Infectious Bursal disease virus: ribonucleoprotein complexes of a double-stranded RNA virus

Abstract

Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some double-stranded DNA (dsDNA) viruses, and the nucleocapsid proteins of single-stranded RNA viruses. dsRNA viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T=2 core involved in the metabolism and insulation of the dsRNA genome. The birnaviruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T=13 capsid only. Moreover, as in many negative single-stranded RNA viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease virus, the best characterized member of the Birnaviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at approximately 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNase III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA viruses.

Fig. 1

IBDV model and virion destabilizing treatments to obtain RNP, VPg–dsRNA complexes, and dsRNA. The scheme represents the IBDV model with its associated stoichiometry for the major structural components. This scheme is adapted from the work of Ahlquist. A polyploid virion with four packaged dsRNA segments is shown as E5 IBDV particles. Dialysis of IBDV virions against low-salt buffer in the presence of EDTA renders structurally preserved RNP (constituted by dsRNA, VP3, and VP1/VPg) and soluble capsid protein subunits of VP2/pVP2 (left arrow); treatment of these RNP crude preparations with SDS releases VP3 and noncovalently bound VP1 subunits as soluble components and VPg–dsRNA complexes. Treatment of these crude VPg–dsRNA preparations with proteinase K would eventually render dsRNA molecules. Incubation of E5 IBDV virions with SDS and proteinase K releases dsRNA segments (right arrow).

Fig. 2

Electron microscopy of disrupted IBDV particles by low-salt treatment. E5 IBDV virions were dialyzed against low-salt buffer and directly processed for electron microscopy by (a) negative staining with 2% uranyl acetate or (b) air drying and metal shadowing. Arrows indicate unaltered virions. Insets show general views of virions dialyzed against PES buffer and therefore structurally well-preserved particles. Bar represents 100 nm.

Fig. 3

Electrophoretic characterization of IBDV-derived RNP complexes. (a) Agarose gel electrophoresis in the absence (left) or in the presence (right) of 0.1% SDS of IBDV-derived complexes: 1, structurally unaltered virions; 2, dsRNA genome molecules; 3, unpurified VPg–dsRNA complexes; and 4, disassembled virions with structurally preserved RNP. (b) Cartoon illustrating the different assemblies analyzed in this study (1–4, as described above). This scheme was adapted from the work of Ahlquist.

Fig. 4

Purification of RNP and VPg–dsRNA complexes. (a and b) RNPs from dissociated virions by low-salt dialysis were purified by glycerol gradient centrifugation; 11 fractions were collected, analyzed by SDS-PAGE, and developed by (a) Coomassie staining or (b) Western blotting with αVP1 (top, αVP1), αVP2 (middle, αVP2), or αVP3 (bottom, αVP3) antibodies. (c) Typical profile of VPg–dsRNA complexes purified from dissociated virions in the presence of SDS. The direction of sedimentation was from right to left, with fraction 11 (or 12) representing the top of each gradient.

Fig. 5

Biochemical characterization of purified IBDV RNP. (a) Agarose gel electrophoresis of (1) virions, (2) genome dsRNA molecules, (3) VPg–dsRNA complexes, and (4) RNPs visualized by bromide ethidium staining (top) or by Western blotting with αVP1 (top, αVP1), αVP2 (middle, αVP2), or αVP3 (bottom, αVP3) antibodies. (b) RNase III accessibility for IBDV dsRNA: (1) dsRNA in intact full E5 particles, (2) purified dsRNA, (3) dsRNA from VPg–dsRNA complexes, and (4) dsRNA from purified RNP were treated with increasing amounts of RNase III (from left to right). Lane c− shows controls for each experiment without RNase III treatment. Schematic cartoons are the same as those used in Fig. 2.

Fig. 6

Lengths of dsRNA molecules, VPg–dsRNA complexes, and RNPs. (a and b) Electron micrographs of metal-shadowed purified dsRNA molecules and the histogram of length measurements made from these micrographs (n = 62). (c and d) Electron micrographs of shadowed purified VPg–dsRNA complexes and the corresponding histogram (n = 50). The arrow indicates a circularized VPg–dsRNA complex, occasionally visualized. (e and f) Electron micrographs of shadowed purified RNP and the corresponding histogram (n = 55). Inset shows immunogold-labeled and shadowed RNPs after incubation with αVP3 antiserum and protein A conjugated to 5-nm colloidal gold particles. Bar represents 200 nm.

Fig. 7

IBDV RdRp activity. RNA polymerase activity was determined on the [α-³²P]UTP incorporated to the reaction products that were separated on 0.7% agarose gel. Mixture reactions were supplemented with full virions (IBDV), RNP complexes, VPg–dsRNA complexes, or purified dsRNA molecules. Each measurement corresponds to the mean value from four independent experiments such that the highest value obtained corresponds to 100%. Insets show autoradiography of the reaction products for a single experiment performed with RNP complexes or full virions (IBDV).