Investigation of the role of the e2 endodomain in sindbis virus assembly

by (John Allen). West, John

WEST, JOHN ALLEN. Investigation of the Role of the E2 Endodomain in Sindbis Virus Assembly. (Under the direction of Dr. Dennis T. Brown) Sindbis virus (SV) is the prototype member of the alphavirus genus belonging to the family Togaviridae. SV is a mosquito-borne virus that can be transmitted to both humans and other animals via mosquito bloodmeal. Structurally, SV is composed of three proteins; two envelope glycoproteins (E1 and E2) and capsid protein (C). The outer shell (envelope) of the virus particle is composed of E1 and E2 proteins and is stabilized by E1-E1 protein interactions. Between the outer shell and the inner shell (nucleocapsid), which is composed of capsid protein and the viral RNA, is a host derived membrane bilayer. Connecting the outer shell to the nucleocapsid core is a critical step in virus maturation without which a mature virus particle could not be formed. The proteins involved in this interaction are E2 (specifically a 33 amino acid cytoplasmic domain) and the capsid protein. The research presented here provides new information on this interaction and sheds light on the role of two specific regions within the E2 endodomain. We have determined that two residues within the conserved TPY domain (aa 398-400) in the E2 endodomain are dispensable for virus assembly, however they are critical for the production of infectious virus. Additionally, a domain encompassing amino acids 409-417 in the E2 endodomain has been determined to be critical for both virus assembly and function. These observations identify two specific regions in the E2 endodomain that play different roles in virus assembly. The conserved TPY domain is critical for infectivity, while the domain from amino acids 409-417 is essential for virus assembly. The identification of amino acids 409-417 as a critical domain for nucleocapsid binding is an important step in understanding how these viruses assemble. In the future this domain could be a target for vaccine development. Investigation of the Role of the E2 Endodomain in Sindbis Virus Assembly By John West A dissertation submitted to the Graduate Faculty of North Carolina State University In partial fulfillment of the Requirements for the degree of Doctor of Philosophy In Biochemistry Raleigh, NC 2005 Approved by: _________________________ _________________________ __________________________ _________________________ Co-chair of Advisory Committee Chair of Advisory Committee