Abstract
Viral capsids are metastable structures that perform many essential processes; they also act as robust cages during the extracellular phase. Viruses can use multifunctional proteins to optimize resources (e.g., VP3 in avian infectious bursal disease virus, IBDV). The IBDV genome is organized as ribonucleoproteins (RNP) of dsRNA with VP3, which also acts as a scaffold during capsid assembly. We characterized mechanical properties of IBDV populations with different RNP content (ranging from none to four RNP). The IBDV population with the greatest 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 results suggest that RNP dimers are the basic stabilization units of the virion, provide better understanding of multifunctional proteins, and highlight the duality of RNP as capsid-stabilizing and genetic information platforms.
Highlights
ribonucleoprotein particles (RNP)-A has two open reading frames (ORF)
RNP-B is monocistronic and encodes the RNA-dependent RNA polymerase (RdRp) VP1, which is packaged within the virion as a free protein or is covalently linked to the 5′ ends of the dsRNA molecules
To characterize the IBDV capsid mechanical reinforcement mediated by VP3/dsRNA RNP, we studied capsid response to mechanical fatigue that consisted of repeated loading forces below the breaking force (~150 pN), which allow controlled peeling of the virus shell and direct access to the internal RNP core[32]
Summary
RNP-A has two open reading frames (ORF). The short ORF encodes VP5, a nonstructural protein involved in virus egress, and the large ORF codes for a polyprotein that is cotranslationally processed by the VP4 viral protease[13] to yield the precursor capsid protein pVP2 as well as VP3 and VP414,15. pVP2 can be further processed by a host protease[16] and/or by VP2 itself[17] to yield mature VP2 and several C-terminal fragments. VP3 (258 residues) is a multifunctional protein that, in addition to its RNA-binding activity, interacts with itself[18,19], with pVP220,21, or with VP122–24. VP3 acts as a scaffolding protein during capsid morphogenesis and, through electrostatic interactions of its final five, mainly acidic residues, co-assembles with pVP220. AFM mechanical fatigue experiments at low force indicated that RNP organized as dimers within the capsid, which increased viral particle stability in the harsher extracellular environment. Our results indicate once VP3 is bound to the viral genome, it mediates capsid stability through a new set of interactions with the mature VP2 protein. These analyses support simultaneous multifunctionality as a mode of VP3 action. Identification of these physical features using a relatively simple viral system susceptible to manipulation indicates new approaches to the study of moonlighting proteins
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