Publication Date

2013-06-14

Availability

Open access

Embargo Period

2013-06-14

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2013-04-19

First Committee Member

Yanbin Zhang

Second Committee Member

Yanbin Zhang

Third Committee Member

Richard S. Myers

Fourth Committee Member

Feng Gong

Fifth Committee Member

Antoni Barrientos

Sixth Committee Member

Rebeca Geffin

Seventh Committee Member

Mathias G. Lichtenheld

Abstract

HIV-1 hijacks our cellular machinery to complete its life cycle. A better understanding of the interactions between cellular proteins and viral components will certainly lead to the discovery of new ways to inhibit viral replication. My primary research goals are directed toward understanding the basis of molecular interactions between viral and host factors (proteins or organelles) during infection of human cells. In my thesis, the role of mitochondria in the early stage of HIV infection has been evaluated by using a VSV-G pseudotyped HIV-based lentiviral vector. I have observed that cells lacking mitochondrial DNA (ρ0 cells), isolated from an established human osteosarcoma cell line, are defective in the ability to support virus infection when compared to their parental cells. This infection deficiency in ρ0 cells can be repaired by reintroducing mitochondria from ρ+ human cells (293T cells) using transmitochondrial technology. Inhibition of oxidative phosphorylation by mitochondrial inhibitors did not inhibit HIV infection in HOS cells (ρ+ cells) indicating that the reduced infection efficiency in ρ0 cells is not simply the result of reduced oxidative phosphorylation. Further analysis indicates that virus infection in ρ0 cells is blocked at steps after reverse transcription and before nuclear import. Confocal fluorescence microscope analysis shows the subcellular location of the majority of virus nucleoprotein particles (identified by the presence of the viral capsid protein) to be near or in contact with mitochondria. Co-fractionation of viral complexes with mitochondria from infected cells also supports the hypothesis of association of viral complexes with mitochondria. In conclusion, in my study I have shown that ρ0 cells are defective in the ability to support HIV infection and viral infection is inhibited at steps after reverse transcription and before nuclear import. Mitochondria may play an important role in the intracellular transport by directly association with viral complexes. A role for mitochondrial association of viral intracellular complexes has not been previously explored. The elucidation of this role will open new avenues for investigation of the early steps in HIV infection.

Keywords

HIV-1 infection; Mitochondrial DNA; Mitochondrial OXPHOS; ρ0 cell

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