Publication Date

2009-02-17

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2008-12-19

First Committee Member

Charles Luetje

Second Committee Member

Carlos Moraes

Third Committee Member

Sandra Lemmon

Fourth Committee Member

Beatriz Fontoura

Fifth Committee Member

Michael Roth

Abstract

Proper mRNA nuclear export is essential for harmonious growth and maintenance of a cell. An effective weapon influenza virus employs to hijack a host cell is its ability to inhibit such export. Exactly how influenza virus achieves this inhibition is not fully known. Here, we demonstrate that upon infection, influenza virus degrades two nucleopore proteins (Nup98 and Nup96), which play a key role in mRNA nuclear export. Also, a main virulence factor of influenza virus (non-structural protein 1, NS1) binds directly to NXF1 and E1B-AP5, two key constituents of the mRNA export pathway (NXF1/NXT pathway) responsible for exporting bulk (~70%) mRNA from the nucleus. By increasing the expression levels of members of the NXF1/NXT pathway, we were able to reverse NS1-mediated inhibition of gene expression. On the other hand, by decreasing the levels of members of the NXF1/NXT pathway, we demonstrated that host cells become more sensitive to influenza virus infection and produce more viral particles. These results demonstrate undiscovered influenza-mediated host interactions that may be used to medicinally inhibit influenza virus. To this end, high-throughput screens were designed to identify small molecule antagonists of both NS1-mediated inhibition of gene expression and influenza virus-mediated cell death. Seventy-one compounds were identified, and the most potent molecule (named compound #8) was examined further. We found that compound #8 releases influenza virus-mediated mRNA nuclear export blockage and decreased viral replication and viral gene expression. Thus, the bulk mRNA nuclear export machinery is vital to antiviral response, and compound #8 enhances its ability to fight the cytopathic effects of NS1 and influenza virus. In conclusion, our data demonstrate that the mRNA export machinery is disrupted by influenza virus, and that this machinery also facilitates an antiviral function. We have also shown that these two events can be manipulated chemically to attenuate the negative effect of the virus and enhance the positive antiviral effect of the mRNA export machinery, thereby providing a powerful, new strategy against the ever-present, global threat of influenza virus.

Keywords

MRNA Nuclear Export; Influenza Virus; Antivirals; Ns1

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