The structural and functional studies of enveloped (+)ssRNA viruses: The structural proteins and their functions in viral assembly

I-Mei Yu, Purdue University

Abstract

The causative agent of severe acute respiratory syndrome is the SARS-associated coronavirus, SARS-CoV. The viral nucleocapsid protein plays an essential role in viral RNA packaging. In the present study, SARS-CoV N protein was expressed and purified. Its oligomeric state was studies using various biochemical and biophysical techniques and the results suggested that the N protein forms a homodimer in solution. Dimeric N proteins have a tendency to self-associate into tetramers and higher molecular weight oligomers at high concentrations. The dimerization domain of N was mapped to the C-terminal 285-422 amino acids through studies with truncated mutants. When in excess, this C-terminal domain inhibits the homodimerization of full length N protein by forming a heterodimer with the full length N protein. In addition, we describe the crystallization and the structural determination of the dimerization domain, consisting of residues 270 to 370, of SARS-CoV N protein. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin. Dengue virus is one of the most important flaviviruses that cause global public health problems. Flaviviruses first assemble into an immature form containing prM and E protein on the surface of the virions. This thesis describes studies of the maturation pathway of flaviviruses. Immature dengue viruses generated in the presence of NH4Cl contain prM protein that is believed to interact with surface E protein to prevent fusion. In vitro furin cleavage of prM in immature dengue particles results in 1000 fold increase in infectivity, demonstrating the protective role of prM in the maturation process. In addition, the furin cleavage occurs only at pH 5.0-6.0, a pH value that resembles the acidic environment of the trans-Golgi network (TGN). This low pH environment triggers a structural change on immature dengue virions leading to the exposure of the furin recognition site and subsequently the cleavage of prM. Furthermore, the reversibility of the low pH induced conformational change was demonstrated. CryoEM images show distinct features and morphologies of immature virions at low pH and neutral pH. The structure of the immature particles at low pH is proposed to serve as an additional intermediate state of the viral assembly process.

Degree

Ph.D.

Advisors

Chen, Purdue University.

Subject Area

Virology

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