The Effects of HCV Core Protein on Mitochondrial Respiration 公开

Jiao, Shiyin (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/00000064j?locale=zh
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Abstract

Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, which may contribute to cirrhosis, culminating in liver failure and hepatocellular carcinoma. According to the World Health Organization (WHO), more than 170 million people worldwide are estimated to be affected. Many promising antiviral drugs are under investigation, but the mechanisms of pathogenesis have not been completely understood. The most common regimens of treatment have not reached a stably high viral response, whereas newly approved drugs are prohibitively expensive, and no vaccine against HCV is currently available. Therefore, new therapeutic approaches to treat HCV infection would be greatly beneficial. The HCV is an enveloped positive-stranded RNA virus. HCV genome encodes a polyprotein precursor of 3012 amino acid residues, which is then processed by cellular and viral proteases to produce 10 active proteins for viral replication and assembling. These include structural proteins Core, E1 and E2, 6 nonstructural proteins, and p7. The HCV Core gene is highly conserved, and previous studies have shown that the Core protein is involved in various cellular processes, including cell proliferation and apoptosis. It performs specific functions upon localizing to important organelles such as the nucleus, ER, and mitochondria. However, the effects and mechanisms of the Core protein on mitochondrial functions are not clear. In this project, I constructed the HCV Core gene with a mitochondrial targeting sequence, cloned into a CMV plasmid vector, and transformed into bacteria cells. The amplified recombinant plasmids were then transfected into human HepG2 cells, translated into recombinant Core protein, and translocate to the mitochondria. Measurements of mitochondrial respiration in intact cells show that cells expressing the Core protein have higher rates of basal respiration, ATP turnover, maximal respiration, and spare respiratory capacity. These observations are not evidence of mitochondrial dysfunction as reported by others, but I propose that the cells may have evolved mechanisms to cope with the stress imposed by the Core protein, and there is selection for cells with higher mitochondrial respiration efficiency and capacity. These results provide new insight into the pathogenesis of hepatitis C, and further investigation is required to confirm my hypothesis.

Table of Contents


I. Introduction 1
II. Materials and Methods 4
Part I. Cloning and Expression of HCV Core Protein in HepG2 Cell 4
Part II. Measurement of Mitochondrial Respiration 7
III. Results 8
PCR Amplification and Cloning 8
Presence of Core Protein in HepG2 Cells 11
Effects of HCV Core Protein on Mitochondrial Respiration 13
IV. Discussion 15
V. Figures
Figure 1. Viral structure and genome composition of HCV 3
Figure 2. Construct of MTS-HCV Core gene. 8
Figure 3. Gel electrophoresis of PCR products amplified from HCV Core gene. 9
Figure 4. (A) Recombinant plasmid CMV-MTS-Core. 10

(B) Transformation of the recombinant plasmid into competent E. coli cells.
Figure 5. PCR genotyping selecting for clones transformed with MTS-Core plasmid. 10
Figure 6. Expression of HCV Core protein in HepG2 cells. 12
Figure 7. Change in oxygen consumption rate (OCR) as the cell respiration control experiment proceeds. 15
Figure 8. A representative cell respiratory control experiment. 18
Figure 9. Electron Transport Chain (ETC) in the mitochondrion and the effects of ETC disrupting drugs. 19
Figure 10. Measurements of oxygen consumption in the cell respiratory control experiment. 22
VI. Works Cited 25

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