%0 Generic
%A Veenstra, Robin
%C Heidelberg
%D 2025
%F heidok:36107
%R 10.11588/heidok.00036107
%T Orchestration of viral replication by norovirus NS4 via host membrane rearrangement and interaction with viral NS1-2
%U https://archiv.ub.uni-heidelberg.de/volltextserver/36107/
%X Norovirus is the leading cause of acute gastroenteritis worldwide with over half a billion infections each year. Belonging to the positive-sense, single-stranded RNA viruses, norovirus is categorized in the order of Picornavirales in the family of Caliciviridae. Similar to other positive-sense, single-stranded RNA viruses, norovirus infection is characterized by the vesicular rearrangement of intracellular membranes. As these vesicular structures colocalize with viral nonstructural (NS) proteins and double-stranded RNA, these membranous alterations harbor the norovirus replication complex (RC). Although norovirus RC biogenesis remains poorly understood, the membrane-associated nonstructural proteins NS1-2, NS3 and NS4 are involved in this process. In particular, NS4 has been shown to induce a variety of membrane rearrangements, hinting at a key role for this protein in membrane remodeling during norovirus infection. However, little is known about the exact mechanism by which NS4 induces membrane alterations or how NS4 interacts with other nonstructural proteins in the norovirus RC.   Therefore, this thesis aimed to unravel the molecular determinants of the membrane-rearranging and membrane-associating properties of NS4 as well as to explore protein-protein interactions between NS4 and other norovirus nonstructural proteins.   The first objective of this thesis was to identify amino acid residues within NS4 essential for viral replication. To this end, an amino acid alignment was performed to identify residues which are conserved among multiple genogroups. The reverse genetics model of GV murine norovirus (MNV)-1.CW1 was used to assess the importance of these residues for viral replication. Several of these amino acids were shown to be indispensable for viral replication. However, single mutation of these residues in the context of GII.4 New Orleans (NO) NS4 or ORF1 did not significantly impact membrane remodeling upon expression.  The second objective of this thesis was to identify and to characterize the NS4 domain responsible for the membrane-associating and membrane-rearranging abilities of NS4. First, in silico analyses predicted three distinct regions in NS4: an N-terminal structured region (SR), a large alpha helix (AH4) and a C-terminal nonstructured region (NSR). GFP fusion proteins of these regions were expressed in Huh7-T7 Lunet cells and subsequent immunofluorescence identified AH4 as the major determinant of membrane association of NS4. Since membrane-associating alpha helices are often amphipathic, the amphipathicity of AH4 of GII.4 NO and GV MNV-1.CW1 was examined in silico. Interestingly, AH4 displayed comparable amphipathicity in both noroviruses. To explore the importance of the amphipathicity of AH4 for viral replication and the induction of membrane alterations, amphipathic mutants of GII.4 NO NS4 were designed and expressed in Huh7-T7 Lunet cells, followed by immunofluorescence and electron microscopy (EM). GII.4 NO NS4 mutants with a decreased amphipathic moment lost membrane association and did not induce membrane enwrapment of lipid droplets, a hallmark of membrane remodeling observed upon NS4 expression. However, deletion of AH4 from GII.4 NO ORF1 did not seem to impact membrane remodeling, challenging the view that AH4 is the main determinant of membrane rearrangements. Nevertheless, mutations in MNV.CW-1 which impaired the amphipathicity of AH4 abolished membrane association of NS4 as well as viral replication, highlighting the importance of the amphipathicity of AH4 for norovirus replication.  The third and final aim of this thesis was to identify and to characterize the protein-protein interactions between NS4 and other nonstructural proteins. Co-expression and immunoprecipitation revealed a strong interaction between GII.4 NO NS1-2 and NS4. Further mapping of NS4 pointed to the C-terminus of AH4 as the minimal binding region for NS1-2. Moreover, alanine scanning of this minimal binding region identified five specific amino acid residues that facilitated the interaction with NS1-2. Since many of these amino acid residues are conserved among multiple genogroups, corresponding sites were mutated in the MNV-1.CW1 genome to study the effect on the NS1-2-NS4 interaction during MNV infection. In line with the findings for GII.4 NO, mutation of corresponding amino acid residues in MNV-1.CW1 NS4 indeed abrogated the interaction between NS1-2 and NS4 and abolished viral replication. Finally, guided by AlphaFold predictions of the NS1-2-NS4 interaction, the C-terminal hydrophobic region of NS1-2 was found to bind to NS4 via specific residues within this region.  In conclusion, this thesis provides evidence that a large alpha helix in norovirus NS4 is essential for viral replication. The amphipathicity of this alpha helix determines the membrane association and is critical for the membrane-rearranging properties of NS4. In addition, this alpha helix interacts through specific residues with norovirus NS1-2. Breaking the NS1-2-NS4 interaction renders MNV replication-deficient, indicating that this interaction is pivotal for viral replication. Future research should therefore examine the use of this interaction as a druggable target against norovirus infection.