| dc.description.abstract | Infectious bursal disease virus (IBDV), an avian double-stranded (ds)RNA virus of the
Birnaviridae family, encompasses features of positive and negative ssRNA viruses. IBDV has a
polyploid, bisegmented dsRNA genome, organized as ribonucleoprotein particles (RNP),
enclosed within a single, non-enveloped ~70-nm-diameter capsid with a T=13l lattice. The
capsid structural units are trimers of a single protein, VP2. The VP2 precursor (pVP2) is
encoded as part of the polyprotein NH2-pVP2-VP4-VP3-COOH; VP4 is the viral protease, able
to cleave its own N and C termini. VP3 is a multifunctional protein that, in addition to its RNAbinding
activity, interacts with itself, with pVP2, or with VP1 (the viral polymerase). Indirect
analyses suggested that VP3 acts as a scaffolding protein during capsid morphogenesis, recruits
VP1, and also encapsidates the viral genome. VP1 is found as a free protein or is covalently
linked to the 5’ ends of the two genome segments (the so-called VPg).
In IBDV-infected cells, the assembly pathway gives rise mainly to virions that package four
RNP, although minor discrete viral populations with lower RNP content are also formed. We
analyzed these IBDV populations (E1-E6, ranging from none to four RNP). Atomic force
microscopy (AFM) analysis of E1 empty T=13 particles, in combination with cryo-electron
tomography (cryo-ET) and three-dimensional single-particle cryo-electron microscopy (3D
cryo-EM) showed that VP3 is organized as a second, partially organized protein layer beneath
the T=13 layer of (p)VP2. This is the first observation in which VP3 is caught acting as a
scaffold protein in these procapsid-like particles. Interactions between VP2/pVP2 and VP3 were
located mainly on hexameric positions of the T=13 capsid.
IBDV virions dialyzed against a low ionic strength basic buffer rendered structurally preserved
RNP (comprised of dsRNA, VP3 and VP1/VPg), which were functionally competent for capsidindependent
RNA synthesis, and also effectively propagated the virus in transfection
experiments. We purified VPg-dsRNA complexes in non-denaturing conditions. Titration of
VPg-dsRNA with VP3 (monitored in electrophoretic mobility shift assays) allowed us to
determine the VP3 ratio at which RNP properties are restored. Negative stain EM and cryo-EM
analysis indicated that RNP are dsRNA molecules wrapped with VP3 monomers. Hybrid
methods (EM combined with high-resolution X-ray structures) were used to analyze the RNP
ends, in which one or two copies of VP1/VPg were found.
Finally, we analyzed the mechanical properties of the IBDV populations. The IBDV population
with the largest RNP number (and best fitness) showed greatest capsid rigidity. When bound to
dsRNA, VP3 reinforces virus stiffness. These contacts involve interactions with capsid
structural subunits that differ from the initial interactions during capsid assembly. Our analysis
suggest that RNP dimers are the basic stabilization units of the virion, which provides better
understanding of multifunctional proteins and highlights the duality of RNP as capsidstabilizing
and genetic information platforms. | en_US |
