The Role of NS3 Helicase Domain in Hepatitis C Virus Particle Assembly
Caroline Bouter
Doctoral thesis
Date of Examination:
2012-11-27
Date of issue:
2013-11-22
Advisor:
Hufert, Frank Torsten Prof. Dr.
Referee:
Hufert, Frank Torsten Prof. Dr.
Referee:
Doenecke, Detlef Prof. Dr.
Persistent Address: http://hdl.handle.net/11858/00-1735-0000-001F-837C-0
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Abstract
English
Hepatitis C virus non-structural protein 3 (NS3) is a multifunctional protein that obtains serine protease, RNA helicase and NTPase activities. The enzyme is essential for viral RNA replication. Its protease domain cleaves the viral polyprotein at multiple sites. Furthermore, NS3 has recently been implicated to play a role in virus particle assembly. But its function in the assembly process is unknown so far. To determine the role of NS3 in HCV particle assembly alanine scanning mutagenesis within the NS3 helicase domain of chimerical genotype 2a full length replicons was performed. 25 mutants with changes of surface residues within the helicase domain were tested. 16 mutants showed moderate or severe defects in replication. 6 mutants were able to replicate and produce infectious virus as efficiently as wild type Jc1. 3 mutants efficiently replicated but had severe defects in virus particle assembly. Residues T612, H613 T537, T540, H541 and A217 within helicase subdomain 1 and 3 were identified being essential for HCV virion assembly. Domain 3 could be shown to be involved in particle assembly for the first time. Furthermore, RT-PCR and freeze-thawing experiments were used to determine the step within the particle assembly process affected by the NS3 helicase domain. The involvement of NS3 helicase domains 1 and 3 in virion assembly could be identified at an early step of the process prior to the formation of intracellular infectious particles. To determine interactions with NS2, protein complexes containing biotinylated NS2 were affinity captured with streptavidin magnetic beads. Previously described physically interactions between NS2 and NS3 were confirmed and 2 of the assembly defective NS3 helicase mutants were shown to influence these interactions but not completely disrupt NS2-NS3 protein complexes. The remaining mutant did not influence these interactions. Data within this thesis reveal that NS3 helicase is involved in an early step of HCV particle assembly. Helicase domain 1 and 3 could be identified to be essential in this process. Furthermore, interactions with NS2 seem to play a central role in the assembly process.
Keywords: Hepatitis C; non-structural protein 3; NS3; virus particle assembly; helicase domain
Schlagwörter: Hepatitis C; non-structural protein 3; NS3; virus particle assembly; helicase domain
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Human- und Zahnmedizin [2270]