Macromolecular interactions required for alphavirus assembly

Katherine Einterz Owen, Purdue University

Abstract

Alphaviruses are small, enveloped viruses found in worldwide distribution. The virion organization of this genus of viruses is quite elegant. The single stranded, genomic RNA is surrounded by multiple, symmetric layers which interact with each other. The inner layer is composed of 240 identical copies of the nucleocapsid protein, whereas the outer layer consists of 240 copies each of two different viral glycoproteins, E1 and E2. Although the assembly of these layers is an integral part of the alphavirus life cycle, relatively little is known about the requirements for this process. This project has made progress in defining in molecular detail the interactions which are necessary for assembly of the virion. Using mutational analyses and structural studies, specific amino acids and motifs which are essential for alphavirus assembly have been identified. On the basis of this experimental evidence, a model which encompasses the steps involved in the assembly pathway has been developed. This model traces the fate of at least three interactions required for alphavirus assembly: specific recognition and binding of the viral RNA by the NCP, interaction of the NCP subunits to form the intact core, and binding of the assembled NC to the glycoproteins which results in budding of the nascent virion from the host cell. The data suggest that specific RNA recognition of the viral RNA occurs through the binding of ten amino acids from the nucleocapsid protein to a specific sequence of viral RNA. Subsequent assembly of the nucleocapsid core probably occurs through discrete assembly intermediates, either dimers or capsomeres. The amino acids involved in maintaining these assembly intermediates have been identified, and mutational analyses have confirmed their importance. Additional studies have suggested that budding of the assembled core is driven by the binding of the outer glycoproteins to the inner nucleocapsid core. This is achieved through the interaction of two hydrophobic amino acids on the cytoplasmic domain of E2 and a hydrophobic pocket found on the surface of the assembled core. The interaction results in wrapping of the membrane and glycoproteins around the inner core and budding.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Molecular biology|Biophysics|Microbiology

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