The Formation and Function of Birnaviridae Virus Factories.

The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid-liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes.

NMR structure of a viral peptide inserted in artificial membranes: a view on the early steps of the birnavirus entry process.

Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5-8 and 2-4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by (1)H NMR studies provide structural insights of the pep46 domain interaction. In CDCl(3)/CD(3)OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.

Sequence analysis of the bicistronic Drosophila X virus genome segment A and its encoded polypeptides.

Drosophila X virus represents the entomobirnavirus genus of the Family Birnaviridae. Segment A of this bisegmented dsRNA containing virus was cloned and sequenced. The 3360-bp-long nucleotide sequence revealed the presence of two open reading frames (ORFs). A large ORF of 3096 nucleotides, which is flanked by a 107-bp 5' and a 157-bp 3'-untranslated region, and a 711-nucleotide-long small ORF located within the carboxy half of the large ORF but in a different reading frame. The large ORF encodes a 114-kDa polyprotein which is cotranslationally processed by the virus-coded protease VP4 to generate preVP2, VP3, and VP4 (VP1 is encoded by genome segment B). N-terminal amino acid sequencing of VP3 and VP4 established the order NH2-preVP2-VP4-VP3-COOH within the polyprotein. The small ORF straddles the VP4/VP3 junction and is capable of encoding a basic, arginine-rich 27-kDa polypeptide which so far has not been detected in infected cells. The amino acid sequences specified by the two ORFs were compared to those of infectious pancreatic necrosis virus (IPNV) and infectious bursal disease virus (IBDV) that represent the two other genera (aquabirnavirus and avibirnavirus) of the Birnaviridae family. Significant sequence homology among the three viruses was found to be restricted to the amino and carboxy regions of preVP2 and to a small 21-residue-long domain near the carboxy terminal region of VP3. Significant sequence homology is exhibited by the small ORFs of the three viruses.