Characterization of the Escherichia coli exopolyphosphatase and production of antibodies against the purified recombinant E3 protein of Venezuelan Equine Encephalitis virus

Anita Ghosh, Purdue University

Abstract

The Escherichia coli Exopolyphosphatase plays a critical role in the adaptation of bacterial cells to environmental starvation. Under normal conditions, the protein maintains basal levels of inorganic polyphosphate in the cells by the processive hydrolysis of long-chain polyphosphates. When the stringent response is evoked as a result of amino-acid deprivation in the media, Ppx is competitively inhibited by guanosine 5’-triphosphate 3’-diphosphate and guanosine 5’-diphosphate 3’-diphosphate. This leads to the accumulation of inorganic polyphosphate, which is required by bacterial cells to adapt to the depleted environment. When conditions revert to the relaxed state, Ppx resumes the processive hydrolysis of the long-chain polyphosphates. The mechanism used by the guanosine nucleotides to regulate Ppx activity is not known. In this thesis, we report the identification of the active site for polyphosphate and guanosine 5’-triphosphate 3’-diphosphate hydrolysis. Additionally, the structure of Ppx bound to guanosine 5’-diphosphate 3’-diphosphate is presented, which provides a better understanding of the events leading to the inhibition of polyphosphate hydrolysis. Alphaviruses are arthropod-borne RNA viruses that cause encephalitis, arthritis, fever, rashes etc. in a broad range of vertebrate hosts. The virus enters a susceptible host cell by receptor-mediated endocytosis. Entry and membrane fusion is initiated by the glycoproteins E2 and E1, respectively. The glycoproteins are arranged in a heterotrimeric spike on the surface of the virus. Formation of the spike is initiated in the endoplasmic reticulum, where E1 and E2 are synthesized as part of a polyprotein. E2 is synthesized as a precursor PE2 that is cleaved by furin-like proteases. E3 is predicted to stabilize the E2/E1 heterodimer under the low pH conditions of the secretary pathways and prevent the premature exposure of the fusion peptide. However, the mechanisms driving these effects remain unknown, partly due to the lack of tools to directly detect E3 in biochemical assays. Here we report the purification of the recombinant E3 protein of Venezuelan Equine Encephalitis virus from E. coli and the generation of anti-E3 polyclonal antibodies. We have also attempted to characterize the recombinant protein structurally.

Degree

Ph.D.

Advisors

Sanders, Purdue University.

Subject Area

Biochemistry|Immunology

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