Skip to main content
Duke University Libraries
DukeSpace Scholarship by Duke Authors
  • Login
  • Ask
  • Menu
  • Login
  • Ask a Librarian
  • Search & Find
  • Using the Library
  • Research Support
  • Course Support
  • Libraries
  • About
View Item 
  •   DukeSpace
  • Theses and Dissertations
  • Duke Dissertations
  • View Item
  •   DukeSpace
  • Theses and Dissertations
  • Duke Dissertations
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

Novel mechanisms of antiviral innate immune regulation by the hepatitis C virus NS3-NS4A protease

Thumbnail
View / Download
2.7 Mb
Date
2019
Author
Vazquez, Christine
Advisor
Horner, Stacy M
Repository Usage Stats
137
views
90
downloads
Abstract

Hepatitis C virus (HCV) evasion of the host immune system is largely mediated by the actions of the HCV NS3-NS4A protease complex, which consists of the serine protease and RNA helicase NS3 and its membrane targeting co-factor NS4A. NS3-NS4A has multiple functions in the HCV life cycle, with roles in both HCV replication and regulation of innate immune signaling. To regulate innate immune signaling, NS3-NS4A inactivates multiple signaling proteins, including MAVS, an adaptor protein in the RIG-I antiviral signaling pathway, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Inactivation of these host proteins results in an inhibition of downstream signaling through the transcription factor IRF3 and inhibition of the subsequent induction of IFN-β. What directs the multiple functions of NS3-NS4A throughout the HCV life cycle is largely undetermined. Here, we identify a tyrosine residue within the transmembrane domain of NS4A that uncouples the various function of NS3-NS4A.

First, to uncouple the roles of NS3-NS4A in replication and immune evasion, I focused on the NS4A transmembrane domain and generated an NS4A mutant (Y16F) in a full-length HCV infectious clone, a subgenomic replicon, and an over-expression construct. I then assessed viral replication of HCV wild-type (WT) and Y16F viruses by measuring replication of a subgenomic HCV replicon in two related liver hepatoma cell lines: Huh7, which have functional RIG-I signaling, and Huh-7.5 cells, which lack functional RIG-I signaling. The HCV Y16F virus replicated to similar levels as WT HCV in Huh-7.5 cells. However, in Huh7 cells, replication of HCV Y16F was decreased compared to the HCV WT. I used CRISPR-Cas9 gene editing to delete proteins in the RIG-I pathway, including RIG-I, MAVS, and IRF3, in Huh7 cells, infected these cells with HCV WT or Y16F viruses, and then measured virus replication. I found that Y16F viral replication was not restored to the levels of WT in Huh7-RIG-I KO cells or the Huh7-MAVS KO cells, but it was restored to the levels of WT in the Huh7-IRF3 KO cells. I also found that the HCV NS3-NS4A Y16F mutation reduced the ability of over-expressed NS3-NS4A to block IRF3 activation, as measured through nuclear translocation via immunofluorescence microscopy. Further the NS3-NS4A Y16F mutation also had a reduced ability to block the induction of interferon-stimulated genes during both HCV replication and infection. This reveals that HCV NS4A Y16 can regulate a RIG-I-independent, yet IRF3-dependent, signaling pathway that limits viral replication.

Second, to further characterize this RIG-I-independent, IRF3-dependent signaling pathway, I examined the interactions of HCV NS3-NS4A with two of its known host substrates, MAVS and Riplet. To test whether the Y16F mutation prevented NS3-NS4A cleavage of MAVS, I performed a MAVS cleavage assay during both overexpression of NS3-NS4A and MAVS and also during infection with HCV WT and Y16F viruses. NS3-NS4A Y16F was able to cleave MAVS just like WT during both conditions. Next, I found that over-expression of NS3-NS4A WT changed Riplet intracellular localization and that NS4A interacted with Riplet. However, the NS4A Y16F mutation prevented NS4A-Riplet interactions in both of these contexts. Interestingly, I found that Huh-7.5 cells express lower levels of Riplet protein and mRNA compared to Huh7 cells. When full-length Riplet was added exogenously to Huh-7.5 cells, HCV Y16F virus replication was reduced compared to WT. However, when a Riplet construct missing the RING domain, which is essential for Riplet signaling, was added exogenously to Huh-7.5, both WT and Y16F viruses now replicated similarly. Taken together, these data identify NS3-NS4A Y16 as important for regulating a previously uncharacterized Riplet-mediated signaling pathway that limits HCV infection.

Description
Dissertation
Type
Dissertation
Department
Molecular Genetics and Microbiology
Subject
Virology
Immunology
HCV
Immune evasion
Innate Immunity
MAVS
RIG-I
Riplet
Permalink
https://hdl.handle.net/10161/20109
Citation
Vazquez, Christine (2019). Novel mechanisms of antiviral innate immune regulation by the hepatitis C virus NS3-NS4A protease. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/20109.
Collections
  • Duke Dissertations
More Info
Show full item record
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.

Rights for Collection: Duke Dissertations


Works are deposited here by their authors, and represent their research and opinions, not that of Duke University. Some materials and descriptions may include offensive content. More info

Make Your Work Available Here

How to Deposit

Browse

All of DukeSpaceCommunities & CollectionsAuthorsTitlesTypesBy Issue DateDepartmentsAffiliations of Duke Author(s)SubjectsBy Submit DateThis CollectionAuthorsTitlesTypesBy Issue DateDepartmentsAffiliations of Duke Author(s)SubjectsBy Submit Date

My Account

LoginRegister

Statistics

View Usage Statistics
Duke University Libraries

Contact Us

411 Chapel Drive
Durham, NC 27708
(919) 660-5870
Perkins Library Service Desk

Digital Repositories at Duke

  • Report a problem with the repositories
  • About digital repositories at Duke
  • Accessibility Policy
  • Deaccession and DMCA Takedown Policy

TwitterFacebookYouTubeFlickrInstagramBlogs

Sign Up for Our Newsletter
  • Re-use & Attribution / Privacy
  • Harmful Language Statement
  • Support the Libraries
Duke University