Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus.

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo.IMPORTANCE Despite intensive study, human virus sampling is still sparse, especially for viruses that cause mild or asymptomatic disease. Human picobirnavirus (hPBV) is a double-stranded-RNA virus, broadly dispersed in the human population, but its pathogenicity is uncertain. Here, we report the hPBV structure derived from cryo-electron microscopy (cryo-EM) and reconstruction methods using three capsid protein variants (of different lengths and N-terminal amino acid compositions) that assemble as virus-like particles with distinct properties. The hPBV near-atomic structure reveals a quasisymmetric dimer as the structural subunit and tetramers as possible assembly intermediates that coassemble with nucleic acids. Our structural studies and atomic force microscopy analyses indicate that hPBV capsids are potentially excellent nanocages for gene therapy and targeted drug delivery in humans.

Multiple divergent picobirnaviruses with functional prokaryotic Shine-Dalgarno ribosome binding sites present in cloacal sample of a diarrheic chicken.

Picobirnaviruses (PBVs) of family Picobirnaviridae have bisegmented (S1 and S2 segments), double-stranded RNA genomes. In this study a total of N = 12 complete chicken PBVs (ChPBV) segments (N = 5 of S1 and N = 7 of S2, Acc. Nos.: MH425579-90) were determined using viral metagenomic and RT-PCR techniques from a single cloacal sample of a diarrheic chicken. The identified ChPBV segments are unrelated to each other and distant from all of the currently known PBVs. In silico sequence analyses revealed the presence of conserved prokaryotic Shine-Dalgarno-like (SD-like) sequences upstream of the three presumed open reading frames (ORFs) of the S1 and a single presumed ORF of the S2 segments. According to the results of expression analyses in E. coli using 6xHis-tagged recombinant ChPBV segment 1 construct and Western blot these SD-like sequences are functional in vivo suggesting that S1 of study PBVs can contain three ORFs and supporting the bacteriophage-nature of PBVs.

Extensive conservation of prokaryotic ribosomal binding sites in known and novel picobirnaviruses.

Currently, the Leviviridae and Cystoviridae are the only two recognized families of prokaryotic RNA viruses. Picobirnaviruses, which are bisegmented double-stranded RNA viruses commonly found in animal stool samples, are currently thought to be animal viruses, but have not been propagated in cell culture or in an animal model. We hypothesize that picobirnaviruses are prokaryotic RNA viruses. We identified and analyzed the genomes of 38 novel picobirnaviruses and determined that a classical bacterial sequence motif, the ribosomal binding site (RBS), is present in the 5' untranslated regions (5' UTRs) of all of the novel as well as all previously published picobirnavirus sequences. Among all viruses, enrichment of the RBS motif is only observed in viral families that infect prokaryotes and not in eukaryotic infecting viral families. These results will enable future studies to more accurately understand the biology of picobirnaviruses.

Genogroup I picobirnavirus in diarrhoeic foals: can the horse serve as a natural reservoir for human infection?

Picobirnaviruses (PBV) are small, non-enveloped viruses with a bisegmented double-stranded RNA genome. In this study a PBV strain, PBV/Horse/India/BG-Eq-3/2010, was identified in the faeces of a 10 month old weaned female foal with diarrhoea in January 2010 from Kolkata, India. Surprisingly, sequence comparison and phylogenetic analysis of a short stretch of the RNA dependent RNA polymerase gene revealed close genetic relatedness (> 98% nucleotide identity) to a human genogroup I PBV strain (Hu/GPBV1) detected earlier from the same part of India. Our observations together with earlier findings on genetic relatedness between human and animal PBV warrant further studies on zoonotic potential.