Genome organization and translation products of Providence virus: insight into a unique tetravirus.

Providence virus (PrV) is a member of the family Tetraviridae, a family of small, positive-sense, ssRNA viruses that exclusively infect lepidopteran insects. PrV is the only known tetravirus that replicates in tissue culture. We have analysed the genome and characterized the viral translation products, showing that PrV has a monopartite genome encoding three ORFs: (i) p130, unique to PrV and of unknown function; (ii) p104, which contains a read-through stop signal, producing an N-terminal product of 40 kDa (p40) and (iii) the capsid protein precursor (p81). There are three 2A-like processing sequences: one at the N terminus of p130 (PrV-2A1) and two more (PrV-2A2 and PrV-2A3) at the N terminus of p81. Metabolic radiolabelling identified viral translation products corresponding to all three ORFs in persistently infected cells and showed that the read-through stop in p104 and PrV-2A3 in p81 are functional in vivo and these results were confirmed by in vitro translation experiments. The RNA-dependent RNA polymerase domain of the PrV replicase is phylogenetically most closely related to members of the families Tombusviridae and Umbraviridae rather than to members of the family Tetraviridae. The unique genome organization, translational control systems and phylogenetic relationship with the replicases of (+ve) plant viruses lead us to propose that PrV represents a novel family of small insect RNA viruses, distinct from current members of the family Tetraviridae.

Evolution in action: N and C termini of subunits in related T = 4 viruses exchange roles as molecular switches.

The T = 4 tetravirus and T = 3 nodavirus capsid proteins undergo closely similar autoproteolysis to produce the N-terminal beta and C-terminal, lipophilic gamma polypeptides. The gamma peptides and the N termini of beta also act as molecular switches that determine their quasi equivalent capsid structures. The crystal structure of Providence virus (PrV), only the second of a tetravirus (the first was NomegaV), reveals conserved folds and cleavage sites, but the protein termini have completely different structures and the opposite functions of those in NomegaV. N termini of beta form the molecular switch in PrV, whereas gamma peptides play this role in NomegaV. PrV gamma peptides instead interact with packaged RNA at the particle two-folds by using a repeating sequence pattern found in only four other RNA- or membrane-binding proteins. The disposition of peptide termini in PrV is closely related to those in nodaviruses, suggesting that PrV may be closer to the primordial T = 4 particle than NomegaV.

Providence virus: a new member of the Tetraviridae that infects cultured insect cells.

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia beta virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.

The palm subdomain-based active site is internally permuted in viral RNA-dependent RNA polymerases of an ancient lineage.

Template-dependent polynucleotide synthesis is catalyzed by enzymes whose core component includes a ubiquitous alphabeta palm subdomain comprising A, B and C sequence motifs crucial for catalysis. Due to its unique, universal conservation in all RNA viruses, the palm subdomain of RNA-dependent RNA polymerases (RdRps) is widely used for evolutionary and taxonomic inferences. We report here the results of elaborated computer-assisted analysis of newly sequenced replicases from Thosea asigna virus (TaV) and the closely related Euprosterna elaeasa virus (EeV), insect-specific ssRNA+ viruses, which revise a capsid-based classification of these viruses with tetraviruses, an Alphavirus-like family. The replicases of TaV and EeV do not have characteristic methyltransferase and helicase domains, and include a putative RdRp with a unique C-A-B motif arrangement in the palm subdomain that is also found in two dsRNA birnaviruses. This circular motif rearrangement is a result of migration of approximately 22 amino acid (aa) residues encompassing motif C between two internal positions, separated by approximately 110 aa, in a conserved region of approximately 550 aa. Protein modeling shows that the canonical palm subdomain architecture of poliovirus (ssRNA+) RdRp could accommodate the identified sequence permutation through changes in backbone connectivity of the major structural elements in three loop regions underlying the active site. This permutation transforms the ferredoxin-like beta1alphaAbeta2beta3alphaBbeta4 fold of the palm subdomain into the beta2beta3beta1alphaAalphaBbeta4 structure and brings beta-strands carrying two principal catalytic Asp residues into sequential proximity such that unique structural properties and, ultimately, unique functionality of the permuted RdRps may result. The permuted enzymes show unprecedented interclass sequence conservation between RdRps of true ssRNA+ and dsRNA viruses and form a minor, deeply separated cluster in the RdRp tree, implying that other, as yet unidentified, viruses may employ this type of RdRp. The structural diversification of the palm subdomain might be a major event in the evolution of template-dependent polynucleotide polymerases in the RNA-protein world.

Analysis of the capsid processing strategy of Thosea asigna virus using baculovirus expression of virus-like particles.

Thosea asigna virus (TaV), a putative member of the genus Betatetravirus of the family Tetraviridae, is predicted to have a novel capsid expression strategy compared with other characterized tetraviruses. The capsid precursor protein is cleaved twice to generate three proteins. Two of the proteins, L (58.3 kDa) and S (6.8 kDa), are incorporated into the TaV virion. The third, non-structural protein, produced from the N terminus of the precursor protein, is up to 17 kDa in size and is of unknown function. The TaV capsid precursor protein sequence without the 17 kDa N-terminal region was modelled against the solved structure from Nudaurelia omega virus (N omega V) using SwissModel. The TaV model was very similar to the solved structure determined for subunit A of N omega V and had features that are conserved between tetraviruses and nodaviruses, including the positioning of the cleavage site between the L and S capsid proteins. The production of virus-like particles (VLPs) using the baculovirus expression system was used to analyse the capsid processing strategy employed by TaV. VLPs were formed in both the presence and absence of the 17 kDa N-terminal region of the capsid precursor. VLPs were not formed when the L and S regions were expressed from separate promoters, indicating that cleavage between the L and S capsid proteins was an essential part of TaV capsid assembly. Expression of the TaV 17 kDa protein in bacteria did not produce intracellular tubules similar to those formed by bacterial expression of the p17 protein from Helicoverpa armigera stunt virus.