IDENTIFICATION OF A PROTEIN-BINDING SITE ON THE SURFACE OF THE ALPHAVIRUS NUCLEOCAPSID AND ITS IMPLICATION IN VIRUS ASSEMBLY

Background: Many enveloped viruses exit cells by budding from the plas ma membrane, The driving force for budding is the interaction of an in ner protein nucleocapsid core with transmembrane glycoprotein spikes, The molecular details of this process are ill defined, Alphaviruses, s uch as Sindbis virus (SINV) and Semliki Forest virus (SFV), represent some of the simplest enveloped viruses and have been well characterize d by structural, genetic and biochemical techniques. Although a high-r esolution structure of an alphavirus has not yet been attained, cryo-e lectron microscopy (cryo-EM) has been used to show the multilayer orga nization at 25 Angstrom resolution, In addition, atomic resolution stu dies are available of the C-terminal domain of the nucleocapsid protei n and this has been modeled into the cryo-EM density. Results: A recom binant form of Sindbis virus core protein (SCP) was crystallized and f ound to diffract much better than protein extracted from the virus (2. 0 Angstrom versus 3.0 Angstrom resolution), The new structure showed t hat amino acids 108 to 111 bind to a specific hydrophobic pocket in ne ighboring molecules, Re-examination of the structures derived from vir us-extracted protein also showed this 'N-terminal arm' binding to the same hydrophobic pocket in adjacent molecules, It is proposed that the binding of these capsid residues into the hydrophobic pocket of SCP m imics the binding of E2 (one of two glycoproteins that penetrate the l ipid bilayer of the viral envelope) C-terminal residues in the pocket, Mutational studies of capsid residues 108 and 110 confirm their role in capsid assembly. Conclusions: Structural and mutational analyses of residues within the hydrophobic pocket suggest that budding results i n a switch between two conformations of the capsid hydrophobic pocket. This is the first description of a viral budding mechanism in molecul ar detail.