Structural investigation of alphaviruses and geminiviruses by cryo -electron microscopy

Wei Zhang, Purdue University

Abstract

My thesis work involves the coordinated application of cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction techniques to study virus structures and to understand questions about how viruses are assembled and how they infect a wide range of hosts including plants and animals. Structures of four viruses and their mutants were studied: (1) Alphaviruses (family Togaviridae), including Aura virus (AURA), Ross River virus (RRV) and Sindbis virus (SINV); (2) Maize streak virus (MSV) (family Geminiviridae). Alphaviruses are a group of enveloped, positive-stranded RNA viruses. Although previous cryo-EM studies demonstrated the T = 4 icosahedral organization of these viruses, there is ambiguity concerning the locations of the surface glycoproteins E1 and E2. Biochemical experiments showed that each of these glycoproteins has two glycosylation sites. The structures of native SINV, RRV, AURA and five SINV deglycosylation mutants were reconstructed at 22 Å resolution, and the locations of glycosylation sites on the E1 and E2 glycoproteins of these viruses were determined by means of difference mapping techniques. The locations of the carbohydrate sites show that the E1 monomers lie in an orientation parallel to the virus surface, and the E2 molecules project radially outwards from the viral membrane. The heparin binding site on RRV was also located. Heparan sulfate molecules are cellular surface moieties that function as an initial receptor for many viruses. Wild type RRV does not bind heparin, whereas an E2-N218R mutant virus binds heparin and infects a new host type. The locations of heparin binding sites, determined from a difference map computed between the RRV mutant-heparin complex and the RRV mutant, were found at the distal end of the spikes, overlapping a known RRV E2 monoclonal antibody Fab binding sites. AURA efficiently encapsidates both genomic RNA (11.8kb) and subgenomic RNA (4.2kb) to form virus particles. Cryo-EM reconstructions indicated that the T = 4 organization is not determined by its RNA contents. MSV, one member of the ssDNA geminiviruses, has an unusual, two-headed morphology. We report the first structure of a geminivirus particle determined from EM images of frozen-hydrated MSV samples. The particle, of dimensions 220 x 380 Å, has a capsid that is organized with 52 point-group symmetry. Each half particle ‘head’ consists of the coat proteins arranged with quasi-icosahedral symmetry.

Degree

Ph.D.

Advisors

Baker, Purdue University.

Subject Area

Microbiology

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