A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production

Abstract

The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2A␣ cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process. IMPORTANCEDespite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified a basic cluster of amino acids in the N terminus of YFV NS2A which inhibited virion assembly upon mutation. The defect was rescued by a spontaneously occurring mutation in NS3. Our study proves an interaction between NS2A and NS3, which, remarkably, was maintained for the NS2A mutant in the presence and absence of the NS3 mutation. This suggests a role for other viral and/or cellular proteins in virion assembly. Residues important for YFV virion production reported here only partially coincided with those reported for other flaviviruses, suggesting that the determinants for particle production are virus specific. Reconstruction of a YFV encoding tagged NS2A paves the way to identify further NS2A interaction partners. Y ellow fever virus (YFV) belongs to the genus flavivirus within the family Flaviviridae. Clinical disease associated with this prototype flavivirus can result in acute viral hemorrhagic fever (1). Other important human flaviviruses include dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus, and tick-borne encephalitis v...

Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus

“…Remarkably, an adaptive mutation, K127KK, in NS2B could compensate for the defects in virion assembly, as well as NS2A-NS2B interaction. These results indicate that NS2B participates in virion assembly through interaction with NS2A, a protein known to be important for flavivirus assembly (17)(18)(19)(20). Overall, our data demonstrate, for the first time, that flavivirus NS2B participates in virion assembly.…”

“…NS2A is the first identified NS protein engaged in both processes. Mutations within NS2A of YFV, DENV-2, and the Kunjin subtype of West Nile virus (KUNV) were found to affect viral RNA synthesis and/or the production of infectious particles (18,19,26). Additionally, the second-site mutations in NS3 helicase could compensate for the defect in infectious YFV production caused by NS2A mutations (17,19), which suggested that the genetic interaction between NS2A and NS3 is critical for virion production (17,19).…”

“…Mutations within NS2A of YFV, DENV-2, and the Kunjin subtype of West Nile virus (KUNV) were found to affect viral RNA synthesis and/or the production of infectious particles (18,19,26). Additionally, the second-site mutations in NS3 helicase could compensate for the defect in infectious YFV production caused by NS2A mutations (17,19), which suggested that the genetic interaction between NS2A and NS3 is critical for virion production (17,19). In addition to the evidence from the compensatory mutations in the NS3 helicase domain (17,19), the role of NS3 in virion assembly has also been directly demonstrated by a mutation in YFV NS3 that selectively blocked infectious-virus production (27).…”

“…Additionally, the second-site mutations in NS3 helicase could compensate for the defect in infectious YFV production caused by NS2A mutations (17,19), which suggested that the genetic interaction between NS2A and NS3 is critical for virion production (17,19). In addition to the evidence from the compensatory mutations in the NS3 helicase domain (17,19), the role of NS3 in virion assembly has also been directly demonstrated by a mutation in YFV NS3 that selectively blocked infectious-virus production (27). During viral RNA synthesis, NS3 interacts with NS2B (7), NS4A (28), NS4B (29), and NS5 (30)(31)(32) to exert its enzymatic activities, facilitating viral RNA replication.…”

Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments

“…For example, mutation of amino acid 190 (K190S) in YFV NS2A had no effect on viral RNA synthesis but blocked the production of infectious virus (24). A basic cluster in the N terminus of YFV NS2A was identified as responsible for infectious-particle production (25). A mutation at amino acid position 59 (I59N) in WNV KUN blocked the production of secreted virus particles but not RNA replication (26).…”