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Genomic Screening Identifies Novel Human Kinases As Suppressors Of HCV Infection

Lee, Aram (2013) Genomic Screening Identifies Novel Human Kinases As Suppressors Of HCV Infection. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

The Human kinome includes between 500-600 known kinases and open reading frames (ORFs) that play key roles in regulating many cellular processes. Some of these cellular processes include, signal transduction, metabolism, transcription, cell cycle progression, cell movement, apoptosis, differentiation and protein phosphorylation. Various protein and lipid kinases have previously been shown to control viral life cycles. The lipid kinase PI4KIIIA was found to be required for hepatitis C virus (HCV) replication in several genomic screens using small interference RNAs that target human kinases. Although such a loss-of-function approach has led to the identification of host cell factors that are required for HCV replication, no kinases have been found with respect to antiviral activity. Here we adopt a genomic approach to identify human kinases that negatively regulate HCV infection. To this end, a library of activated kinases, which consists of 192 human kinases and kinase-related ORFs, was cloned into a retroviral vector, which adds a myristoylation sequence and flag-epitope tag to each ORF. Using an overexpression screening approach, we identified that several kinases, including Cyclin-dependent kinases regulatory subunit 1 (CKS1B), Mitogen-activated protein kinase kinase 5 (MAP2K5) and Protein kinase C and casein kinase substrate in neurons 1 (PACSIN1), potently suppressed HCV infection. The expression of these active kinases did not activate the nuclear factor-kappaB (NF-κB) pathway or the production of type I interferon and interferon-stimulated genes (ISGs), instead they acted at different post-entry stages. Specifically, CKS1B significantly inhibited viral RNA replication; MAP2K5 also suppressed virus replication and appeared to exert additional inhibition after replication. PACSIN1, by contrast, inhibited HCV infection via a yet-to-be-defined mechanism.

Public Health significance: Hepatitis C occurs worldwide and is the major cause of liver diseases. Approximately three quarters of people infected with HCV become chronic carriers of the virus and of these chronically infected, majority of the patients develop liver cirrhosis and hepatocellular carcinoma. Current treatments available to patients are costly and cause severe side-effects that result in poor patient compliance. Thus, the inhibitory effects as shown here by targeting kinases, shows the potential of combating HCV infection by activating specific kinase-mediated pathways, which would be an alternative method of treatment of these patients.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Lee, Aramarl75@pitt.eduARL75
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorWang, Tianyitywang@pitt.eduTYWANG
Committee MemberReinhart, Todd A.reinhar@pitt.eduREINHAR
Committee MemberAyyavoo, Velpandivelpandi@pitt.eduVELPANDI
Committee MemberSluis-Cremer, Nicolasnps2@pitt.eduNPS2
Date: 29 January 2013
Date Type: Publication
Defense Date: 30 November 2012
Approval Date: 29 January 2013
Submission Date: 27 November 2012
Access Restriction: 3 year -- Restrict access to University of Pittsburgh for a period of 3 years.
Number of Pages: 82
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Hepatitis C virus, kinases, myristoylation, CKS1B, MAP2K5, PACSIN1
Date Deposited: 29 Jan 2013 22:31
Last Modified: 15 Nov 2016 14:07
URI: http://d-scholarship.pitt.edu/id/eprint/16499

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