue, purple, light pink, cyan, brown and green, respectively. (C) Surface representation of the ArCV-1 3D polymerase showing a donut shaped structure with a central catalytic cavity and the top channel. Few of the representative motifs of ArCV-1 3D with residues known to interact with the RNA and the incoming NTP substrate were identified close to the catalytic center (GDD cyan). (D) ArCV-1 3D^pol, turned 90° to the front showing view from the top, with three subdomains, Fingers, Thumb and Palm farming putative RNA binding cleft. The N-terminal (1–55 residues) over layered on the enzyme surface to show the fingers region tether to the thumb region (shown as yellow coil with residues Arg-24, I-29, S-36 binding to the thumb subdomain). (E) Structural details of the altered conformation of motif A and B side view of the RdRp surface representation exposing the three channelsArCV-1 polymerase that are described for the picornoviruses. Positioning of channels as template channel (top) NTPs channel (rear) and product exiting channel (front) were denoted. The subdomain motifs A, B, and C in part, as cartoon was over layered to exact position. Figures were developed with PyMol.

Fig 5. Comparison of cryptoviral and picornaviral polymerase structures.

(A) Cartoon representation showing ArCV-1 3Dpol; (B) Fragaria chiloensis cryptic virus (FcCV) (C) Sapporo virus (SV; PDB ID, 2wk4A) and (D) Norwalk virus (NV; PDB ID 1SH0). The molecules are shown in conventional right hand orientation with spectrum colors. The N-terminus colored blue and red for C-terminus for all the molecules. The three subdomains the fingers (shades blue and green) palm and thumb domains are in yellow and red. Carboxyl-terminal ends of the ArCV-1, FcCV, and SV were seen protruded out and interestingly in NV C-terminus was found to be located in the catalytic region of the central cavity. (E) ArCV-1, 3Dpol (blue) in a cartoon representation was superimposed by FcCV (green) polymerase. (F) in a backbone representation ArCV-1 3Dpol (blue), with every 40th residues numbered was superimposed by FcCV (grey) polymerase and (G) by Sapporo virus (green) polymerase (PDB ID, 2wk4A) and (H) by Norwalk virus (brown) polymerase (PDB ID, 2b43D) A high degree of alignment was noticed ArCV-1, 3Dpol with FcCV and SV followed by NV especially in the palm region. Structural comparison analysis provided for SV and NV an RMSD value of 2.10 Å and 2.15 Å, respectively.

Fig 6. Conserved residues the RdRp B motif of ds RNA cryptovirus and ss (+) RNA picornaviruses.

The RdRp multifunctional B motif, comprising of conserved residues, forms structurally important entity containing B-loop (see Fig 5F) as demonstrated in picornaviruses. Shown here is a similar motif in cryptovirus RdRps with the conserved gly306 residue that acts as axis for the conformational changes, highlighted in green block. The positions of the catalytic Asn (in α-helix11, predominantly substituted by gly314 in dsRNA viruses) and of the Ser and Thr residues of picornaviruses interacting with the incoming NTP substrate also are common to cryptovirus RdRps (details in Text). ArCV-1 (HG797710), FcCV (DQ093961), RmCV (EU024675), RoCV-1 (EU413666), RsCV-2 (DQ218036), Phi6 (PDB 4A8W), Reovirus (RV; PDB 1MUK), SV (PDB 2WK4), NV (PDB 2B43), RHDV (PDB 1KHW), FMDV (PDB 1WNE). Middle = middle finger of the fingers subdomain; Loop = structures in palm subdomain.

Fig 7. Phylogenetic analysis of ArCV-1 dsRNA1 (RdRp) and dsRNA-2A.

(A) Phylogenetically though all members originate from common ancestor as they diverge from a single clade formed five distinct groups corresponding to their genera (Group 1- Alphapartitivirus; Group 2- Betapartitivirus; Group 3- Gammapartitivirus; Group 4- Deltapartitivirus and Group 5- Cryspovirus). Members of group 1 and 2 clustered separately but originate from common point however origin of members of group 3 were different. Group 4 and 5 originate from common sub-clade, which is different from other groups. ArCV-1 RNA-1 clusters with tripartite members of the genus Deltapartitivirus and is closely related to FcCV and RsCV-2. *Beet cryptic virus-2 was an assigned member of Deltapartitivirus but it was noticed to fit in with Alphapartitivirus. (B) Phylogenetic analysis of dsRNA-2A (P4 protein), displays its proximity with bipartite PepCV-2 and RsCV-3. The bar represents base substitution per site. Sequences of different partitiviruses from NCBI database were used in the analysis (S1 Table). RdRps of hPBV (AB186898) and oPBV (JQ776552) and CP sequence (hPBV: AB186897 and Q776551) were used to provide an out group root in the analysis.