Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

Abstract

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

Establishment of a CPER Reverse Genetics System for Powassan Virus Defines Attenuating NS1 Glycosylation Sites and an Infectious NS1-GFP11 Reporter Virus

“…Currently, there are no established mechanisms for attenuating POWVs or defining determinants of POWV neurovirulence. To generate recombinant POWVs (revPOWVs), we used a CPER approach (36,(59)(60)(61)(62)(63)(64) to develop a robust POWV reverse genetics system (Fig. 1B-D).…”

“…NS1 is translocated into the lumen of the ER where it both orchestrates cytoplasmic replication complex assembly and ER-Golgi virion maturation (31,35,36) . NS1 is modified by N-linked glycosylation with high-mannose carbohydrates that direct dimerization (31,35,38,39) , and Golgi compartment processing of complex oligosaccharides facilitates NS1 secretion (39,79) .…”

“…In contrast to Env, translocated NS1 is glycosylated and secreted from cells, and required for cytoplasmic FV replication (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) . Env and NS1 proteins both contain conserved and FV specific glycosylation sites, and changes in the glycosylation of either protein can impact FV virulence (31,35,38) .…”

“…Analysis of Japanese encephalitis virus (JEV) NS1 mutants distinguished NS1 residues required for replication (AA160) from viral particle formation (AA273) (36) . However, mutating specific NS1 N-linked glycosylation sites (NxS/T) has been reported to either enhance or inhibit the replication and neurovirulence of discrete FVs (34,45,46,48) .…”

“…Typical reverse genetics approaches are based on cloning viral RNA genomes into bacterial plasmids or artificial chromosome vectors with the toxicity and stability issues of large viral inserts that magnify the complexity of genetic virus modification (50)(51)(52)(53)(54)(55)(56) . The circular polymerase extension reaction (CPER) was originally devised to clone complex genes (57,58) and subsequently used to bypass the plasmid instability of large viral inserts and to rapidly generate mutant and reporter viruses (36,(59)(60)(61)(62)(63)(64) . CPER has now been used to investigate virulence determinants in West Nile virus (WNV), Zika virus (ZIKV), Yellow Fever and Dengue virus (DENV) and JEV (36, 59-63, 65, 66) , and has the potential to be applied to other positive stranded RNA viruses (61,67) .…”