Polycipiviridae: a proposed new family of polycistronic picorna-like RNA viruses

Abstract

Solenopsis invicta virus 2 is a single-stranded positive-sense picorna-like RNA virus with an unusual genome structure. The monopartite genome of approximately 11 kb contains four open reading frames in its 5' third, three of which encode proteins with homology to picornavirus-like jelly-roll fold capsid proteins. These are followed by an intergenic region, and then a single long open reading frame that covers the 3' two-thirds of the genome. The polypeptide translation of this 3' open reading frame contains motifs characteristic of picornavirus-like helicase, protease and RNA-dependent RNA polymerase domains. An inspection of public transcriptome shotgun assembly sequences revealed five related apparently nearly complete virus genomes isolated from ant species and one from a dipteran insect. By high-throughput sequencing and in silico assembly of RNA isolated from Solenopsis invicta and four other ant species, followed by targeted Sanger sequencing, we obtained nearly complete genomes for four further viruses in the group. Four further sequences were obtained from a recent large-scale invertebrate virus study. The 15 sequences are highly divergent (pairwise amino acid identities of as low as 17 % in the non-structural polyprotein), but possess the same overall polycistronic genome structure, which is distinct from all other characterized picorna-like viruses. Consequently, we propose the formation of a new virus family, Polycipiviridae, to classify this clade of arthropod-infecting polycistronic picorna-like viruses. We further propose that this family be divided into three genera: Chipolycivirus (2 species), Hupolycivirus (2 species) and Sopolycivirus (11 species), with members of the latter infecting ants in at least 3 different subfamilies.

Fig. 1.

Genome map of SINV-2. The genome is represented by a black line. ORFs are indicated in pale blue with vertical offsets indicating the reading frame (−1, 0, +1) relative to ORF5. IGR, intergenic region.

Fig. 2.

Genome maps of proposed polycipiviruses. ORFs are represented by coloured rectangles with vertical offsets indicating reading frames relative to the Hel-Pro-RdRp ORF. Green rectangles indicate ORFs encoding jelly-roll capsid protein domains as predicted by HHpred; hatched green lines indicate uncertain capsid domain identifications. Pink rectangles indicate a conserved overlapping ORF encoding a transmembrane helix as predicted by TMHMM. A dark green rectangle in the F. exsecta TSA map indicates an additional ORF, 3a. Replication protein domains, identified by their conserved characteristic motifs, are indicated as Hel (helicase), Pro (protease) and RdRp (RNA-dependent RNA polymerase). TSA indicates sequences obtained from the NCBI Transcriptome Shotgun Assembly database. Two horizontal lines (on the right side) separate the three proposed genera.

Fig. 3.

RNA sequencing coverage of the MsaV-1 and LneV-1 genomes. The total coverage at each nucleotide position is indicated in red (positive sense) or blue (negative sense; note the different axis scale). Black lines show the mean coverage in a 1000-nt sliding window.

Fig. 4.

Phylogenetic tree of the proposed family Polycipiviridae. ORF5 amino acid sequences were aligned with muscle, and a Bayesian Markov chain Monte Carlo-based phylogenetic tree was produced with MrBayes. All posterior probabilities were equal to 1.00. The tree was mid-point rooted and visualized with FigTree. TSA indicates sequences obtained from the NCBI Transcriptome Shotgun Assembly database. Proposed Polycipiviridae genera – Chipolycivirus, Hupolycivirus and Sopolycivirus – are delineated with boxes. For sopolyciviruses, the host ant subfamilies are indicated on the right-hand side.

Fig. 5.

Phylogenetic tree for polycipiviruses and representative members of the order Picornavirales. Core RdRp amino acid sequences from representative Picornavirales viruses were obtained from Koonin et al. [7] and combined with the equivalent regions from the 15 sequences in Table 1 with RdRp coverage. Sequences were aligned with muscle, and a Bayesian Markov chain Monte Carlo-based phylogenetic tree was produced. Posterior probabilities are indicated for family root nodes, and elsewhere if P<1.00. Abbreviations: ALSV, apple latent spherical virus; BBWV1, broad bean wilt virus 1; CPMV, cowpea mosaic virus; CRLV, cherry rasp leaf virus; CrPV, cricket paralysis virus; DCV, Drosophila C virus; DWV, deformed wing virus; EMCV, encephalomyocarditis virus; FMDV, foot-and-mouth disease virus; GFLV, grapevine fanleaf virus; HaRNAV, Heterosigma akashiwo RNA virus; HAV, hepatitis A virus; HplV-81, Hubei picorna-like virus 81; HplV-82, Hubei picorna-like virus 82; HRV1A, human rhinovirus 1A; IFV, infectious flacherie virus; LneV-1, Lasius neglectus virus 1; LniV-1, Lasius niger virus 1; MsaV-1, Myrmica scabrinodis virus 1; NIMV, navel orange infectious mottling virus; PnPV, Perina nuda picorna-like virus; PV, polio virus; PYFV, parsnip yellow fleck virus; RTSV, rice tungro spherical virus; SDV, satsuma dwarf virus; ShiV-8, Shuangao insect virus 8; SINV-2, Solenopsis invicta virus 2; SINV-4, Solenopsis invicta virus 4; TRSV, tobacco ringspot virus; TrV, Triatoma virus; TSV, Taura syndrome virus.