Defining the Role of the Nonstructural Protein 4B C-Terminal Domain in Hepatitis C Virus Replication
Open Access
- Author:
- Aligo, Jason Alan
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 17, 2009
- Committee Members:
- Craig Eugene Cameron, Dissertation Advisor/Co-Advisor
Kouacou Konan, Committee Member
Craig Eugene Cameron, Committee Member
Joseph C. Reese, Committee Chair/Co-Chair
Avery August, Committee Member
Leslie Joan Parent, Committee Member - Keywords:
- HCV
NS4B
Virus
Replication
CTD - Abstract:
- Like most positive-strand RNA viruses, the Hepatitis C Virus (HCV) replication complex is associated with rearranged cytoplasmic membranes. For HCV, a novel membrane structure, termed the membranous web, has been observed in HCV-infected cells or in cells expressing the mature NS4B. However, we still do not understand the role of NS4B protein in HCV genome replication. NS4B could contribute by forming the web structure, providing the scaffold for other replicase proteins, or in binding HCV RNA. To better understand the role of NS4B in HCV genome replication, we introduced mutations in the poorly characterized C-terminal domain (CTD) in the context of the HCV subgenomic replicon. Interestingly, these mutants all resulted in a significant decrease in HCV replication as determined by colony formation assay. Transient expression of mutant replicons, followed by immunofluorescence indicated that certain NS4B CTD point mutantss resulted in reticular staining rather than punctate foci shown in cells expressing the WT replicon. These same mutants caused redistribution of NS5A to these reticular structures, indicating that the CTD may play a role in membranous web formation or in recruitment of other replicase proteins (or both). To test the hypothesis that the NS4B CTD might play a role in membranous web formation, we deleted the entire CTD in the context of NS4B protein alone. Deletion or removal of parts of this domain resulted in loss of webs as visualized via electron microscopy. However, expression of the CTD alone did not result in web formation, arguing that the NS4B CTD was required but not sufficient to form the web structure. Further, our data indicate that deletion of the NS4B CTD prevents NS4B from co-localizing with Rab5, an early endosomal GTPase that our laboratory has previously found to be required for HCV replication. Finally, studies with an NS4B protein from an HCV-related pestivirus (Bovine Viral Diarrhea Virus - BVDV) indicated that a chimeric protein consisting of the HCV N-terminus of NS4B and the C-terminus from BVDV NS4B induces the formation of foci. However, these foci were of a different morphology compared to those formed after expression of HCV NS4B. The chimeric NS4B protein also failed to co-localize with Rab5. These results suggest that although the NS4B CTD from related flaviviruses play a role in the formation of NS4B-asociated foci, that these structures may have alternate functions or utilize different host factors. These factors may also be unique to the host cell utilized by these viruses. The significance of these findings and the ramifications for the flavivirus family is discussed.