Characterization of Human Norovirus Nonstructural Protein NS1.2 Involved in the Induction of the Filamentous Endoplasmic Reticulum, Enlarged Lipid Droplets, LC3 Recruitment, and Interaction with NTPase and NS4.

Human noroviruses (HuNVs) are the leading cause of gastroenteritis worldwide. NS1.2 is critical for HuNV pathogenesis, but the function is still unclear. The GII NS1.2 of HuNVs, unlike GI NS1.2, was localized to the endoplasmic reticulum (ER) and lipid droplets (LDs) and is accompanied by a distorted-filamentous ER morphology and aggregated-enlarged LDs. LC3 was recruited to the NS1.2-localized membrane through an autophagy-independent pathway. NS1.2, expressed from a cDNA clone of GII.4 norovirus, formed complexes with NTPase and NS4, which exhibited aggregated vesicle-like structures that were also colocalized with LC3 and LDs. NS1.2 is structurally divided into three domains from the N terminus: an inherently disordered region (IDR), a region that contains a putative hydrolase with the H-box/NC catalytic center (H-box/NC), and a C-terminal 251-330 a.a. region containing membrane-targeting domain. All three functional domains of NS1.2 were required for the induction of the filamentous ER. The IDR was essential for LC3 recruitment by NS1.2. Both the H-Box/NC and membrane-targeting domains are required for the induction of aggregated-enlarged LDs, NS1.2 self-assembly, and interaction with NTPase. The membrane-targeting domain was sufficient to interact with NS4. The study characterized the NS1.2 domain required for membrane targeting and protein-protein interactions, which are crucial for forming a viral replication complex.